Photoprotection and Anti-inflammatory Properties of Non–cytotoxic Melanin from Marine Isolate Providencia rettgeri strain BTKKS1

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 97.87μg/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.

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 , 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.
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 UVprotective 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.
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.

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.

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 (OD 600 = 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±2 o C for 180 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.

Anti-inflammatory potential of melanin
RAW 264.7 cells were then grown to 60% confluence followed by activation with 1µL Lipopolysaccharide (LPS) (1µg/mL). LPS  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 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 (l) = erythmogenic effect of radiation with wavelength k; Abs (l) = spectrophotometric absorbance value of the solution; and I = solar intensity spectrum. EE (l) ×I is constant and was determined (Sayre et al. 1979).

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.

Strain identification
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 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 µg/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% H 2 O 2 ) and reducing (Na 2 SO 3 ) 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). 1 H 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 (100µg/mL) tested (Fig. 4). 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-inflammatory 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 IC 50 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 (IC 50 = 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 IC 50 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)  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 and Osmanthus fragrans. Later Tarangini and Mishra (2014) also reported the profound enhancement in SPF value by Bacillus 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