Melanin: insights into structure, analysis, and biological activities for future development

Abstract

Melanin, a widely distributed pigment found in various organisms, possesses distinct structures that can be classified into five main types: eumelanin (found in animals and plants), pheomelanin (found in animals and plants), allomelanin (found in plants), neuromelanin (found in animals), and pyomelanin (found in fungi and bacteria). In this review, we present an overview of the structure and composition of melanin, as well as the various spectroscopic identification methods that can be used, such as Fourier transform infrared (FTIR) spectroscopy, electron spin resonance (ESR) spectroscopy, and thermogravimetric analysis (TGA). We also provide a summary of the extraction methods of melanin and its diverse biological activities, including antibacterial properties, anti-radiation effects, and photothermal effects. The current state of research on natural melanin and its potential for further development is discussed. In particular, the review provides a comprehensive summary of the analysis methods used to determine melanin species, offering valuable insights and references for future research. Overall, this review aims to provide a thorough understanding of the concept and classification of melanin, its structure, physicochemical properties, and structural identification methods, as well as its various applications in the field of biology.

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1. Introduction

Melanin, the most enigmatic of the major pigments, has been the subject of slow and gradual research progress over the past century, despite being ubiquitous in nature. Derived from the Greek word “melanos”, the term “melanin” was first coined by Swedish chemist Berzelius in 1840 to describe the dark brown pigment isolated from the retina.¹ Formal research on melanin began in 1856, and in 1895, Bourquelot discovered an enzyme capable of converting tyrosine into a melanin-like substance under aerobic conditions.²

While melanin is a heteropolymer and lacks a uniform definition, it is a high molecular bio-pigment formed by oxidative polymerization of polyhydroxyphenols or polyhydroxyindole substances.^1,3 Although the structure and chemical composition of melanin remain incompletely described,⁴ this family of natural pigments is widely distributed in animals, plants, and microorganisms, and exhibits remarkable diversity in terms of its huge number and rich variety. Melanin extracted from different organisms has varying shapes, such as round, granular, round and slender, flat and hollow, and there are currently five types of melanin classified in detail, including eumelanin (found in humans and mammals), pheomelanin (found in humans and mammals), allomelanin (found in plants), neuromelanin, and pyomelanin (found in fungi and bacteria).^3,5 Melanin also has rich types of exterior colors such as black, yellow, red, brown, etc.¹ These applications span various fields, including the food industry, hairdressing industry, microbiology, nanotechnology, chemistry, and drug delivery.^6–10 Despite the progress made in recent years, the structure of melanin remains a complex and intriguing area of research.^11–15

This comprehensive review article aims to delve into the intricate world of melanin, with a specific focus on eumelanin and its functional structure. We will provide an in-depth exploration of the different types of melanin, their unique properties, and their critical role in biological processes. In addition, we will discuss the various cutting-edge techniques employed for the identification and characterization of melanin, including advanced spectroscopic, microscopic, and biochemical methods. Our examination will also encompass the diverse biological activities of melanin and its potential applications in numerous fields, from food to nanotechnology and drug delivery. By synthesizing and analyzing the current state-of-the-art knowledge of melanin, our review aims to inspire and pave the way for further ground-breaking research into this fascinating and enigmatic pigment.

2. Classification, origin, and formation of melanin

2.1 Eumelanin

Eumelanin is produced by the oxidative polymerization of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA).¹⁶ The synthesis of vertebrate melanin¹⁷ is closely associated with tyrosinase (Fig. 1a). In vivo tyrosine is catalyzed by tyrosinase to produce 3,4-dihydroxyalanine or dopa, which is further oxidized to produce dopaquinone, which is polymorphed and oxidized to produce DOPA pigment. Under the action of dopachrome isomerase, dopachrome is hydroxylated to 5,6-dihydroxyindole carboxylic acid (DHICA), decarboxylated to 5,6-dihydroxyindole, and then oxidized to 5,6-indolequinone under the catalytic action of tyrosinase, and finally combined with other intermediates to form eumelanin.

Fig. 1 Synthetic pathway of eumelanin (a), pheomelanin (b), allomelanin (c), pyomelanin (d) and neuromelanin (e).

Eumelanin is widely acknowledged as a pivotal factor in providing effective photoprotection. The pigmentation of the skin arises from the accumulation of two distinct types of melanin granules within keratin-forming cells: eumelanin and pheomelanin. The significance of melanin in safeguarding against harmful ultraviolet radiation (UVR) is further complicated by the existence of these two melanin granule types. Eumelanin, known for its ability to absorb UVR, plays a crucial role in shielding the deeper layers of the skin from UVR-induced damage. Conversely, pheomelanin, the other type of melanin granule, not only exhibits instability when exposed to light but may even contribute to the development of skin cancer.¹⁸

2.2 Pheomelanin

Pheo (formerly “phaeo”) means “dark” in ancient Greek. Pheomelanin is derived from the combination of dopaquinone spontaneously with the amino acid cysteine to form cysteine-based dopa,¹⁷ which undergoes oxidation, cyclization and rearrangement to yield 1,4-benzothiazine (Fig. 1b). It then eventually polymerizes to produce pheomelanin. Pigment production may be related to physiological limitations. Nevertheless, little is known about the structure and function of melanin, so elucidating this melanin further is an essential challenge.

