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BIOSCIENCES BIOTECHNOLOGY RESEARCH ASIA, December 2017. Vol. 14(4), p. 1475-1484
Protoprotection and Anti-inflammatory Properties of
Non–cytotoxic Melanin from Marine Isolate
Providencia rettgeri strain BTKKS1
Noble Kiriyachan Kurian and Sarita Ganapathy Bhat*
Department of Biotechnology, Cochin University of Science and Technology,
Kalamassery, Cochin-22, Kerala, India.
http://dx.doi.org/10.13005/bbra/2594
(Received: 15 December 2017; accepted: 22 December 2017)
Photoprotection and Anti-inflammatory properties of characterized melanin
produced by marine proteobacterium Providencia rettgeri strain BTKKS1 was explored in
the study. Characterization of melanin was carried out by chemical, FTIR, proton NMR and
EPR analysis. The radical scavenging property was estimated using DPPH assay and Fe 2+
chelating potential was also evaluated. Effect of melanin on the activities of Cyclooxygenase,
Lipoxygenase, Myeloperoxidase and Cellular Nitrite is used to evaluate anti-inflammatory
potential. Enhancement of Sun Protection Factor (SPF) is evaluated to study its effectiveness in
photoprotection. Cytotoxicity of melanin was estimated using MTT assay.The chemical, FTIR,
proton NMR and EPR characterization were typical of eumelanin.. The pigment also showed
profound radical scavenging activity (63.73%) and metal chelating potential (97.09%). Melanin
significantly inhibited the activity of the inflammatory enzymes in a dose dependent manner and
enhanced the SPF value of commercial sunscreens at an average of 2.64 factors. This melanin was
also less cytotoxic with an IC50 value of 97.87¼g/mL. The immense Anti-inflammatory property
of the pigment can be utilized in therapeutic applications. The photoprotection potential of
melanin can be utilized in cosmetic formulations, UV protection devices etc.
Keywords : Melanins, Providencia rettgeri, anti-inflammatory, SPF, bioactivity.
The dark colored biopolymer complex
melanin is widely distributed in nature, in all
living forms, having diverse biological functions
including photo protection, thermoregulation, as
free radical sinks, cation chelators and antibiotics.
In plants it is incorporated as strengtheners
in the cell walls (Riley 1997), whereas it not
only determines the skin color in humans, but
also plays a significant role in protecting skin
against UV damage (Huang and Chang 2012). In
microorganisms, they protect against environmental
stresses, with instances of increased resistances
toward antibacterials in melanin producers (Lin
et al. 2005), besides being involved in fungal
pathogenesis (Butler and Day 1998). According
to Nicolaus (1968), melanins can be sub grouped
into three namely eumelanin, a brown to black
pigment derived by the oxidative polymerization of
precursors like tyrosine, dihydroxyphenylalanine
(DOPA), dopamine and tyramine; pheomelanin, a
cysteine containing yellow to red pigment with a
biosynthetic pathway similar to eumelanin and the
heterogeneous allomelanins, which are formed by
the polymerization of di- or tetrahydrofolate via
pentaketide pathway.
*Corresponding author E-mail: saritagbhat@gmail.com
This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike
4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted Non
Commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Published by Oriental Scientific Publishing Company © 2017
1476 KURIAN & BHAT, Biosci., Biotech. Res. Asia, Vol. 14(4), 1475-1484 (2017)
The common commercial application of
melanin is in cosmetics such as sunscreen lotions
where it acts as a photo protective component due
to its UV-protective and free radicals scavenging
properties (Riley 1997). Melanins act as UV-
protective agents in bioinsecticide preparation
like the Bacillus thuringenesis (Bt) insecticidal
crystals (Wan et al. 2007; Zhang et al. 2007). The
melanin producing organism can also be used
in bioremediation of radioactive waste such as
uranium (Turick et al. 2008) and so on. Due to the
diverse application possibilities not restricted to
any particular field, the study of melanins is the
demand of the hour.
Numerous bacteria like Vibrio cholerae,
Shewanella colwelliana (Kotob et al. 1995) and
Alteromonas nigrifaciens (Ivanova et al. 1996)
produce melanins, including pyomelanin producers
like Pseudomonas aeruginosa (Eiko and Ohyama
1972), Shewanella. colwelliana, Vibrio cholerae,
Hyphomonas sp. (Ruzafa et al. 1995; Kotob
et al. 1995) and Alcaligenes eutrophus (David
et al. 1996). Marine actinomycetes including
Streptomyces strains reportedly use tyrosinases in
melanin synthesis. Another melanin-synthesizing
microbe which produces black eumelanin from
L- tyrosine is Marinomonas mediterranea (Solano
and Sanchez-Amat 1999). Most of these melanin
producers are terrestrial in origin, while marine
bacteria remain unexplored.
