Biocompatible melanin from the marine black yeast Hortaea werneckii R23 with antioxidant and photoprotection property

Abstract   

Pigments from diverse sources have a great deal of interest due to its multifaceted applications. Hence, this study reports the physicochemical and functional characterization of the black pigment melanin from the marine black yeast Hortaea werneckii R23. In the present study, Hortaea werneckii R23, produced a black pigment in the yeast biomass. The pigment was extracted from the harvested yeast biomass and followed by pigment purification, characterization and identification was done. Physicochemical characterization of the pigment showed acid precipitation, alkali solubilization, insolubility in most organic solvents and water. The black pigment was confirmed as melanin based on ultraviolet–visible spectroscopy, Fourier-transform infrared, and Nuclear magnetic resonance spectroscopy analyses. Furthermore, the analyses of the elemental composition indicated that the pigment possessed a moderately high percentage of nitrogen and also detectable proportion of sulfur. All these Physicochemical properties indicated that H. werneckii melanin (HwM) mostly consisted of eumelanin. HwM exhibited strong antioxidant potential as reactive oxygen species (ROS) scavenger by in vitro DPPH (1,1-diphenyl-2-picryl hydrazyl) and ABTS (2,2-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid) radical scavenging assay, and lipid peroxidation assay. The photoprotectant role of HwM on UV-irradiated human epithelial cells (HEp-2) revealed its potential effect in photoprotection. In addition, cytotoxicity study by XTT and SRB assay confirmed its biocompatibility with HEp-2 cells. From these findings, it is evident that the HwM from the marine black yeast possesses strong antioxidant and photoprotectant activity, moreover, it is biocompatible to human epithelial cells. So HwM could be used as a protective agent against oxidative stress associated disorders in an environment-friendly perspective.

Introduction

Melanin is an important class of natural pigment receiving great attention currently due to their biological role and technological applications. Melanins are pigments of high molecular weight formed by oxidative polymerization of phenolic or indolic compounds and usually are dark brown or black. Melanins are produced by a wide variety of microorganisms including several species of yeasts, fungi and bacteria [7, 15, 36]. They are negatively charged, hydrophobic molecule formed by oxidative polymerization of phenolic or indolic compounds usually found as complexes with proteins and carbohydrates in plants, animals, bacteria and fungi [2]. Melanin, in general exists in three forms viz., eumelanin, pheomelanin and allo-melanin that share some common physicochemical properties [37]. Melanin has variety of functions such as photoprotection (against UV and visible light) [26], free radical scavenging [29], antioxidant [42, 45], antitumor [43] and immunostimulant activity [31]. It is also used in various biomedical fields, such as biological imaging, photothermal therapy, and drug delivery systems [13]. Antioxidant potential of pigments from natural sources including melanin have already been proved apart from conventional usage as a coloring agent [7, 45].

Even though melanin is not an essential biomolecule for the growth and development of an organism, it enhances the competitive abilities and survival of organisms in the environment [1]. However, the marine environment is an untapped resource and particularly the information regarding biodiversity and production of bioactive substances from marine yeasts is limited. Their characteristic features are meristematic growth, thick and darkly pigmented cell wall, and ability to survive in extreme habitats [11]. These ascomycetous black yeasts produce dark brown to black pigments viz., melanin, one of the largest classes of natural pigments. Usually, fungal melanins are complex pigments that are produced by two different pathways, known as the DHN (1,8-dihydroxynaphthalene) and L-DOPA (L-3, 4-dihydroxyphenyl-alanine), depending on the species [1, 18].

Synthetic antioxidants are widely used; but due to their undesirable effects, search for natural antioxidants is in the upsurge. Interest in melanin has been prompted because of their biological origin and activity profile. In this perspective, the present work is focused on the extraction, physicochemical characterization and potential applications of the melanin from marine black yeast, H. werneckii R23.

Materials and methods

Microorganism used for melanin production

Black yeast Hortaea werneckii R23 isolated from the Arabian Sea and maintained in the Microbiology Laboratory of the Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology (CUSAT), India was used for the study (Fig. 1A and 1B).

Fig. 1.

A Black yeast Hortaea werneckii R23 on malt extract agar plates B) Micromorphology of Hortaea werneckii R23 C) Melanin extracted from black yeast Hortaea werneckii R23 (lyophilized)

Extraction of melanin

The black yeast, H. werneckii R23 was swab inoculated on to malt extract agar plates, incubated at 28 ± 2 °C for five days and harvested with sterile saline (30‰). The cell suspensions were centrifuged at 10,000 × g for 30 min in a refrigerated centrifuge to separate the yeast biomass. Melanin was extracted from the yeast biomass as per Gadd [10]. Briefly, 1N NaOH was added to the yeast biomass and autoclaved for 20 min at 121 °C, centrifuged at 6000 × g for 10 min and the supernatant containing melanin was separated. Melanin was precipitated by adding concentrated HCl to the supernatant until the pH is reduced to 2. This solution was centrifuged at 10,000 × g for 10 min and the pellet was washed repeatedly with distilled water, dried in a lyophilizer and stored at -20 °C in a Freezer (MDF U334 Panasonic, Japan) till use. The dry weight of the melanin was noted and the melanin yield was recorded per gm wet weight of yeast biomass.

