Deeper Than Skin:

An Exploration into the Antioxidant Properties of Melanin

Sidney Cromwell

Intern Mentor

Beth Dungey

5/7/2018
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Abstract

Various sources have indicated that melanin has a free radical scavenging activity
comparable to antioxidants and may be used as a viable natural antioxidant. Research on
melanin’s antioxidant properties is important as the results could provide insight on why diseases
associated with free radical damage. Furthermore, melanin may be a an alternative to artificial
antioxidants added to food which have been proven to have adverse health effects such as cancer.
It was hypothesized that melanin displays antioxidant properties when exposed to free radicals.
A meta-analysis was conducted to disprove the hypothesis and to determine how effective
melanin is as a free radical scavenger. Due to limitations on human inquiries, in addition to a
lack of access to a lab equipment and samples, a meta-analysis was the most accurate, ethical,
and reasonable method to test them hypothesis. Results from the meta-analysis shows that fungal
melanin displays free radicals scavenging activity comparable to that of artificial melanin, at
least. One study on ​Auricularia auricula​ in particular concludes that AAFB melanin is more
effective at scavenging superoxides than BHT, an antioxidant. Nonetheless, there are limitations
in melanin’s free radical scavenging activity. The conclusion of this meta-analysis is that
melanin though shows some promise as an antioxidant, more studies must be conducted in order
to determine the best extent of this function on homeostasis in the body. Does melanin provide
superficial protection only or does it protect visceral organs from free radical that can cause
deterioration? Future studies may be able to answer this question and establish a definite
correlation between one’s melanin concentration and his or her risk of developing
neurodegenerative disease.
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Introduction

Melanin is a pigment produced by melanocytes which are located in the epidermis of the

skin. It has long been established that melanin is responsible for the prevention of skin cancer.

However, many are not aware of melanin’s antioxidant properties. Melanin has a distinct history.

Melanin if produced by melanocytes which are considered a nerve-related cell. During embryo

formation, some melanocytes migrate from the neural crest to the epidermis, hair follicles, inner

ear, and iris, while the rest remain in the brain (Olsson, 2006). The dispersion of melanin allows

it to complete its main function: protect the body from oxidative damage. Melanin’s

antioxidative properties can possibly lower one’s risk for degenerative disease.

Literature Review

Antioxidants stabilize free radicals, and melanin can behave in a similar manner, thus

preventing tissue damage. Previous studies and reports have established a correlation between

the presence of free radicals and degenerative disease. One report written by Dr. Florence

(1995), in the journal article “The Role of Free Radicals in Disease” concluded that “the

inflammatory reaction stemming from free radicals are at least partially responsible for the the

development of degenerative and autoimmune diseases such as asthma, Alzheimer’s, diabetes,

and arthritis” (para. 20). The accumulation of free radicals, such as unpaired oxygen, initiates an

inflammatory response and robs tissue and bones of key compounds. Furthermore, the heat and
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moisture resulting from the inflammatory response exacerbates the damage. When soft neural

tissue is damaged, it can lead to neurodegenerative disease of the brain and sensory organs.

Antioxidants defend tissues against oxidative damage by stabilizing these free radicals

through the transfer of electrons. Scientists have attempted to establish a correlation between an

antioxidant’s ability to transfer electrons and a lower risk of degenerative disease. A study

published in the ​Arthritis and Research Therapy​ ​Journal​ highlighted the ability of antioxidants

to inhibit catabolic reactions, the breakdown of organic compounds. For this reason, it was

hypothesized that antioxidants effectively prevent the degeneration of musculoskeletal tissue.

Therefore, it was concluded that antioxidants effectively prevent the degeneration of

musculoskeletal tissue (Suzuki, Fujita, Hosogane, & Matsumoto, 2015). By donating electrons,

antioxidants prevent free radicals from snatching necessary electrons away from body tissues. As

a result, the inflammatory response is not triggered, and tissue damage is avoided. If free radicals

and the inflammatory disease stimulated by free radicals are the main cause to degenerative

disease, in theory, antioxidants cwould prevent degenerative disease. Due to its properties, the

compound melanin would have a similar effect.