Pheomelanin has been found in mammalian yellow to red hairs, fair-skinned individuals, bird feathers, dinosaur fossils, some insects and fungi.¹⁹ Changes in pheomelanin synthesis could be a side effect of maintaining cysteine homeostasis.²⁰ This may help explain the variability in pigment phenotype expression. Notably, pheomelanin is seldom detected in its pure form since it is rarely seen in a distinct isolated condition.²¹ Many research studies have relied on synthetic pheomelanin, which is not identical to natural pheomelanin.³

2.3 Allomelanin

Allomelanin are a group of heterogeneous polymers that appear through the oxidation or polymerization of 1,8-DHN (1,8-dihydroxynaphthalene) or THN (1,3,6,8-tetrahydroxynaphthalene) to produce DHN-melanin or allosteric melanin in various colors²² (Fig. 1c). Various enzymes are engaged in the synthesis of allomelanin. Allomelanin found in plants and fungi is featured by the existence of phenolic, catechol and 1,8-dihydroxynaphthalene (1,8-DHN).¹⁵ The first intermediate in the pathway for the synthesis of allomelanin is THN, which is formed by the action of polyketide synthase.²² Mutations in enzyme genes lead to similar results in various microorganisms, suggesting that the mutations may have occurred in the common ancestor of these microorganisms, which means the DHN melanin pathway is an ancient and evolutionarily conserved survival mechanism.²³

2.4 Pyomelanin

Pyomelanin, which is predominantly present in bacteria, is formed by the breakdown of tyrosine into p-hydroxyphenylpyruvic acid (HPP) and homogentisic acid (HGA), and is characterized by the presence of homogentisic acid units²⁴ (Fig. 1d). Many strains produce pyomelanin by oxidation of HGA during the catabolism of tyrosine or phenylalanine, including Legionella pneumophila, Burkholderia cepacia, etc.²⁵ Furthermore, pyomelanin is also a key corrosion factor, and it was found that even after Pseudomonas solanacearum was damaged by Cu, the pyomelanin produced by this strain still hastened the corrosion of Cu.²⁶

2.5 Neuromelanin

Neuromelanin (NM) is synthesized by oxidative polymerization of dopamine or norepinephrine, possibly involving cysteine derivatives, starting from the center of pheomelanin and proceeding to eumelanin after depletion of cysteine levels²⁷ (Fig. 1e).

Neuromelanin (NM), a group of chemicals, is prominently present in catecholaminergic neurons, specifically in the substantia nigra (SN) and locus coeruleus (LC), throughout the human and animal brain,²⁸ which are early targets in Parkinson's disease (PD).²⁹ NM in SN is thought to be mainly a non-enzymatic product from dopamine (DA) iron-mediated autoxidation.³⁰ What's more, NM's pigmentation diminishes significantly in Parkinson's disease.¹² The question of whether NM confers protection or contributes to harm in various disorders, given its presence in susceptible neurons, has long been a subject of debate.^31,32 Recently, the structure of neuromelanin has been elaborated as a melanin–protein core model.¹²

2.6 Chemical synthetic melanin

The synthesis of melanin, especially water-soluble melanin, is now a hot research direction (Fig. 2). Ju et al.³³ found that melanin could be synthesized when dopamine hydrochloride solution was added to NaOH solution under vigorous stirring. Directly inspired by the color production by self-assembled melanosomes in bird feathers, Xiao et al.³⁴ set out to synthesize and assemble synthetic melanin nanoparticles based on polydopamine to create colored films. Synthetic melanin-like nanoparticles (MelNPs) resembling natural tan melanin can be used as contrast agents for magnetic resonance imaging (MRI),³³ creating new opportunities for potential applications in clinical MRI diagnostics.

Fig. 2 Chemical synthesis of melanin. (a) Schematic illustration of the development of efficient T₁-weighted MRI contrast agents using synthetic melanin-like nanoparticles (MelNPs).³³ Copyright © 2013, American Chemical Society. (b) Synthesis of synthetic melanin nanoparticles.³⁴ Copyright © 2015, American Chemical Society. Created with https://BioRender.com.

3. Structure analysis and physicochemical properties of melanin

Melanin binds to scaffold proteins through covalent bonds, resulting in a highly cross-linked heteropolymer. The analysis and simulation of the melanin structure is shown in Fig. 3. An understanding of the optical properties of melanin with different compositions and morphologies remains incomplete and challenging, even if the analysis is fundamental from a technical point of view.

Fig. 3 Cellular construction and structure analysis of melanin. (A) Transient spectral hole burning of DOPA melanin (eumelanin) and putative chromophore structures.³⁷ Copyright © 2020, The Author(s). (B) Structural disassembly of synthetic melanin-like nanoparticles (MelNPs) under deoxygenated alkaline conditions.³⁵ Copyright © 2018, American Chemical Society. (C) Different orientations of DHI (D1-4) and the directing hydrogen bonds observed in the single crystal.³⁶ Copyright 2022, Royal society of chemistry. (D). Snapshots of 27 eumelanin protomolecules in an aqueous solution at 1600 ns.¹¹ Copyright © 2022, American Chemical Society.

3.1 Cellular construction

The structural unit of melanin is a popular topic of research interest. The simulated and speculated structure of partial melanin is presented in Fig. 3. Ju et al.³⁵ investigated the complicated layered assembled structure of eumelanin and its unique broad absorption bands by pump–probe or transient absorption microscopy (TAM), revealing a relationship between the highly unusual nonlinear dynamics. Although many theoretical studies have explored the structure of eumelanin, precise mapping of the large space of eumelanin remains elusive. In contrast, pheomelanin has been a neglected melanin species. In pheomelanin, the existence of such equivalent lamellar structures for benzothiazine and benzothiazole has not been resolved.