Physicochemical characterization of H. werneckii melanin

Solubility

The solubility of the yeast melanin in various solvents was tested as per Wang et al. [40] Melanin (100 mg) was added in 10 ml of various solvents viz., distilled water, aqueous acid, organic solvents (acetic acid, acetone, chloroform, ethanol, methanol, petroleum ether, hexane) and dilute alkali (sodium hydroxide, aqueous ammonia), stirred for 1 h and filtered. The absorbance was recorded at 400 nm using TU-1901 UV–VIS double beam Spectrophotometer (Hitachi, Japan) to measure the solubility of melanin.

UV–Visible (UV–VIS) absorption spectra of black yeast melanin

Melanin (0.05 mg) was dissolved in 1 ml alkaline distilled water (pH 9) prepared by adding 25% of 0.1 ml aqueous ammonia to 10 ml distilled water. The solution was scanned for absorption spectra using UV–VIS Spectrophotometer (Hitachi, Japan) in the wavelength range 230–580 nm [28]

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectrum of H. werneckii melanin was recorded at 4,000–600 cm⁻¹ using a FTIR spectrum 100 (Perkin Elmer FTIR spectrophotometer, USA) [9]. The spectrum of H. werneckii melanin was compared with that of sepia and synthetic melanin standards.

Nuclear magnetic resonance (NMR) spectroscopy

Proton one-dimensional NMR spectra of H. werneckii melanin and synthetic melanin (Sigma-Aldrich, USA) were carried out as per Leyden and Cox [20]. Melanin samples were dissolved in 1 M NaOD in D₂O and then transferred to 5 mm NMR tubes. The 1 M NaOD in D₂O was prepared by diluting 0.25 ml NaOD (40% NaOD in D₂O) with 3.75 ml D₂O. Proton one-dimensional NMR spectra were collected on a Bruker Avance III 600 NMR Spectrometer using a CH cryoprobe operating at 298°K (25 °C) and 345°K (72 °C). Spectral data were collected and processed as follows: 256 scans, 2 pre-scans, 65,536 points, 20.6 ppm sweep width centred at 6.18 ppm, exponential apodization with 0.2 (298°K) and 1.0 (345°K) Hz broadening, and 1 s pulse delay. Chemical shift reference was sodium trimethyl silylpropionate-2, 2, 3, 3-d₄ (TMSP-d₄) at 0.0 ppm. Spectra were processed using the JEOL DELTA software package (version 5.0.3) on a MacBook Pro operating system (version 10.9.4).

Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES)

ICP-AES analysis was used to determine the metal element composition of H. werneckii melanin by atomic emission spectrum [30]. The wavelength at which emission occurs identifies the metal element, while the intensity of this emission is indicative of the concentration of the metal element within the sample. Synthetic melanin was used as standard. The analysis was carried out using an ICP-AES Thermo Electron IRIS INTREPID II XSP DUO at STIC, CUSAT, India.

Elemental analysis of H. werneckii melanin

Elemental analysis of H. werneckii melanin was performed as per Sajjan et al. [29] to determine the percentage of elements such as carbon, hydrogen, nitrogen and sulphur over a wide range of sample matrices and concentrations with Elementar Vario EL III (Elementar Analysis systems, Inc., Germany). Synthetic and sepia melanin standards were used for comparison.

Scanning Electron Microscope-Energy dispersive spectroscopy (SEM–EDS)

SEM–EDS analysis of H. werneckii melanin was performed to detect the topography and qualitative elemental composition of the compound [22]. SEM observations and elemental analysis were performed on gold-coated samples that had been previously air-dried on glass slides using an analytical SEM (JEOL JSM-6390LV) equipped with EDS (JEOL JED–2300). Synthetic and sepia melanin standards were used for comparison.

Antioxidant activity

The antioxidant activity of H. werneckii melanin was assessed using the following assays: ABTS radical scavenging assay; DPPH radical scavenging assay; Lipid peroxidation inhibition assay.