Bacteria of the Morganella-Proteus-
Providencia group produce a yet uncharacterized
brownish melanin - like- pigment on agar containing
L-form of aromatic amino acids (Müller 1985).
In this work the melanin produced by marine
proteobacteria Providencia rettgeri strain BTKKS1
is characterized and its various biological properties
of therapeutic and cosmetological importance were
explored.
MATERIALS AND METHODS
Chemicals, cell lines and bacterial isolates
Synthetic melanin (Sigma Chemicals Co,
St Louis, USA), L-tyrosine (Himedia chemicals,
Mumbai, India) and all other chemicals used were
of analytical reagent grade.
RAW 264.7 and L929 cell lines were
maintained in Dulbecco’s modified eagles media
(Himedia, India) supplemented with 10 % FBS
(Fetal Bovine serum) (Invitrogen, USA) and
grown to confluence at 37°C at 5 % CO2 in a CO2
incubator (Eppendorf, Germany).
The melanin prod ucing Providencia
rettgeri strain BTKKS1 was isolated from marine
sediments from Kanyakumari (8° 5’N, 77° 32’E)
coast of southern India. Screening for melanin
production was initially by a plate based assay
(Kurian et al. 2014) and then in tyrosine basal
broth (Eiko and Ohyama 1972). The bacterium
was identified by biochemical and 16SrDNA
sequencing (Mac Faddin 1976; Ausbel et al. 1995;
Sambrook et al. 1989; Shivaji et al. 2000).
Production, Extraction and Purification of
melanin
Tyrosine basal broth (Eiko and Ohyama
1972) containing 0.2% tyrosine was used for
melanin production.5 mL of this culture suspension
(OD600 = 1) was used as primary inoculums for
50 mL of production medium and kept in an
environment shaker (Orbitek, Scigenics, India) at
140 rpm at 37±2oC for180 h. Melanin production
kinetics was studied by sampling at 12 h intervals
and estimating bacterial growth and melanin
production spectrophotometrically (Turick et al.
2002).
After 180 h of incubation, the cell free
supernatant was acidified to pH 2 using 1 N HCl.
Black precipitate of melanin can be visualized at the
bottom of the flask at lower pH . Further treatment
with acid, water and ethanol simultaneously
according to Sajjan et al (2013) helped to get pure
melanin.
Physicochemical characterization of melanin
Reactivity of melanin with various organic
solvents, acidic and basic solutions, oxidising
and reducing agents were evaluated (Fava et al.
1993). Spectroscopic techniques such as FT-IR
(Ravishankar et al. 1995), Proton NMR (Guo et al.
2014) and EPR spectroscopy (Enochs et al. 1993)
were used to evaluate the biophysical properties of
the pigment, as also elemental analysis (Sajjan et al.
2013). Antioxidant and metal chelating properties
of the pigment was evaluated using standard
procedures (Liyana-Pathirana and Shahidi 2005;
Dinis et al. 1994).
Anti- inammatory potential of melanin
RAW 264.7 cells were then grown to
60% confluence followed by activation with
1µL Lipopolysaccharide (LPS) (1µg/mL). LPS
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KURIAN & BHAT, Biosci., Biotech. Res. Asia, Vol. 14(4), 1475-1484 (2017)
Table 1. Sun protection factors (SPFs) for commercial sunscreen
preparations before and after supplementation with BTKKS1 melanin
Commercial SPF value stated SPF value determined +BTKKS1
Sunscreen by the manufacturer empirically during Melanin
the current study (0.005% w/w) SPF
Sunscreen 1 15 14.24±0.007 17.09±0.06
Sunscreen 2 15 14.61±0.01 17.74±0.02
Sunscreen 3 15 14.77±0.05 17.57±0.05
Sunscreen 4 17 16.47±0.04 19.52±0.01
Sunscreen 5 30 26.26±0.04 28.90±0.05
Fig. 1. Time course of melanin production by Providencia rettgeri strain BTKKS1
Fig. 2. 1H NMR spectra of Providencia rettgeri BTKKS1 melanin
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KURIAN & BHAT, Biosci., Biotech. Res. Asia, Vol. 14(4), 1475-1484 (2017)
Fig. 3. Electron paramagnetic resonance spectrum of BTKKS1 melanin
Fig. 4. Radical scavenging (a) and metal chelating activity (b) of melanin
stimulated RAW cells were exposed to different
concentration (6.25, 12.5, 25, 50, 100 µg/mL) of
melanin solution. Diclofenac sodium, a standard
anti-inflammatory drug, in varying concentration
corresponding to the sample was also added and
incubated for 24 hours. After incubation the anti-
inflammatory assays were performed using the cell
lysate. Activities of three inflammatory enzymes
namely Cyclooxygenase (COX) (Walker and
Gierse. 2010), Lipoxygenase (LOX) (Axelrod et
al. 1981), Myeloperoxidase (MPO) (Bradley et
al. 1982) and Cellular nitrite levels (Lepoivre et
al. 1990) were assayed using standard protocols.