DHI diffraction mass single crystals obtained in chloroform solution show zig-zag helical stacking.³⁶ The first computational study of structural characteristics of DHI-eumelanin polymers in aqueous solution was reported by Soltani et al., and it was shown that eumelanin aggregated by forming nanoscale stacks due to the reduction of hydrogen bonds between eumelanin and water, and the results showed non-covalent stacks with interlayer distances less than 3.5 Å.¹¹

3.2 Solubility

Generally, melanin is insoluble in acidic solutions, water, and other organic solvents such as acetone, chloroform, ethyl acetate, hexane, methanol, and petroleum ether, but melanin is slightly soluble in alkaline solutions.³⁸ Therefore, many studies have been conducted to improve the water solubility of melanin by chemical modifications, such as adding chemical modification methods to fungal melanin or performing synthetic melanin analogs to increase its solubility and expand the application areas.³⁹

But there is also dissolved melanin. One study found that bacterial cultures of Streptomyces cavourensis SV 21 produced two different forms of melanin: (1) a granular insoluble form and (2) a water-soluble form that is rarely observed.⁴⁰ Moreover, the difference in solubility between soluble and insoluble melanin in aqueous solution may be due to the difference in the species of adjacent cations.

3.3 Melanin storage stability

Eumelanin subunits are more stable and moderately reactive than free monomers due to their capacity to scavenge and block free radicals, especially under high temperature and photo-oxidation conditions, making them superior for technical applications.¹⁶ The melanin species in red hair is pheomelanin, which contains a 2-S-cysteinyldopa component that prevents ROS attack or UV-induced degradation during hair growth, thus exhibiting intrinsic stability.⁴¹ Pyo et al.⁴² attempted to synthesize artificial pheomelanin nanoparticles and found that in various media artificial pheomelanin nanoparticles exhibited high dispersion stability. Nowadays, most of the studies on allomelanin stability are synthesized and fabricated into materials to further investigate its activity.⁴³ Synthesized spherical allomelanin nanoparticles partially dissolved in ethanol can reach a stable state in solution after aging, polymerization and darkening.⁴⁴ The absorption spectrum of pyomelanin secreted by Shewanella sp. remained stable after 30 min of continuous exposure to UV light,¹⁴ and the pyomelanin synthesized by HGA autoxidation has an ultra-high thermal stability.⁴⁵ Neuromelanin extracted from the human brain shows highly stable and consistent morphology under AFM, SEM, etc.²⁷ Moreover, neuromelanin has a protective effect by scavenging reactive metals, pesticides, and other toxins forming stable adducts and thus exerting protective effects.⁴⁶ Overall, melanin is an extremely stable substance with superior storage stability.

3.4 Structure analysis

Accurate characterization of melanin using structure analytical methods has proven difficult because of its heterogeneity, insolubility over a wide range of pH values and a wide range of solvents.⁴⁷ However, there are many analytical methods for identifying the basis structure of melanin, and in this paper, the structure of melanin was analyzed using high technology such as infrared and ultraviolet, etc.Fig. 4 displays the identification methods of melanin. The characteristics of different melanin under different identification methods were classified and organized, such as Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (Uv-vis) and High-Performance Liquid Chromatography (HPLC) (Table 1); elemental analysis (Table 2).

Fig. 4 Melanin identification and analysis method. Created with https://BioRender.com.

Table 1 FTIR spectroscopy, UV-vis spectral characteristics and HPLC markers of melanin

Type	FTIR	Uv-vis	HPLC
Eumelanin	3500–3000 cm^−1: broad absorption; stretching vibrations of carboxyl, phenols and free OH and –NH groups, as well as intermolecular hydrogen bonding.		Pyrrole-2,3,5-tricarboxylic acid (PTCA) & pyrrole-2,3-dicarboxylic acid (PDCA).^55–58
	1710 cm^−1: significant unique peak, the C=O stretch;^50,51 1650–1500 cm^−1: C=C stretching and COO stretching of aromatic groups	Broad absorption overall.
		220 nm: maximum absorbance.^48,53
	1400–1300 cm^−1: C–H in-plane bending vibrations, C–O stretching vibrations and C–C single bond skeleton vibrations^52,53	300–450 nm: show strong absorption, extending into the infrared region.^54
Pheomelanin	1535 cm^−1: strong absorption; 1280 cm^−1: weak, corresponds to the (COH) phenolic stretch.	300–450 nm: show strong absorption, extending into the infrared region.^54	4-Amino-3-hydroxyphenylalanine (AHP) & 3-amino-l-tyrosine (AT).^56,57,60
	1124 cm^−1, 1172 cm^−1 and 1213 cm^−1: extremely strong, (S–O symmetric stretching vibration);^50,54 800 cm^−1 and 678 cm^−1: bands of aromatic rings and sulfur.^54,59	233 nm: maximum absorbance, higher absorption in the full spectrum.^42
Allomelanin	1020 cm^−1: a distinct peak, associated with the stretching vibration of the (C–O–C) group;^61 1200–1400 cm^−1: broad band, the presence of C–O residues;^62 1381 cm^−1: associated with the aromatic C–C linear stretching or C–N stretching of the indole structure; 1593 cm^−1: attributed to the C=C bond of the indole structure;^62 1720 cm^−1: no absorption, the absence of free carboxyl groups.^61	200–300 nm: maximum absorbance & absorption decreases within the visible region.^63 270–280 nm: a typical small protein absorption. The absorbance value decreases with increasing wavelength.^64	Dimer of DHN^65,66
Pyomelanin	3300–3420 cm^−1: broad absorption, stretching vibrations of –NH and –OH.^14,67	250 nm: start an absorbance; 250–280 nm: reach the maximum absorbance, and then decrease.^24,49	Precursor melanin acid (HGA).^69
	2952 cm^−1 and 2925 cm^−1: stretching vibration of aliphatic bound to –CH.^67
	1631 cm^−1 and 1388 cm^−1: strong, bending vibrations of aromatic rings and aliphatic groups, respectively.^14
	1000 cm^−1 and 500 cm^−1: occurs in the fingerprint region.^68
Neuromelanin	2950 cm^−1 and centered at 1060 cm^−1: unique feature with three band absorption peaks around.^51	200–300 nm: maximum absorbance, showing a monotonic absorption curve decreasing gradually to the visible range.^70	Thiazole-4,5-dicarboxylic acid (TDCA)/pyrrole-2,3,5-tricarboxylic acid (PTCA) ratio.^71
	Typical quinone structure, an aromatic structure and aliphatic moieties.^51