ABTS radical scavenging assay

Free radical scavenging activity of H. werneckii melanin was measured in accordance with Miller and Rice Evans [24] with slight modifications. ABTS radical cation (ABTS⁺) was produced by reacting 8 mM ABTS solution with 3 mM potassium persulphate and the mixture was allowed to stand for 12–16 h in dark at room temperature (RT) for the accomplishment of free radical generation. The (ABTS⁺) solution was diluted with phosphate buffer (pH 7.4) in order to obtain an absorbance of 0.8 ± 0.01 at 734 nm. 50 µl of different concentrations (5–100 µg ml⁻¹) of melanin solution were mixed with 200 µl of diluted (ABTS⁺) solution and incubated at 30 °C. Absorbance in terms of reduction of ABTS by melanin was read at 734 nm after 10 min using a Microplate Reader (TECAN Infinite Tm, Austria). Trolox was used as a standard. The ability to scavenge the ABTS radical was reported as inhibition percentage (I%) of the test sample at various concentrations.

DPPH radical-scavenging activity

DPPH scavenging activity of H. werneckii melanin was determined as per Chen et al. [5] based on the capacity of the antioxidant to donate hydrogen or the radical scavenging ability. Melanin (50 µl) of different concentrations (10–100 µg ml⁻¹) in triplicates, was thoroughly mixed with 100 µl of freshly prepared DPPH solution (0.1 mM in 95% ethanol) and 100 µl of 95% ethanol. The mixture was shaken vigorously and left to stand for 30 min in the dark. The reduction of DPPH radical by melanin was measured as absorbance at 517 nm using Microplate Reader (TECAN Infinite Tm, Austria). Trolox was used as standard. The ability to scavenge the DPPH radical was reported as inhibition percentage (I%) of the test sample at various concentrations.

Lipid peroxidation inhibition assay

A modified thiobarbituric acid reactive substances (TBARS) assay was done based on Dasgupta and De [6] to measure the lipid peroxides formed using egg yolk homogenate as lipid-rich media. Briefly, 0.5 ml of 10% (v/v) egg homogenate and H. werneckii melanin of different concentrations (75–750 µg ml⁻¹) were added to a test tube and made up to 1 ml with distilled water; 0.05 ml of FeSO₄ (0.07 M) was added to induce lipid peroxidation and the mixture was incubated at 37 °C for 30 min. Then 0.5 ml of 20% trichloroacetic acid and 0.5 ml of 0.8% (w/v) thiobarbituric acid in 50% glacial acetic acid was added to quench the reaction. The resulting mixture was vortexed and heated at 95 °C for 60 min. After cooling, 2.0 ml of butanol was added to each tube and the mixture was centrifuged at 1000 × g for 10 min. Melanin-induced reduction in lipid peroxidation, as an index of antioxidant activity, was measured by a decrease in absorbance at 532 nm of the pinkish-red chromogen derived from malondialdehyde (MDA) and thiobarbituric acid (TBA). Butylated hydroxytoluene (BHT) was used as positive control. The results were reported as inhibition of lipid peroxidation (I%) by test sample at various concentrations.

Biocompatibility of black yeast H. werneckii melanin

Biocompatibility and cytotoxicity study of melanin was performed in HEp-2 cells. Biocompatibility and cytotoxicity of H. werneckii melanin were evaluated by estimating mitochondrial dehydrogenase activity in terms of reduction of 2, 3-Bis [2- methoxy 4 nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and protein synthesis by sulforhodamine B dye reduction (SRB) assay.

For above sequential assays, ~ 1 × 10⁶ HEp-2 cells were inoculated into each well of a 96 well tissue culture plate containing MEM (minimal essential medium) supplemented with 10% fetal bovine serum (FBS) and incubated for 12 h at 37 °C. After incubation, the cells were copiously washed with phosphate buffered saline (PBS), and the medium was exchanged with MEM containing different concentrations (0.05–6.4 mg ml⁻¹) of melaninin in triplicates. Following incubation for 24 h at 37 °C, the wells were observed under Inverted phase contrast microscope (Leica, Germany) and sequential cytotoxicity assays were performed following manufacturer’s instruction (Cytotox-PAN I, Xenometrix, Germany). For XTT assay, melanin treated cells were treated with 50 µl pre-warmed XTT at 37 °C for 4 h, mixed the formazan formed in each well and the absorbance was measured as a function of reduction of XTT to soluble formazan by healthy cells at 480 nm in a Microplate Reader (TECAN Infinite Tm, Austria) with a reference wavelength at 690 nm and the results were reported as percentage of cell viability [25, 38].  

For SRB assay, XTT solution from each well was discarded and the cells remaining attached to the bottom of the wells were washed with 300 μl wash solution, added 250 μl fixing solution and incubated the plate for 1 h at 4ºC (Cytotox-PAN I, Xenometrix, Germany). Then the cells were washed, added 50 μl labeling solution and incubated for 15 min at RT. The cells were washed twice with 400 μl rinsing solution and air dried. The air-dried cells were dissolved in 200 μl solubilization solution, and incubated for 1 h at RT and the absorbance was read at 540 nm (reference filter 690 nm) as a measure of protein synthesis [39]. The results were reported as percentage of cell viability at each concentration of melanin.