Melanin displays properties similar to antioxidants; therefore, it can effectively combat

oxidative damage to soft tissues. Melanin has a function that goes beyond preventing skin

cancer. Researchers at the University of Chicago have found that the pigment protects soft neural

tissue, such as retinal pigment epithelial cells free- radical damage by “absorbing the destructive

energy of free radicals and converting it into heat” (20015, para. 2). In this way melanin behaves

much like an antioxidant in that it possesses the biological capability to stabilize the destructive
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free radicals, thus preventing oxidative damage to soft neural tissue. Therefore, if antioxidants

are granted the title as the defenders against degeneration, melanin would partake in that role.

Macular degeneration, caused by free radicals in the eye and described as the gradual loss

of vision, can be prevented by increased melanin in the system. Individuals most susceptible to

this degenerative disease include the elderly and those with light- colored eyes (Hu, Simon, &

Sarna, 2008). It is believed that free radicals cause irreparable damage to the retinal epithelial

cells, thus resulting in vision loss over time. In the back of the eye, “Oxygen concentrations [...]

are very high. At the same time the eye is constantly bombarded with light energy, which

interacts with oxygen and can lead to the production of harmful free radicals – which can

damage cell membranes and DNA” (University of Chicago Medical Center, 2005, para. 7).

Unpaired oxygen is the most prevalent free radical in the human body, yet oxygen is essential

for human survival. When it interacts with other molecules, such as light photons, the oxygen

molecule becomes highly reactive. It behaves as an electron in the sense that it strips ocuclear of

electrons, which initiates an inflammatory response and subsequent destruction of retinal tissue.

Consequently, as the retinal tissue deteriorates, one experiences gradual vision loss. Once the

process begins, it cannot be reversed; however, it can be prevented.

Melanin produced in the eye behaves as an antioxidant to prevent damage to optical

tissues. In a study conducted by researchers Dungel and Zimmer (2016), it was found that

“Fifty-one of 61 eyes (84%) of 43 patients with AMD were determined to have melanin

disruption in their MSI images” (para. 3). The results of Dungel and Zimmer’s study suggests a

correlation between melanin and the incidence of macular degeneration (AMD). 84%, the

majority of the patients, displayed abnormal (low) melanin production. Although it is unknown
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whether low melanin production is the cause or the effect of macular degeneration, the fact is a

correlation exists. Another experiment discovered a correlation between macular degeneration

and SHI (hearing loss). It found that patients with macular degeneration were more likely to

develop hearing impairment (​Ghasemi, Pourakbari, Entezari, & Yarmohammadi, 2016)​. The

only similarity between the ear and the eye is the presence of the neural-related melanocytes. The

positive correlation between the two conditions suggests that melanin produced by the

melanocytes may play a role in age-related neurodegenerative disease. The presence of melanin

is important as the melanin in retinal epithelial cells absorb “the destructive energy of free

radicals and [convert]ing it into heat” (University of Chicago Medical Center, 2005, para. 12).

Energy stemming from the reaction between melanin and oxygen can lead to the destruction of

delicate nerve tissue. By absorbing, neutralizing, and deflecting this harmful energy, melanin

inhibits the inflammatory response, and thus the deterioration of RPE tissue. Consequently, the

presence of melanin indirectly prevents the development of macular degeneration and the

degeneration of sensory organs overall.

By behaving as an antioxidant, melanin can potentially reduce the symptoms of Multiple

Sclerosis, a neurodegenerative disease. Melanin concentration can affect one’s likelihood of

developing Multiple Sclerosis. This is supported by research conducted by Constantinescu

(1995), who discovered that ethnicity and geography are determining factors in the incidence

rate of Multiple Sclerosis, which is lower among individuals of African descent compared to

those of European descent . Melanin contributes to this difference in incidence rate as individuals

of African descent produce more melanin than those of European descent. Since melanin protects

neuronal cells by shielding them from oxidative damage, the higher the concentration of
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melanin, the better protection the brain tissue receives and, as result, the likelihood of developing

MS is greatly reduced. Besides race, oxidative stress has been cited as a factor of demyelination

and axonal damage in MS (Gilgun-Sherki, Melamed, & Offen, 2004). Free radicals, which are

the cause of oxidative damage, can strip axons of their protective membrane, myelin, thus

causing axonal damage. Melanin can prevent this damage by using its antioxidant properties to

neutralize the free radicals. If the free radicals are neutralized, the myelin on the axons will

remain intact consequently, MS would not develop.