Photo protective nature of melanin
Photoprotective nature of melanin
was expressed by its ability to enhance the Sun
Protection Factor (SPF) of commercial sun screens.
Sun Protection Factor (SPF) was estimated by
a modified protocol (Suryawanshi et al. 2015).
Commercial sunscreens of (0.1 g) was added each
1479 KURIAN & BHAT, Biosci., Biotech. Res. Asia, Vol. 14(4), 1475-1484 (2017)
Fig. 5. Effect of BTKKS1 melanin on inflammatory enzymes (a) Cyclooxygenase (COX) (b) Lipoxygenase (LOX)
(c) Myeloperoxidase (MPO) (d) Cellular Nitrite Levels
to 10 mL of absolute ethanol, as also melanin at
0.005% concentration. Absorbance of the mixture
in the UV range (290–320 nm) was taken at 5 nm
intervals using ethanol as the blank.
SPFs were calculated, according to
Mansur et al. 1986, using following formula
where CF (correction factor) = 10; EE ( )
= erythmogenic effect of radiation with wavelength
k; Abs () = spectrophotometric absorbance value
of the solution; and I = solar intensity spectrum.
EE () ×I is constant and was determined (Sayre et
al. 1979).
Cytotoxicity of melanin
Different concentrations (6.25, 12.5,
25, 50 and 100 µg/mL) of melanin were added
to L929 cells at and incubated for 24 hours.
The percentage difference in viability was
determined by standard 3-(4, 5dimethythiazol-2-
yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay
(Arung et al. 2009) after 24 hours of incubation.
Statistical analysis
All the experiments were repeated thrice.
The statistical analysis was done by ANOVA using
GraphPad Prism. Ver.6 computer program, where
p values<0.05 were considered significant.
RESULTS
Strain identication
Following preliminary screening, bacteria
from marine sediment sample producing a clearing
zone on tyrosine agar plates were selected as
melanin producers. Strain BTKKS1 selected for
further characterization after secondary screening
was identified as Gram negative rod, indole, methyl
red and citrate positive and Voges– Proskauer
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KURIAN & BHAT, Biosci., Biotech. Res. Asia, Vol. 14(4), 1475-1484 (2017)
negative. The bacterium was catalase positive but
oxidase negative and could utilize sugars such
as glucose, adonitol and manitol in the medium.
It was identified further as Providencia rettgeri
(KF515633) by 16S rDNA sequence analysis.
Pigment production
Strain BTKKS1 produced considerable
amount of pigment in the tyrosine broth from third
day until the eight day, when pigment concentration
was 30.31±0.69 ¼g/mL, with no further increase
in production thereafter (Fig. 1).
Physicochemical characterization of melanin
Melanin from strain BTKKS1 was
soluble in alkaline solvents like sodium hydroxide,
potassium hydroxide and Dimethyl sulfoxide
(DMSO). However, the pigment showed least
solubility in water and common organic solvents.
Oxidizing (30% H2O2) and reducing (Na2SO3)
agents decolorized the pigment.
The IR spectrum showed characteristic
peaks (Laxmi et al. 2016) showing similarity with
those in earlier reports (Selvakumar et al. 2008). 1H
NMR peaks of melanins (Fig.2) showed similarity
with earlier reports (Arun et al. 2015; Guo et al.
2014) with signals in both the aromatic (7.03-7.32
ppm) and aliphatic regions (0.8 ppm). Sharp peaks
in the EPR spectra (Fig. 3) of melanins indicated
the presence of unpaired electrons, which can trap
free radicals. This was further confirmed by the
immense radical scavenging activity (63.73%) and
metal chelating potential (97.09%) of the pigment
at its higher concentration (100¼g/mL) tested
(Fig. 4).
Fig. 6. Cytotoxicity BTKKS1 melanin (a) Phase contrast micrographs (×20 magnification) showing the cytotoxic
effect of Providencia rettgeri BTKKS1 melanin (1) Control (2) melanin treated (100ìg/mL) (b) Graph showing the
percentage inhibition of growth of L929 cells
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Elemental composition of Providencia
rettgeri strain BTKKS1 melanin showed 47.48%
carbon, 4.10% hydrogen, 12.73% nitrogen and
0.89% sulfur. Typical elemental composition of this
bacterial melanin was similar to those obtained in
earlier reports (Hong and Simon 2006).