Photoprotection assay of H. werneckii melanin

Various concentrations (50, 100, 200 µg ml⁻¹) of H. werneckii melanin were treated with HEp-2 cells prepared in 96 well microplate containing MEM (minimal essential medium). The cells were exposed to 4.9W UV-C irradiation at a wavelength of 254 nm (TUV 15W/G15T8, Philips, Holland) for 10 min by keeping in an ice chest. Cells were washed copiously with phosphate buffered saline and reactive oxygen stress was analyzed by nitroblue-tetrazolium (NBT) reduction assay [32]. Briefly, NBT solution (2 mg ml⁻¹) in 0.05 mol l⁻¹ Tris HCl buffer (pH 7.6) was added to the melanin exposed cells and incubated for 1 h at 10 °C. The samples were then centrifuged at 2500 × g for 5 min, discarded the supernatant and quenched the reaction by adding 500 µl absolute methanol. Following incubation for 10 min, the insoluble formazan residue was fixed by rinsing thrice with 500 µl of 50% methanol. The formazan residue was solubilized with 120 µl of 2 M KOH and 140 µl dimethyl sulphoxide (DMSO), and absorbance was read at 620 nm in a microplate reader (TECAN Infinite, Tm Austria) as a function of the reduction of NBT to soluble formazan. Control cells (HEp-2) were also maintained without melanin under UV exposure and without UV exposure. Synthetic melanin (50 µg ml⁻¹) was used as positive control. All experiments were performed in triplicates.

Statistical analysis

The data of all the experiments were recorded as mean ± SD and were analyzed with SPSS (version 22.0 for Windows, SPSS Inc.). The IC₅₀ was calculated by Probit analysis of SPSS.

Results

Production and extraction of melanin from black yeast

Yeast inoculated on malt extract agar plates exhibited melanin production after 74 h of incubation. Melanin extracted from H. werneckii was brownish black in colour (Fig. 1C). The yield of melanin was 0.012 g dry weight/g wet weight of the yeast biomass.

Physicochemical properties of black yeast melanin

Solubility of black yeast melanin

The H. werneckii melanin was insoluble in water and most of the organic solvents tested, viz., acetic acid, acetone, chloroform, ethanol, methanol, petroleum ether and hexane. The melanin was soluble only in sodium hydroxide and aqueous ammonia (25%). Moreover, it also exhibited precipitation in acidic aqueous solution (0.1 M HCl) below pH 3. This solubility profile indicates the properties of a typical melanin.

UV–VIS absorption spectra of H. werneckii melanin

H. werneckii melanin exhibited a maximum absorption at a wavelength 230 nm in the UV region and an additional absorption at 275 nm. The absorption spectrum of H. werneckii melanin revealed that absorption peak was at UV region, which is a characteristic property of typical melanin (Fig. 2).

Fig. 2.

UV–VIS spectra of H. werneckii melanin. The UV–VIS wavelength scan showed that absorption was maximum in the UV region between 230–300 nm, but absorption diminished monotonically with increasing wavelengths from 300 to 580 nm. The images were taken from triplicates (n = 3) and provided representative images in the text

Functional groups in H. werneckii melanin

The FT-IR spectra of of the extracted marine yeast pigment along with the standard synthetic and sepia melanin were analyzed to confirm that the extracted yeast pigment was melanin. Peak assignments of H. werneckii melanin is OH or NH stretching at 3283.34 cm⁻¹, CH₂ asymmetrical stretching (2919.08 cm⁻¹), CH₂ symmetrical stretching (2850.83 cm⁻¹), C = C stretching or C = O stretching (1631.3 cm⁻¹), NH bending (1532.48 cm⁻¹), CH₂CH₃ bending (1456.46 cm⁻¹), CN stretching (1411.12 cm⁻¹), phenolic COH stretching (1228.12 cm⁻¹) and CO stretching (1043.23 cm⁻¹). Signal peaks detected at 3283.34, 2919.08 and 2850.83 cm⁻¹ are attributed to the stretching vibrations (O–H and N–H) of the carboxylic acid, phenolic acids and the aromatic amino functions present in the indolic and pyrrolic systems. Moreover, a C = C stretching or C = O stretching detected at 1631.3 cm⁻¹ is representative of aromatic C = C and/or carboxylate groups as well as nitrogen-containing heterocycles. Additionally, the two peaks at 1532.48 and 1411.12 cm⁻¹ strongly imply a pyrrole or indole NH group. Furthermore, the peak suggestive of the free carboxylic group found at 1707.9 cm⁻¹ in H. werneckii melanin and synthetic melanin, was not detected in sepia melanin. The most noticeable differences between H. werneckii melanin with sepia melanin and synthetic melanin are the peaks at 2919.08, 2850.83 and 1456.46 cm⁻¹, which also imply the presence of a considerable amount of aliphatic groups in the H. werneckii melanin structure (Fig. 3). These FTIR features have a close similarity to the typical structure of melanin.