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Table 2 Elemental analysis of melanin

Type	Main characterization	Ref.
Eumelanin	• No or very little sulfur.	56 and 82
	• Eumelanin extracted from squid ink: C (52.25%), H (3.40%), N (7.88%), and S (0.20%).	83
Pheomelanin	• Sulfur content of about 6–16%.	56 and 59
Allomelanin	Low nitrogen content (<2%), C/N ratio was 34 or 35.	84
Pyomelanin	• A lower carbon content compared to eumelanin (polycyclic structure).	85
	• Pyomelanin extracted from Shewanella sp. has a unique elemental composition: C (29.2%), H (8.23%), N (6.41%), and S (1.58%), C/H equals 4.55 and C/N ratios equals 3.55.	14
	• Chemically synthesized pyomelanin has no nitrogen atoms.	86
Neuromelanin	• High sulfur content (2.5–2.8%), a lower C/H molar ratio than synthetic melanin.	51
	• 2.3% sulfur and 8.1% nitrogen.	71

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Fourier transform infrared (FTIR) spectroscopy analysis. Infrared spectroscopy is a commonly used spectroscopy for the detection of organic structures. It doesn’t destroy the structure of the sample and can reveal detailed information about the functional groups of the sample. FTIR is a valuable technique for the identification and characterization of different melanin structures and is the most widely used spectroscopic technique for the identification of melanin today. The analysis of infrared spectra is related to the interpretation of the thermal properties of the compounds, which allows a detailed study of the structural changes of melanin during heating.⁴⁸ A common method of operation using FTIR is to scan in the range of 4000–500 cm⁻¹ with a resolution of 4 cm⁻¹.⁴⁹ C–H, C–N, C–O and C=C functional groups are the key functional groups in the indole/pyrrole structure.⁴⁸ The functional groups can be identified by FTIR.

Eumelanin has three typical absorption peaks: (1) broad absorption at 3500–3000 cm⁻¹ corresponding to stretching vibrations of carboxyl, phenols and free OH and –NH groups, as well as intermolecular hydrogen bonding. (2) C=C stretching and COO stretching of aromatic groups are seen at 1650–1500 cm⁻¹. (3) 1400–1300 cm⁻¹ corresponds to C–H in-plane bending vibrations, C–O stretching vibrations and C–C single bond skeleton vibrations.^52,53 And the C=O stretch in the carboxyl group shows a significant unique peak at around 1710 cm⁻¹.^50,51

Pheomelanin is composed of sulfur-containing units, mainly phenprothiazine and benzothiazole, which are shown to have a strong absorption of 1535 cm⁻¹ on the infrared spectrum, and there are characteristic bands of aromatic rings and sulfur at both 800 cm⁻¹ and 678 cm⁻¹.^54,59 The region from 1000 cm⁻¹ to 1300 cm⁻¹ can only be characterized by pheomelanin, and pheomelanin has extremely strong peaks at 1124 cm⁻¹, 1172 cm⁻¹ and 1213 cm⁻¹ (S–O symmetric stretching vibration).^50,54 A weak peak at about 1280 cm⁻¹ is also characteristic of melanopsin, which corresponds to the (COH) phenolic stretch, respectively.