However, when prevention is not possible, Melanin can reduce the symptoms of MS and

perhaps delay its onset. Just because individuals of African descent have a lower likelihood of

contracting MS does not mean that they possess a magical immunity to the disease. There are

cases where individuals with higher melanin concentrations develop MS. In those cases, what is

the role of melanin? Results from a study conducted on Australians “demonstrated that those

with greater sun exposure were sixty percent less likely to experience MS symptoms” (Chan,

2011, para. 7). Greater sun exposure means tanner skin, which means greater melanin production

since the sun stimulates melanin. As a result, vulnerable neuronal tissue receives greater

protection from oxidative damage. Likewise, a study conducted on mammalian animals supports

this aforementioned claim. The study found that the longer the mice were restricted to a dark

environment, the more severe their symptoms of MS were. From data, the researchers claim that

melatonin has the ability to indirectly prevent demyelination, and thus reduces symptoms of MS

(Constantinescu, 1995). Darkness suppressed melanin production in the mice. Although mice

are not identical to humans anatomically or biologically, they are mammals. Thus, they can

provide some insight into the effect of melanin on the symptoms on MS. If the lack inhibition of
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melanin production aggravated the symptoms in mice, it is safe of assume that the melanin has a

role in slowing down the progression of MS and possibly other neurodegenerative disorders.

The deterioration of melanin is indirectly linked to the progression of Parkinson’s

Disease. Parkinson’s disease is a neurodegenerative disease caused by insufficient production of

the neurotransmitter dopamine and the death of neurons. Oxidative damage is cited as one factor

leading to the development of Parkinson’s disease with only five percent of all cases stemming

from a genetic mutation (Petroysan, 2015). This suggests that a high concentration of free

radicals is a more accurate indication of one’s likelihood to develop Parkinson’s Disease. As free

radicals are responsible for creating the toxic environment that kills healthy neurons.

Neuromelanin, a pigment present in the brain stem, is considered a neuroprotector for its free

radical scavenging activities. A lack of neuromelanin has been connected to decreased motor

function and an increase in oxidative stress (Pan, 2011). Deterioration of neuromelanin leads to

an increase in free radicals which cause oxidative damage. Likewise, the greater the

concentration of free radicals, the greater the risk of oxidative damage caused of dopaminergic

neurons. Damage to these neurons would result in the development of Parkinson's.

Similar to with incident rates of Multiple Sclerosis, melanin can reduce the severity of

Parkinson’s Disease symptoms. A study conducted on rats highlights the effects of melanin on

symptoms of Parkinson’s Disease. In the experiment, Petrosyan (2005) observed the following:

The recovery of balancing instrumental conditioned reflex after the destruction of SNc
was completed in a very short period of time in rats injected with bacterial melanin
solution, suggesting not only the neuroprotective action of bacterial melanin, but also
possible compensating role of bacterial melanin in the process of motor control and
recovery (para. 11).
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SNc refers to the substantia nigra compacta, an area of the brain which houses

dopamine-producing neurons. As aforementioned, Parkinson’s occurs due to insufficient

dopamine production. Damage to dopamine neurons would introduce Parkinson- like symptoms.

The fact the rats regained their balancing reflex after receiving bacterial melanin injections hints

at melanin’s role in the reversal of acute neurological damage. Melanin’s role as a healer and

protector of cranial neurons permits the prevention of debilitating neurological damage, and thus

neurodegenerative disease, such as Parkinson’s, which are the result of chronic harm to neurons.