Fig. 3.

FT-IR Spectroscopic analysis of melanin samples: A H. werneckii melanin, B synthetic melanin, and (C) sepia melanin. The signals in the 3500–2800 cm⁻¹ area of H. werneckii melanin are attributed to the stretching vibrations (O–H and N–H) of the amide, amine, or carboxylic acid, phenolic and aromatic amino functions present in the indolic and pyrrolic systems. FTIR spectra present variations among samples. The images were taken from triplicates (n = 3) and provided representative images in the text

NMR spectra of H. werneckii melanin

The proton NMR spectra of H. werneckii melanin and standard synthetic melanin are shown in (Fig. 4). The resonance in the aliphatic region of H. werneckii melanin is almost similar to the standard synthetic melanin and some more additional peaks could be detected in the 3.0 to 4.0 ppm. Peaks in the absorption region from 3.0 to 4.0 ppm can be assigned to protons on carbons attached to nitrogen and/or oxygen atoms. Resonance between 0.5 ppm to 2.5 ppm is assigned to CH₃ and CH₂ groups of alkyl fragments. Similarly, the resonance in the aromatic region also exhibits close similarity compared to synthetic melanin. In the aromatic regions of ¹H NMR spectra of H. werneckii melanin, peaks between 6.5 and 8.5 ppm are assigned to the protons attached to indole and/or other substituted aromatic or heteroaromatic rings. These resonances are similar to resonances in the aromatic region with indole or pyrrole structural units previously reported for melanin isolated from human hair.

Fig. 4.

Proton NMR spectra of melanin A) Aliphatic proton spectral regions taken at 600 MHz of synthetic melanin (SyM) and H. werneckii Melanin (HwM) plotted between about 1.0 and 5.0 ppm. Peaks from 3.0 to 4.0 ppm are assigned to protons on carbons attached to nitrogen and/or oxygen atoms in aliphatic region and resonances between 1.0 and 3.0 ppm are assigned to residual protein. B Aromatic proton spectral regions taken at 600 MHz of SyM and HwM plotted between about 5.9 and 9.9 ppm. Peaks between 6.5 and 8.5 ppm are assigned to the protons attached to indole and/or pyrrole repeat units of the melanin polymer in aromatic region. The images were taken from triplicates (n = 3) and provided representative images in the text

Metal composition of Melanin by ICP-AES

Different metal ions were detected in H. werneckii melanin by ICP-AES. It includes Na (I), Ca (II), Mg (II), K(I), and Fe (III). Table 1 shows the metal ions present in H. werneckii melanin and synthetic melanin. When compared to synthetic melanin, an elevated percentage of sodium, calcium and magnesium ions were detected in H. werneckii melanin. Melanin is linked with many metal ions that are subject to various functional groups. As stated in the proposed molecular structures of melanin, the pigment contains phenolic, hydroxyl (OH), carboxyl (COOH) and amine (NH) groups as potential functional binding groups for metal ions.

Table 1.

Chemical composition of various melanins

		Analysis
		ICP-AES					CHNS					EDS
S. No	Sample	Metal ions (%)					Elemental composition (%)					Atom (%)
		Ca	Fe	K	Mg	Na	C	H	N	S	C		O	Na	P	S	Cl	Mg	Ca
1	HwM	5.57	0.33	0.53	1.86	6.54	49.71	9.52	7.83	1.03	85.85		6.61	2.39	0.33	0.52	4.31	-	-
2	SyM	0.23	0.61	0.02	0.03	0.04	49.77	3.39	6.18	-	92.1		7.62	-	-	-	0.28	-	-
3	SeM	-	-	-	-	-	32.61	3.11	5.87	0.33	66.06		10.11	7.66	-	0.25	13.05	1.66	1.22

HwM: H. werneckii Melanin; SyM: Synthetic Melanin; SeM: Sepia melanin

Elemental composition of melanin

The elemental analysis detected the amounts of carbon, nitrogen, hydrogen, and sulphur in the melanin samples (Table 1). H. werneckii melanin showed higher percentages of hydrogen, nitrogen, and sulphur compared to sepia melanin and synthetic melanin. However, the carbon content was almost similar to synthetic melanin but higher to sepia melanin. Higher C:N ratio indicated that the H. werneckii melanin contains aliphatic groups almost closer to sepia melanin than synthetic melanin. The C:H ratio of H. werneckii melanin is also similar to sepia melanin.