The broad band of allomelanin between 1200 and 1400 cm⁻¹ indicates the presence of C–O residues.⁶² Allomelanin from pathogenic black knot fungus has two absorption peaks: the peak at 1381 cm⁻¹ is associated with the aromatic C–C linear stretching or C–N stretching of the indole structure, while the peak at 1593 cm⁻¹ is attributed to the C=C bond of the indole structure.⁶² The allomelanin of C. islandica had a distinct peak in the range of 1020 cm⁻¹ associated with the stretching vibration of the (C–O–C) group; no absorption was recorded at 1720 cm⁻¹, which may indicate the absence of free carboxyl groups.⁶¹

Pyomelanin was found with a broad absorption in the 3300–3420 cm⁻¹ range, which corresponds to the stretching vibrations of –NH and –OH.^14,67 The stretching vibration of aliphatic bound to –CH appears at 2952 cm⁻¹ and 2925 cm⁻¹, respectively.⁶⁷ Moreover, strong bands of pyomelanin were also found at 1631 cm⁻¹ and 1388 cm⁻¹, which correspond to the bending vibrations of aromatic rings and aliphatic groups, respectively.¹⁴ In addition, pyomelanin also occurs in the fingerprint region between 500 cm⁻¹ and 1000 cm⁻¹.⁶⁸

Neuromelanin has a unique IR spectral feature with three band absorption peaks around 2950 cm⁻¹ and centered at 1060 cm⁻¹.⁵¹ Neuromelanin has a typical quinone structure, an aromatic structure and aliphatic moieties.⁵¹ Infrared spectra of neuromelanin show the presence of aliphatic groups and low-intensity aromatic components, while synthetic melanin does not have aliphatic groups.⁵¹ Neuromelanin is a dark brown melanin whose biosynthesis, structure and function have not been well characterized. However, infrared spectroscopy is currently used only as a technical tool for the identification of melanin species and basic group characteristics, and no such spectroscopic technique has been found to be suitable for determining the melanin structure.

Ultraviolet-visible spectroscopy (Uv-vis). The Uv-vis wavelength scan spectrum of melanin has a very distinctive feature: the absorption has its highest value in the UV region between 200 and 300 nm, but the absorption value decreases towards the visible region, due to the actual complex structure of melanin. Melanin showed a broad absorption overall.¹⁶ The relationship between log absorbance and wavelength is a linear curve with a negative slope, which is another important characteristic of melanin.⁶³

When eumelanin is dissolved in alkaline solution and analyzed by UV-vis in the range of 180–900 nm, eumelanin produces a maximum absorbance peak at 220 nm and a broad absorption at other wavelengths.^48,53 Pheomelanin and eumelanin both show strong absorption in the wavelength range of 300–450 nm with attenuation behavior extending into the infrared region.⁵⁴ In general, pheomelanin exhibits higher absorption in the full spectrum, becoming more pronounced in the visible region, with the highest absorption wavelength near 233 nm.⁴² Allomelanin has the highest absorption value at 200–300 nm, but the absorption decreases in the visible region.⁶³ The absorbance value of allomelanin decreases with increasing wavelength and also exhibits a typical small protein absorption peak (270–280 nm).⁶⁴ Pyomelanin produces an absorbance peak at 250 nm and reaches its maximum absorbance value between 250 and 280 nm, and then gradually decreases.^24,49 Neuromelanin, like other melanin, has the highest absorption peak between 200 and 300 nm, showing a monotonic absorption curve decreasing gradually to the visible range.⁷⁰

While UV-vis absorption spectroscopy has proven to be a useful tool for melanin quantification, it is not without its limitations. One of the main challenges in this regard is the potential interference from other pigments and chromophores present in the sample.⁶⁴ To mitigate this issue, a range of strategies could be employed, including sample purification, targeted wavelength selection, and advanced mathematical models for data analysis.

High performance liquid chromatography (HPLC) analysis. The HPLC technique, when applied to melanin analysis, often has two roles: on the one hand, as identification of melanin species. On the other hand, it can be used to determine the melanin content by the concentration of markers after melanin degradation. This HPLC method can be applied to research on biological samples with unknown melanin content in the future.⁵⁸

A specific marker, pyrrole-2,3,5-tricarboxylic acid (PTCA), was identified by HPLC and LC-MS analysis after oxidative degradation of eumelanin mixtures.⁵⁵ Quantification of markers by HPLC provides useful information on melanin concentration and structural diversity in a variety of biological samples.⁵⁸ Pheomelanin was hydrolyzed by hydriodic acid to produce 4-amino-3-hydroxyphenylalanine (AHP) and 3-amino-l-tyrosine (AT), and AHP and AT were separated using ion exchange chromatography, followed by separation and quantification by HPLC and electrochemical detection.⁶⁰ Compared with the commonly used methods for the determination of pheomelanin, the HPLC method has good specificity and higher sensitivity. Ito described a method for the quantitative analysis of eumelanin and pheomelanin in tissues such as hair and melanoma.⁵⁶ They also further compared eumelanin and pheomelanin levels in various tissues obtained from humans, mice and other animals.⁵⁷ The nitrogen-free precursor of allomelanin is 1,8-DHN. Zhou et al.⁶⁵ oxidized the solution (containing 1,8-DHN) by controlling the ratio of potassium permanganate, which could be used to obtain a dimer of DHN for isolation and purification by HPLC. Cecchini et al.⁶⁶ used ultra performance liquid chromatography (UPLC) as well as mass spectrometry data to confirm that the precursor of allomelanin, 1,8-DHN is oxidatively polymerized by C–C coupling of the naphthalene ring. The identification of three bacterial melanin types by HPLC analysis by Singh et al. showed the presence of precursor melanin acid (HGA) in pyomelanin.⁶⁹ After isolation of neuromelanin from human brain cells, the pigment can be oxidized with HI or KMnO₄, and the TDCA/PTCA ratio can be determined by HPLC, which reveals that neuromelanin may be derived from pheomelanin or eumelanin.⁷¹

However, HPLC equipment is expensive and has limitations when used as a common melanin content assay. Therefore, HPLC is recommended for the analysis of complex biological samples to avoid potential false positive identification of melanin.