Considering melanin’s ability to counteract neurological damage in rats and the fact that rats

have been used to test various human products, it is safe to assume that melanin functions

similarly in human.

Melanin’s role in the prevention of melanoma has overshadowed its lesser known

functions. It jurisdiction as protector extends further than the skin. In accordance with it origins,

melanin protects the neurons of the brain and sensory organs from oxidative damage caused by

free radicals. Considering this, melanin functions much like an antioxidant and shares some of its

properties including the stabilization of free radicals through electron donation. Various studies

have explored the antioxidant-like properties of melanin, connecting melanin’s neuro-protective

abilities to various neurodegenerative diseases- macular degeneration, Multiple Sclerosis, and

Parkinson’s Disease. If a definite correlation can be established, the medical community would

receive a better understanding of the body’s natural defenses against neurodegenerative disease,

and possibly, a viable treatment using melanin pigment could be developed.


Research Methods and Data Collection
Antioxidant Activity of the
Melanin Pigment
Extracted from ​Aspergillus
nidulans
Summary The antioxidant activity of
synthetic melanin and a
highly melanized strain
from ​A. nidulans​ (MEL1)
was assessed. The ability
of these two substances
to scavenge oxidants
HOCL and H2O2 were
measured through the
inhibition of the oxidation
of TNB.
Species Studied Aspergillus nidulans
Methods and Materials ● Melanin
extracted by
raising pH until
precipitate forms,
then the melanin
precipitate was
9
Chemical Composition Antioxidant Function Physicochemical
and Radical Scavenging of Fungal Melanin characterization and
Activity of Melanin from antioxidant activity of
Auricularia auricula melanin from a novel
strain of Aspergillus
bridgeri ICTF-201
Melanin extracted from The antioxidant Melanin was extracted
the fungi ​Auricularia capacity of from ​Aspergillus
auricula​ was chemically Cryptococcus bridgeri​, then analyzed
analyzed, The analysis neoformans​ was through various
found 13 metal elements tested and measured. chemical tests. Melanin
present in the compound. Its capacity was found from ​Aspergillus
AAFB also displayed to be 21.3x10 ⁻¹⁵ mole bridgeri ​demonstrated
strong scavenging per cell good antioxidant
activities. scavenging activity.
Auricularia auricula Cryptococcus Aspergillus bridgeri
neoformans
● 80 grams of ● Melanin ● Extracted from
fruiting bodies extracted soil sample of
was purchased from Eucalyptus
● Fruiting body wild-type tree
powder washed, version of ● Cultured in
then centrifuged Cryptococcus potato
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centrifuged ● Melanin mixed neoformans dextrose agar

● Melani purified to with oxidants, ● Grown in a at 4 degrees
remove lipids shaken 10mL culture Celsius
and vigorously and of 2% ● 1mL of DPPH
carbohydrates left to stand for glucose- 2% added to
30 min in the yeast with testing culture
dark was agitated ● Testing culture
at 37°C until with DPPH
stationary shaken
phase is met vigorously and
left to stand in
dark for 30
minutes
● Then,
antioxidant
activity
calculated

Description of Data MEL1 strain is a potential ● Scavenging ● Nonmelanize ● Aspergillus

Results HOCL scavenger. Its activities of d culture bridgeri
protection is comparable AAFB melanin contained demonstrated
to that of the synthetic and BHT little significant
melanin increased with reductant radical
an increase of ● Graphs show scavenging
free radicals that melanin activity
● AAFB melanin neutralized comparable
showed substantial with synthetic
significantly quantities of melanin
higher permanganat ● Ascorbic acid
scavenging e; this exhibited a
activity on suggests that stronger
DPPH than extracellular radical
BHT at an reducing scavenging
identical power lies in activity
concentration melanin
● AAFB had ● Melanin has
higher a protective
superoxide antioxidant
radical function and
scavenging consumes
activity than superoxide
BHT, silky fowl and other
melanin, and oxidants
synthetic ● H2O2
melanin molecules
depending on move freely
the across the
concentration membrane,
suggesting
that melanin
afford no
protection
against
H2O2