Morphological/Elemental characteristics of melanin by SEM–EDS

The morphology and elemental composition pattern of the melanin samples were obtained by SEM–EDS analysis (Fig. 5 and Table 1). The appearance of H. werneckii melanin is in a crystalline form while synthetic melanin appears to be amorphous and sepia melanin in defined spherical forms. The surface elemental composition pattern of H. werneckii melanin revealed that it contains six elements, in which the major amount was carbon followed by moderate amounts of oxygen and chlorine; sodium, sulphur and phosphorous were also detected. However, synthetic melanin contains only three elements including a large quantity of carbon, moderate amounts of oxygen and a trace of chlorine. Whereas, sepia melanin consisted of seven elements, in which carbon is abundant followed by chlorine, oxygen and sodium in moderate amounts; very small amount of magnesium and calcium and trace of sulphur were also observed. The elements detected in H. werneckii melanin are more comparable with sepia melanin (standard) than synthetic melanin.

Fig. 5.

Electron micrographs of melanin samples shown in two different magnifications (3000 and 6000 x). A1 and A2 are melanin from H. werneckii (crystalline). B1 and B2 are synthetic melanin (amorphous). C1 and C2 are melanin from Sepia officinalis (spherical). The images were taken from triplicate samples (n = 3) and representative images are shown. Scale bars are 2 and 5 μm for 6000 and 3000 × magnifications, respectively

Antioxidant property

The scavenging ability of H. werneckii melanin was found to be increased in a dose-dependent manner (Fig. 6A). At a concentration of 5 µg ml⁻¹ the inhibition was 33.3 ± 3.62% and attained an inhibition of 98.59 ± 0.69% at 50 µg ml⁻¹. The concentration of an antioxidant required to reduce the initial ABTS concentration by 50% (IC₅₀) is a parameter generally used to calculate antioxidant activity. Thus, the IC₅₀ of H. werneckii melanin on the ABTS radicals was determined as 9.76 ± 4.10 µg ml⁻¹. The IC₅₀ of standard trolox on ABTS scavenging was found to be 1.042 ± 0.06 µg ml⁻¹.

Fig. 6.

Antioxidant activity of the H. werneckii melanin. A ABTS radical scavenging activity B) DPPH radical scavenging activity C) Lipid peroxidation inhibition which are indirect indications of antioxidant properties of the melanin. Significant differences are indicated by different letters on the data bar (P < 0.05) analyzed by Duncan post-hoc analysis. Data are shown as the mean ± SD of replicates (n = 3)

The scavenging capability of H. werneckii melanin on DPPH radical is shown in Fig. 6B. At a concentration of 10 µg ml⁻¹, the scavenging effect was 29.32 ± 1.97% and at 100 µg ml⁻¹ the scavenging was 87.8 ± 1.84%. The IC₅₀ of H. werneckii melanin on the DPPH radicals was 28.11 ± 10.76 µg ml⁻¹. The IC₅₀ of standard trolox on DPPH scavenging was determined as 1.334 ± 0.20 µg ml⁻¹.

Non-enzymatic peroxidation occurs when egg yolk (lipids) is incubated with ferrous sulphate, resulting in the formation of MDA and other aldehydes that form a pink colour with thiobarbituric acid. The inhibition effect of H. werneckii melanin on non-enzymatic peroxidation was also detected (Fig. 6C). At 75 µg ml⁻¹, the inhibition was 29.87 ± 4.68% and at 750 µg ml⁻¹ the inhibition effect was 81.5 ± 2.53%. The IC₅₀ of H. werneckii melanin was calculated as 222.56 ± 43.49 µg ml⁻¹. The positive control BHT exhibited an IC₅₀ of 8.63 ± 4.38 µg ml⁻¹ on lipid peroxidation.

Biocompatibility of H. werneckii Melanin

The viability of HEp-2 cells treated with different concentration of marine yeast melanin was analyzed as a function of mitochondrial dehydrogenase activity and protein synthesis. Melanin was nontoxic to HEp-2 cells in culture up to a concentration of 0.2 mg ml⁻¹, where only < 8% inhibition in terms of mitochondrial dehydrogenase activity (XTT assay) and protein synthesis (SRB) were observed. The IC₅₀ of melanin in terms of mitochondrial dehydrogenase activity was found to be 1.42 ± 0.56 mg ml⁻¹ and 1.25 ± 1.32 mg ml⁻¹ in terms of protein synthesis. Both XTT and SRB showed < 25% reduction in cell viability at 0.8 mg ml⁻¹, a concentration much higher than the dosage required for bioactivity ( Fig. 7).

Fig. 7.