Liquid chromatography-mass spectrometry (LC-MS). Currently the use of liquid chromatography-mass spectrometry (LC-MS) is a common method for the characterization of melanin content. Liquid chromatography-tandem mass spectrometry allows the detection of oxidative degradation mixtures of melanins, such as the eumelanin markers (PTCA and PDCA), and pheomelanin markers (TDCA and TTCA).⁵⁵ This cutting-edge technique enables the detection of oxidative degradation mixtures of melanins and specific markers, such as eumelanin and pheomelanin markers, providing important insights into the type and quantity of melanin present. LC-MS is particularly useful for analyzing biological samples with unknown melanin content and has the potential to advance our understanding of the roles of melanin in various biological processes and diseases.⁵⁸ As instrumentation and methodology continue to advance, LC-MS is expected to remain a valuable technique for the precise analysis and quantification of melanin in biological samples, paving the way for future research in this field.

Raman spectroscopy. Raman spectroscopy can be applied to identify the differentiation status of melanocyte spectrum cells and to characterize the immediate and transient biochemical changes associated with UV irradiation that vary by cell type, differentiation status, and melanin synthesis capacity.⁷² Yakimov et al.⁷³ showed how to estimate the matrix decomposition of melanin fractions at different depths in human skin in vivo from their Raman spectra (bands at 1380 and 1570 cm⁻¹). de Oliveira Neves et al.¹³ applied Raman spectroscopy in combination with chemometrics to quantify the constituent monomers of melanin, such as benzothiazide (BT) and benzothiazole (BZ).

Raman spectroscopy is a promising technique for the identification and characterization of melanin-related biochemical changes, such as those induced by UV irradiation, in different types of cells and tissues. With further advances in instrumentation and methodology, Raman spectroscopy has the potential to become a valuable tool for the analysis of melanin and its roles in various biological processes and diseases.

Solid state nuclear magnetic resonance (ssNMR). Solid-state nuclear magnetic resonance (ssNMR) is a detection method that does not destroy the structure of substances, and it allows obtaining structural information at the atomic scale to gain further insight into their chemical environment.

Peaks displayed at 10–40 ppm correspond mainly to the aliphatic carbon of lipids, 50–110 ppm to the polysaccharide ring carbon, 110–160 ppm to the aromatic carbon of melanin, and several overlapping peaks within the 165–190 ppm range corresponding to all three constituent types of carbon-based carbons.⁷⁴ Cao et al.⁷⁵ emphasised the elucidation of the biosynthetic pathways and structural characterization methods of melanin. These pathways and structural characterization techniques can be used to study relationships between structure function, including electron paramagnetic resonance (EPR) and ssNMR spectroscopy. The introduction of melanin into the broader chemical community could stimulate new functional synthetic materials.

Fluorescence quantification. The fluorescence quantification method is applicable to all melanin species. The fluorescence content of melanin is easy and quick to obtain. Fluorescence spectroscopy is the most suitable method for melanin quantification because it has been shown to have high specificity and accuracy in detecting even small changes in melanin synthesis. Moreover, the fluorescence signal from melanin is not affected by protein or lipid contamination.⁷⁶ The position of the fluorescence maximum was dependent on the kind of melanin and its reaction environment. The dissolution of melanin significantly enhanced fluorescence emission, and it is hypothesized that the oxidative cleavage of the quinone structure existing in melanin is related to the solubilization of melanin and the enhancement of fluorescence.

X-Ray analysis. There are various methods developed using X-ray technology, and here we summarize the techniques mentioned in previous literature species according to different melanin types. X-Rays can obtain electron density as they pass through the sample, providing better contrast even for unstained samples.

Fast scanning X-ray fluorescence imaging can detect the distribution of zinc and organic sulfur, for example, for the analysis of residual pheomelanin in valuable specimens¹⁹ or feathers⁷⁷ without destroying the sample. Manning et al.¹⁹ used synchrotron rapid scanning-X-ray fluorescence (SRS-XRF) to analyze sulfur in the fur of a 3 million-year-old fossil mouse (Apodemus atavus) and found that resonance of the reduced sulfur oxidation state occurs at 2472.5 eV. Edwards et al.⁷⁷ used sulfur X-ray absorption near edge structure (XANES) spectroscopy to investigate the potential difference in S coordination between eumelanin and pheomelanin, and the spectra showed two sharp peaks at 2472.3 and 2473.5 eV, more pronounced at 2480.4 eV, the reason for the double peak being derived from the abundant disulfide and sulfur–carbon bonds in keratin.

By light and X-ray scattering studies, McCallum et al.⁴⁴ determined the dispersion, morphology and fine structure of allomelanin in solution. Singla et al.⁶² determined by XPS that the nitrogen content of allomelanin extracted from pathogenic black knot fungus was very low (3.5 ± 0.9%).

The XRD spectrum of pyomelanin is characterized by a broad peak centered at approximately 2θ equal to 22°, and its diffraction pattern has a high overlap with the XRD analysis of eumelanin.²⁴ The XRD pattern of pyomelanin produced by Rubrivivax benzoatilyticus JA2 has broad diffraction peaks and is amorphous.⁷⁸

Bush et al.²⁷ studied neuromelanin isolated from the substantia nigra region of human brain by scanning probe and photoelectron emission microscopy. Therefore, nanoscale visualization of neuromelanin in human brain tissue can be achieved using scanning transmission X-ray microscopy (STXM) by characterizing neuromelanin in X-ray absorption spectra.