Variables Control:antioxidant activity Control: synthetic Control: Asparagine Control: Ascorbic acid
of synthetic melanin with culture, no Independent:
Independent: Type of Independent: type of melanin concentration of
melanin oxidant placed in culture Independent: Strain melanin
Dependent:% of TNB Dependent:scavenging phenotype Dependent: % free
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oxidized activity Dependent: radical scavenging

Constant: type of oxidants Constant: Extraction and Resistance factor activity
used; measurement; purification process Constant: Brain heart Constant: preparation
amount of each substance infusion agar; the process; left in dark for
used strain of fungi used 30 min; free radical
(wild-type); cultural species used
days

What is the extent of ● MEL1 showed ● Melanin used ● Purified ABM

melanin’s antioxidant less scavenging in the from
properties? activity in the experiment Aspergillus
presence of did not bridgeri​ found
H2O2 provide any to be an
protection effective
against hydrogen
hydrogen peroxide free
peroxide radical
(H2O2) scavenger
● Melanin
protected
cells against
anions,
hypochlorite,
and
permanganat
e
● H2O2
molecules
allowed to
move across
the cell
membrane
freely and
attack
intracellular
structures

What is fungal ● “Comparable” ● Better ● Free radical

melanin’s oxidizing ● Scavenging scavenger than scavenging
strength compared to ability of MEL1 synthetic activity
synthetic melanin? close to that of melanin at comparable to
synthetic melanin specific that of
concentrations synthetic
melanin

How similar is fungal ● AAFB melanin

melanin to human or is very similar to
animal melanin? that of
pheomelanin
● AAFB melanin
is rich in Ca, Fe,
Cu, and Zn.
Similarly, Ca,
Fe, Mg, and Zn
were found in
melanin
extracted from
the muscle
tissue of
black-bone silky
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fowl

What purpose does ● Behave as ● Serve as a ● Extracellular ● Enhanced

the pigment melanin “fungal armor” reservoir for reductant protection
serve in fungus? ● Not important to reactive metal ● Invading ​C. under certain
fungal growth/ ions that can neoformans stress (ie UV
development bind to other surround radiation, cell
● Protection compounds itself with wall degrading
against oxidative melanin to enzymes,
damage protect itself extreme
from human temperatures,
macrophages and metals)
● Protection ● Increased
against virulence in
leukocyte pathogenic
oxidant fungi

Other useful HOCL is cited as the most ● Concludes that Exogenous melanin ● Melanin may
information abundant and toxic AAFB can serve was injected into an hold promise
oxidant. as a natural albino. Albino cells in treatment of
antioxidant lack melanin, so they AIDS
● Free radicals will not make
are byproducts extracellular
of normal reductant. However,
metabolism after the exogenous
● Artificial melanin was added, it
antioxidants are neutralized
added to food, permanganate (free
however they radicals) and
are known to protected the cell
have adverse
effects on
human health,
like the
development of
cancer
● In China
melanin is
considered an
important
component of
black food

Results and Data Analysis

After analyzing four studies on the free radical scavenging properties of fungal melanin,

it was found that every form of melanin extracted from from the four fungal species exhibited

free radical scavenging comparable to that of synthetic melanin. Although this shows that
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organic melanin is equally, if not more effective as synthetic melanin, it does not illustrate the

scavenging power of melanin compared to that of antioxidants. The studies done on ​Auricularia

auricula ​and ​Aspergillus bridgerim​, however provides some insight into the effectiveness of

melanin as an antioxidant. Results from the study on ​Auricularia auricula​ demonstrated that