Biocompatibility and cytotoxicity assay of melanin. XTT and SRB assay shows the cellular metabolism through mitochondrial dehydrogenase activity and protein synthesis in the presence of H. werneckii melanin. The Y-axis shows the percentage of cell viability (HEp-2) with respect to the concentration of melanin represented in X-axis. Data are shown as the mean ± SD of replicates (n = 3)

Photoprotection

HEp-2 cell line was used for the study and the cells were treated with H. werneckii melanin and synthetic melanin. Exposure of HEp-2 cell lines to UV radiation induced significantly high ROS generation compared to UV unexposed cells. Also ROS production was found to be significantly low in all the melanin treated HEp-2 cells, evidenced by a lower level of formazan in NBT reduction assay. It is evident that melanin could act as a protective barrier and thus reduce ROS generation and its deleterious effect in cells. In melanin treated HEp-2 cells, melanin absorbed the UV rays and thus protected the cells from the UV-induced damage. This photoprotection ability of H. werneckii melanin is obvious from the reduction of ROS generation as the concentration of melanin increased. H. werneckii melanin exhibited higher photoprotection than the positive control, synthetic melanin (Fig. 8).

Fig. 8.

Photoprotection effect of H. werneckii melanin on HEp-2 cells. Absorbance (620 nm) of formazan formed by NBT reduction; which is indirect indications of the ROS generated by UV exposure in HEp-2 cells treated with melanin. There are two controls; Control-1: HEp-2 cells unexposed to UV and Control-2:HEp-2 cells exposed to UV. SyM-50: Synthetic melanin at 50 µg ml⁻¹; HwM-50: H. werneckii melanin at 50 µg ml⁻¹; HwM-100: H. werneckii melanin at 100 µg ml⁻¹; HwM-200: H. werneckii melanin at 200 µg ml⁻¹ (n = 3). The error bars represent the mean ± SD. The bars indicated by different letters represent significant difference (P < 0.05) in one-way ANOVA and Duncan Post-hoc test

Discussion

Microorganisms which produce pigments have a competitive advantage over others in varying environmental conditions [3, 44]. Under hypersaline conditions, melanin was found to play a crucial role in the growth of H. werneckii [15, 17] and provide a protective effect to the organism. The yield of H. werneckii melanin (1.22%) was lower compared to that of melanin from black tea (BT) and about ten fold higher to black soybean (BS) and black-bone silky fowl (SF) i.e., 2%, 0.16% and 0.095%, respectively [12]. The appearance of H. werneckii melanin was found to be black in colour and similar to synthetic melanin. Melanin pigments from microbial sources exhibited a range of brown to black color [16, 21, 34].

H. werneckii melanin was insoluble in most of the solvents tested, except in sodium hydroxide and aqueous ammonia, and displayed solubilization property similar to the Taihe Black-bone silky fowl (TBSF) melanin reported by Tu et al. [37] and Pseudomonas stutzeri melanin by Kumar et al. [19].

Melanin plays a significant role in skin photoprotection, particularly as an optical screen that reduces the penetration of UV light and offers its multi dimensional biological effectiveness in protection from light, oxidative stress and energy transduction. The FTIR absorption spectrum of H. werneckii melanin is similar to the characteristic absorption spectra for fungal melanin. Peaks present in TBSF melanin (at 1539 and 1398 cm⁻¹), were also observed in H. werneckii melanin suggesting the presence of a pyrrole or indole NH group [37].

NMR spectra of H. werneckii melanin and synthetic melanin exhibited similarity in few regions. Additional peaks appeared in the aliphatic region from 3.0 to 4.0 ppm is comparable to that reported by Katritzky et al. [14] and Sun et al. [33]. Aliphatic carbons indicate the presence of proteinaceous material, based on similar assignments already reported [33]. Well-defined peaks present in the aromatic region of H. werneckii melanin were also observed in the synthetic melanin. The aliphatic and aromatic moieties in the H. werneckii melanin include signals from various functional groups as shown by other fungal melanin [4, 27]. The richness of metal ions such as Na, Ca, Mg, K, and Fe in the H. werneckii melanin sample show the binding potential of melanin to various metal ions. Similarly, melanin pigment from Pseudomonas stutzeri was also reported to be rich in Na(I), Ca(II), Al(III), Mg(II), Fe(III), K(I), and Zn(II) ions [19]. Meredith and Sarna [23] also reported the ability of both eumelanin and pheomelanin to bind metal ions, the most distinctive characteristic of this class of pigments. Elemental analysis showed that the H. werneckii melanin mainly contained C, H, N, and S elements. The C:N ratio and C:H ratios were closely related to sepia melanin than synthetic melanin.