Electron spin resonance spectroscopy (ESR). Electron spin resonance spectroscopy (ESR) measures electron spin resonance signals based on the free radicals of melanin, most notably characterized by the presence of unpaired electrons, which define their paramagnetic properties.⁷⁶ The ESR method is an essential tool for recognizing and investigating the nature of pigment radicals.

Panzella reported the purification of black sturgeon caviar pigments and their definitive identification by chemical degradation combined with electron paramagnetic resonance (EPR) evidence as the typical eumelanin.⁵⁵ Chikvaidze et al.⁷⁹ studied the ESR spectra of red hair samples and found that the ESR signal at low power was generated by the superposition of two spectral shapes: single-linear spectra indicating the presence of eumelanin and trilinear spectra indicating the presence of pheomelanin, which means having trilinear ESR spectra is an important feature of pheomelanin. The melanin produced by Actinomyces spp. belongs to pheomelanin, and the EPR spectrum showed a peak of 2.00968.⁵⁹ The EPR signal of allomelanin extracted from oat hulls showed a single peak⁸⁰ without any fine structure similar to the EPR spectra of other synthetic and natural melanin from different sources.^53,81 The g value of this signal was calculated as 2.0051, typical of a carbon-centered organic radical with a conjugated structure.⁸⁰ Mekala et al.⁷⁸ performed ESR analysis of pyomelanin produced by photosynthetic bacteria and found that pyomelanin had a characteristic signal of 325 mT. ESR is a powerful technique for studying the properties of materials with unpaired electrons, such as free radicals, transition metal ions, and paramagnetic molecules. In the context of melanin and iron in nigrostriatal cells, ESR can be used to investigate the interactions between melanin, iron, and other reactive species, and to elucidate their roles in oxidative damage and neurodegeneration.

Thermogravimetric analysis (TGA). The use of thermogravimetric analysis to measure the thermal stability of melanin gives information about physical phenomena such as phase changes, chemical phenomena, thermal decomposition, etc. TGA is a method of thermal analysis in which the mass of a powder or pellet sample is continuously examined as the temperature increases. In addition, the micropyrolysis technique can be combined with mass spectrometry analysis (Py-GC/MS), which allows the detection of pyrolysis products during the thermal degradation of the developed material. An interesting feature of natural melanin is its high thermal stability.^48,87 All melanin species showed three stages of mass loss during heating, for example this effect occurs at temperatures similar to those reported for melanin isolated from black garlic, cuttlefish juice and B. subtilis.⁴⁹

Eumelanin frequently shows three major mass loss phenomena:⁸⁸ loss of weakly bound water, decarboxylation phenomena, covalent bond rupture and decomposition of indole or pyrrole rings. What's more, eumelanin is an extremely stable substance that needs to reach at least 500 °C before its mass is affected even after binding to heavy metals.⁴⁸ During pyrolysis, pheomelanin exhibits three stages of weight loss and is relatively stable in the polymer structure, except for hydrolytic absorption up to about 140 °C, as observed in pheomelanin produced by Bacillus subtilis.⁸⁷ Two exothermic peaks at around 200–300 °C and 500–560 °C are the main characteristics of pheomelanin.¹⁷ High levels of allomelanin are present in date fruits and have a high thermal stability: only 20% weight loss occurs at 220 °C.⁸⁹ Pyomelanin produced by Bacillus subtilis and Bacillus thermophilus have mass reduction (three stages):⁴⁹ the release of intramolecular or intermolecular water bound to melanin, the decomposition of intermolecular aliphatic groups, and the release of aromatic compounds.

Overall, TGA is an important tool for studying the thermal properties of melanin and other materials, and its development and application are likely to continue to advance our understanding of these complex biopolymers in the future.

Elemental analysis. Elemental analysis is a chemical analysis method for studying the composition of elements in organic compounds, and it is divided into two types: qualitative and quantitative. The former is used to identify which elements are present in an organic compound and the latter is used to determine the percentage content of these elements in an organic compound. The carbon to nitrogen molar ratio (C/N ratio) obtained from elemental analysis can be used as a parameter to characterize eumelanin.⁹⁰

The classification of eumelanin and pheomelanin depends mainly on the color, solubility and sulfur content of the isolated melanin. Ito et al.⁸³ found that eumelanin extracted from squid ink contains C (52.25%), H (3.40%), N (7.88%), and S (0.20%). A distinctive feature that distinguishes eumelanin from pheomelanin is the absence of a sulfur group.⁸² Pheomelanin or dopamine melanin chemically modified by amino acids (e.g. cys-DOPA melanin) is known to have a sulfur content of about 6–16%.^56,59

The monomer in pyomelanin is a single benzene ring structure (benzoquinone acetate) and therefore has a lower carbon content compared to eumelanin (polycyclic structure).⁸⁵ Pyomelanin extracted from Shewanella sp. has a unique elemental composition (C/N/H/S of 29.2 : 8.23 : 6.41 : 1.58) and C/H and C/N ratios of 4.55 and 3.55, respectively.¹⁴ Both pyomelanin produced by laccase and pyomelanin produced by bacteria have nitrogen atoms, but the chemically synthesized pyomelanin did not have nitrogen atoms.⁸⁶