AAFB melanin (melanin from the fungus ​Auricularia auricula​) had a stronger scavenging

activity on DPPH than BHT (an antioxidant) at an identical concentration. Evidence from the

study conducted on ​Aspergillus bridgerim​, on the other hand, contradicts the evidence found in

the aforementioned study. Results from the study on ​Aspergillus bridgerim ​Ascorbic acid,

another type of antioxidant exhibited stronger free radical scavenging activity that melanin

extracted from the fungi. Since the antioxidant used differed in both experiments, it is possible

that melanin exhibits stronger radical scavenging activity compared to some weaker antioxidants

and weaker radical scavenging activity compared to stronger antioxidants. Conversely, the

difference may lie in the type of melanin used. Due to evolutionary reasons, some fungal

melanin compounds may possess greater scavenging activity. For example, melanin extracted

from a tropical fungus may exhibit better UV radiation protection since solar radiation is a

common stressor in its native environment. Either way, the effectiveness of melanin as a free

radical scavenger relative to other antioxidants is still inconclusive.

The extent of melanin’s free radical scavenging capability was another are of inquiry

investigated. It was found that the melanin extracted from ​Aspergillus nidulans​ and

Cryptococcus neoformans ​provided cells with little to no protection against hydrogen peroxide

molecules. However, the study on ​Aspergillus bridgeri​ found it to be an effective hydrogen

peroxide scavenger. Perhaps, melanin producing proteins in ​Aspergillus bridgeri​ possess an

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adaptation not found in most fungi. This is a possible explanation for discordance in melanin’s

limitation as a free radical scavenger. Nonetheless, there is agreement between all four studies

that melanin displayed remarkable free radical activity, meaning that melanin can effectively

neutralize most, if not all free radicals that antioxidants can.

The similarity between human and fungal melanin was the last are investigated in the

meta-analysis. The similarity of fungal melanin and human melanin was investigated through

two ways- the chemical structure of melanin and the purpose it serves in fungi. It was found that

fungal melanin served the same purpose as human melanin- protection from oxidant damage.

There is no evidence that demonstrates melanin’s importance in fungal growth and development.

However, multiple studies have collected evidence on melanin’s role in the protection of cells

from extracellular stresses such as UV radiation, metal, cell-wall degrading enzymes, and

oxidants (free radicals). A study conducted on melanin​ ​extracted from​ Auricularia auricula

found that AAFB acts as a reservoir for reactive metals. This discovery relates to the scavenging

activity of melanin as the highly reactive metals stored in the melanocytes can be used to donate

electrons to free radicals, thus stabilizing them. The role of melanin in fungi is comparable to

that of melanin in the human body. The functional similarities suggest that a correlation between

human and fungal melanin exists.

As far as the chemical structure of melanin, one study done on ​Auricularia auricula

found that AAFB is very similar to pheomelanin, which is present in the human epidermis

(Thody, 1991). AAFB is rich in Ca, Fe, Cu, and Zn. Three of which-Ca, Fe, and Zn are elements

that are found in eukaryotic cells. This discovery was made through an analysis of melanin
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extracted muscle tissue from the black-bone silky fowl. Although humans and chickens are not

closely related, they are closer than fungi and humans.

Discussion and Conclusion

Results from the meta-analysis and preliminary research can bring researchers a step

closer to establishing a correlation- whether positive or negative- between melanin concentration

and the one’s risk of degenerative disease. As indicated from the meta-analysis, melanin displays

free radical scavenging abilities comparable to that of synthetic melanin and certain antioxidants.

However, with more tests or studies, a definite, more detailed conclusion about melanin’s free

radical scavenging abilities can be established. Evidence from these future studies can then be

used to explore the correlation between melanin concentration and the incidence of

neurodegenerative disease through the exploration of melanin as an antioxidant. If a positive

correlation between the two is established, then treatments involving the ingestion of melanin-

rich food may follow.

References

Chan, A. (2011, February 7). Life in the sun lower multiple sclerosis risk. ​Live Science.

Retrieved from

https://www.livescience.com/35454-sunlight-lowers-multiple-sclerosis-risk.html

Constantinescu, C. (1995). Melanin, melatonin, melanocyte-stimulating hormone, and the

susceptibility to autoimmune demyelination: A rationale for light therapy in multiple

sclerosis. ​Medical Hypotheses​, 45(5), 455-458. Retrieved from

https://www.ncbi.nlm.nih.gov/pubmed/8748085.
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