SEM imaging of melanin showed that a definite structural order is a property of natural melanin, whereas synthetic melanin exhibited an amorphous nature and lack structural integrity as reported by Tarangini and Mishra [36] and Mbonyiryivuze et al. [22]. Surface elemental composition analysis by EDS showed that H. werneckii melanin showed higher similarity to sepia melanin than synthetic melanin. The compositional variation observed might be due to the change in production and growth medium composition as reported by Tarangini and Mishra [36].

Melanin readily interacts with free radicals and other ROS due to unpaired electrons present in the molecules. The antioxidant efficiency of melanin has been described by previous researchers and this property is mainly attributed to the chemical structure of melanin, especially the phenolic group present in the molecule [41]. Earlier studies have reported the antioxidant potential of melanin from different sources such as Exophiala pisciphila [45], Pseudomonas sp. [36] and Klebsiella sp. GSK [29]. In the present study, IC₅₀ of H. werneckii melanin (9.76 ± 4.104 µg ml⁻¹) on the ABTS radicals was found to be almost nine times lesser than the IC₅₀ of standard trolox (1.042 ± 0.063 µg ml⁻¹). Zhong et al. [46] reported that IC₅₀ of ethyl acetate extract of Streptomyces Eri12 was 172.43 ± 22.19 µg ml⁻¹ for ABTS which was about 220 times lesser than that of the positive control (Trolox; 0.76 µg ml⁻¹). When we compare this data, melanin extracted from the marine yeast, H.werneckii exhibited significant antioxidant potential in terms of radical scavenging activity. The activity of this crude H. Werneckii melanin can be increased by further purification of the compound.

IC₅₀ of H. werneckii melanin on the DPPH radicals was 21 times lesser than that of standard trolox. Recently, melanin extracted from Auricularia auricula fruiting bodies (AAFB) exhibited antioxidant activity on DPPH radical with an IC₅₀ value of 0.18 ± 0.03 mg ml⁻¹ [47]. The DPPH scavenging effect of melanin from Klebsiella sp. GSK revealed a higher scavenging activity at 50 μg ml⁻¹ (74%) than at 25 μg ml⁻¹ (55%) [29]. All the earlier reports have shown that melanin had significant potential in scavenging free radicals. Significant antioxidant property by H. werneckii melanin shows its potential for application in the cosmetic industry for the formulation of creams that protect the skin against oxidative damage. Biomelanins are also potent inhibitors of peroxidative damage. Different fractions of melanin extracted from chestnut shells were found to demonstrate strong lipid peroxidation inhibition [41]. Indeed, H. werneckii melanin demonstrated a lower IC₅₀ value and significant antioxidant property compared to TBSF melanin [37].

Evaluation of the cytotoxic effect of newly isolated compounds is essential for its acceptance in biological applications. Cytotoxicity of H. werneckii melanin was analyzed by XTT and SRB assays. Cytotoxicity assays using HEp-2 cells revealed that up to a concentration of 0.8 mg ml⁻¹, approximately 25% reduction in cell viability was exhibited by both XTT and SRB assays. This is a concentration much higher than the dosage required for antioxidant activity in cells. Therefore, our data supports that melanin from marine black yeast H. werneckii is biocompatible for its use as an antioxidant.

Hortaea werneckii melanin was found to be an effective photoprotectant in UV exposed HEp-2 cells. Melanin acts as a neutral density filter equally reducing all wavelengths of light and inhibit photoinduced damage [8]. A bacterial melanin incorporated cream formulation has showed significant reduction of ROS generated on exposure to direct sunlight [26]. Melanin possess photoprotectant activity against cellular stressors such as UVA, UVB, and H₂O₂ in human melanoma cells [35]. The ROS generated by means of UV irradiation in melanin treated cells were considerably lower than control cells (melanin untreated). This may be due to the competency of melanin to act as a barrier against UV irradiation and thus aid photoprotection to the cells. In this perspective, H. werneckii melanin will be a promising natural bioactive compound for the cosmetic industry for developing skin protection formulations against UV radiation.

Conclusion

From this study, it is apparent that H. werneckii melanin exhibits antioxidant and photoprotection activity, enabling its application as a protectant against UV radiation apart from other industrial applications. The physico-chemical characteristics of H. werneckii melanin are analogous to typical natural melanins. The in vitro assessment of radical scavenging activity and inhibition of lipid peroxidation proved that H. werneckii melanin possesses strong antioxidant potential. Furthermore, the study also proved the role of H. werneckii melanin as a photoprotectant on HEp-2cells in vitro in terms of reduction of ROS production against UV. Further, the cytotoxicity study confirmed its biocompatibility with human cells. H. werneckii melanin can be used as a sunscreen on human skin to absorb or scatter the hazardous solar UV radiation and thus protect the underlying cells from direct or indirect phototoxicity. Also, it could be used as a natural antioxidant in the cosmetic and pharmaceutical formulations in an environment-friendly perspective.

Declarations

Conflicts of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.