Anti- inammatory potential of melanin
P. rettgeri melanin significantly inhibited
the activity of four inflammatory enzymes assayed
in the study in a dose dependent manner. Melanin
inhibited COX at an IC50 of 95.09%, with maximum
inhibition at highest concentration tested (100 µg/
mL) being 52.58% (Fig. 5 a), while it showed
63.62% inhibition (IC50= 78.59 µg/mL) of LOX
enzyme (Fig. 5 b).About 74 % of the MPO activity
was inhibited by BTKKS1 melanin at 100 µg/mL
concentration (Fig.5 c), while the cellular nitrite
level also decreased considerably. (Fig. 5 d),
Photo protective nature of melanin
SPF value of the sun screens tested was
increased by the addition of 0.005% melanin.
BTKKS1 melanin enhanced the SPF value by an
average of 2.64 factors (Table 1).
Cytotoxicity of melanin
Providencia rettgeri BTKKS1 (Fig. 6)
melanin was observed to be less toxic to L929 cells
with an IC50 value of 97.87 µg/mL.
DISCUSSION
Melanin production of BTKKS1 started
from the second day and continued till day eight,
when it stabilized. Earlier reports showed that
melanin production in bacteria usually starts in
24-72 hours after inoculation (Zhang et al. 2007).
The actual period may vary with the genus, but we
don’t have many reports on melanin production
by other Providencia rettgeri so to compare with
BTKKS1 production pattern.
Chemical nature of melanin, especially
its insolubility in most of solvents including water
may be due to aromatic rings and carboxylic acids,
which could get fully protonated when contacted
with water. But it is solubilized in alkaline solvents
and DMSO. Solubility in DMSO may be the result
of thioalkylation of the phenolic units in melanins
(Hansen et al. 2011).
One of the most unusual features of
melanin is its persistent EPR signal (Blois
et al. 1964). Indeed, melanin was among the
first biological materials examined by EPR
spectroscopy (Commoner et al. 1954). Melanin
free radicals are stable, and the content of melanin
free radicals and their corresponding EPR signal
intensity can easily be modified by a number of
physicochemical agents (Sealy et al. 1980) like
metal ions, light etc. Ability of melanin to interact
with stable free radical DPPH indicated further
the scavenging activity of the pigment due to the
presence of paramagnetic centres (PMC). BTKKS1
melanin was proved to bind tightly to reactive
metals like Fe(II) which enables protection from
Femton reactions which cause tissue damages
(Flora 2009). This protective nature can be utilized
in many useful applications.
Classification of melanin as pheomelanin
subclass can be done by CHN(S) elemental
analysis. Pheomelanin (Ito and Fujita 1985)
with cysteine incorporated structure have more
sulfur content (9.78%) compared to other types
like synthetic dopa melanin (Ito and Fujita 1985)
(0.09%) and Klebsiella sp melanin (Sajjan et al.
2013) (0.86%). The low sulfur (0.89%) content of
BTKKS1 pigment contraindicated pheomelanin
class (Sajjan et al. 2013).
BTKKS1 melanin decreased the activity
of all inflammatory enzymes (COX, LOX, MPO
and NO synthase) tested. Kurian et al. (2015)
reported similar effect of Bacillus melanin.
There are no other reports so far though there
are many reports available regarding phenolic
compounds (Masuda et al. 2010; Kato et al. 2003;
Tsao et al. 2005) inhibiting the activity of these
inflammatory enzymes. May be similar mechanism
is also employed here. There are only few reports
regarding the anti-inflammatory properties of
melanin. Avramidis et al. (1998) reported that
grape melanin interfered with the prostaglandin as
well as the leukotriene and/or complement system
mediated inflammation.
Immense improvement of photoprotection
by BTKKS1 melanin supplemented sunscreens,
opens doors for more melanin based cosmetics.
Huang et al. (2011) reported the sun protection
effect of melanin from berry of Cinnamomum
burmannii an d Osmanthus fragrans. La ter
Tarangini and Mishra (2014) also reported the
profound enhancement in SPF value by Bacillus
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KURIAN & BHAT, Biosci., Biotech. Res. Asia, Vol. 14(4), 1475-1484 (2017)
safensis melanin. The less cytotoxic nature
of BTKKS1 melanin also makes it a suitable
candidate for cosmetic formulations.
Thus the characterized melanins from
Providencia rettgeri strain BTKKS1 had shown
immense bioactivities which can be utilized
further in different areas of life activities. Its
anti-inflammatory properties can be utilized for
therapeutic applications. While its property of SPF
enhancement in sun screens makes it an essential
ingredient in cosmetic formulations. More in vivo
and clinical trials were required to confirm its
utility
ACKNOWLEDGEMENTS
First author acknowledges DST (Dept
of Science and Technology, Govt. of India) for
the DST INSPIRE- Junior and Senior Research
Fellowship
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