Identification of allomelanin was typically characterized by a low nitrogen content (<2%) and carbon to nitrogen ratio (34 or 35).⁸⁴ Odh et al.⁷¹ analyzed isolated neuromelanin showing that it contained 2.3% sulfur and 8.1% nitrogen. Elemental analysis of neuromelanin showed a high sulfur content (2.5–2.8%) and a lower C/H molar ratio than synthetic melanin, thus indicating the presence of aliphatic groups.⁵¹

Scanning electron microscopy (SEM). Scanning electron microscopy (SEM) is a means of observation between transmission electron microscopy (TEM) and optical microscopy, which can be used to obtain high-resolution images, thus providing a sense of stereo and realism. In addition, SEM has the advantages of a wide variety of samples that can be measured, little damage and contamination of the original sample and the simultaneous acquisition of morphological, structural, compositional, and crystallographic information.

Melanin exhibits irregular shapes and sizes at different magnifications and is not uniform in shape across different sources, including granular, round, elongated, flat or hollow melanosomes found in animals.⁵² SEM observed the morphology of nanostructures of eumelanin extracted from squid ink, and the particles obtained after extensive washing contained mainly spherical particles with diameters of 150–220 nm.⁴⁷ The nanoparticles of eumelanin, which have a powder density of 1.69 g mL⁻¹, are non-porous and have a smallest subunit diameter of 15 nm on their surface as observed by SEM.⁹¹ Due to the low magnification of SEM observation, only the blocky solid shape of the particles after aggregation was visible.⁸⁷ Therefore, subsequent observations by SEM magnification revealed that pheomelanin are spherical nanoparticles with an unusual size distribution, with diameters of about 90–180 nm.⁵⁴ Natural allomelanin extracted from pathogenic black knotted bacteria was irregularly shaped,⁶² which may result from disruption after passing through acidic and alkaline solutions. To better understand the structure of allomelanin, Zhou et al.⁶⁵ synthesized artificial allomelanin and observed that the dimer could be formed by forming an oval platelet shape and finally assumed a spherical particle shape. Several studies succeeded in synthesizing allomelanin under experimental conditions, and four states of allomelanin are now available. By SEM observation, it is now possible to synthesize solid particles, walnut-shaped particles, lacy particles, and hollow particles.^43,44 By extracting pyomelanin from Shewanella sp., Kuttan et al.¹⁴ found that the shape of the pigment was disrupted after acid and alkaline extraction. Marín-Sanhueza et al.⁴⁹ found that thermophilic Bacillus species from Chilean Hot Spring produced pyomelanin with a morphology similar to that of other melanin produced by other Bacillus species.⁸⁷ It usually appears as a dense, amorphous deposit without distinguishable patterns. Thus, pyomelanin has essentially no distinguishable shape.

Transmission electron microscopy (TEM). Since there are similarities and differences between the observations of TEM and SEM, TEM analysis was carried out. In fact, whether SEM or TEM, the main purpose is to image. When performing pigment observation, the appropriate imaging method can be chosen according to the location (surface, interior) to be observed. TEM observation of eumelanin contains mainly spherical particles with a diameter of 150 nm.⁹² Ju et al.⁹³ observed the state of eumelanin in alkaline solution at different times by TEM and found that chemical oxidation of subunits of eumelanin leads to its stacked layer structure. It was observed using TEM that the size of the nanoaggregates of individual pheomelanin was significantly reduced after prolonged photolysis.⁹⁴

The allomelanin self-assembled by 4-4′ dimers, formed well-defined edges of oriented flakes, while the allomelanin self-assembled by 2-4′ and 2-2′ dimers forming well-defined spherical nanoparticles.⁶⁵

Eskandari et al.⁹⁵ showed that pyomelanin extracted from Pseudomonas koreensis strain UIS 19 accumulates in intracellular and extracellular spaces by TEM studies. Sacchini et al.⁹⁶ found that neuromelanin granules in toothed whale brain cells have striking similarities to human neuromelanin.

Atomic force microscopy (AFM). Atomic force microscopy (AFM) is the most widely used tool for nanoscale measurement and imaging and is becoming increasingly popular in many different scientific and engineering fields. AFM can provide a level of detail of surface texture for melanin visualization and quantify the roughness and dimensions of melanin surfaces,⁹⁷ and the resolution can be less than one nanometer.³ Moreover, AFM can accommodate larger samples and eliminates the need for any destructive sample preparation. The natural melanin particles of both cuttlefish juice and bull's eye melanosomes are aggregates of 10–30 nm substructure.⁹² Both 2D and 3D AFM micrographs of eumelanin can be captured, and melanin nanoparticles in cuttlefish juice have smaller substructural units with sizes of 10–30 nm.⁹⁸ Although the shape of melanin extracted from different sources may vary, many studies have found that melanin is composed of smaller subunits.⁹⁹

Two structures were observed in the imaging of dried NM samples isolated from the human brain using AFM: small spherical particles with a diameter of 30 nm, and larger nanoparticles with an average diameter of 350 nm. These small particles were also visible on the surface of larger particles, suggesting that they were aggregates of these smaller structures.²⁷

4. Extraction method of melanin

Melanin is extracted using a variety of methods, and the traditional methods include ultracentrifugation and water washing. However, these two methods are often unable to further purify and separate melanin, and with the advancement of extraction technology, both methods have been further incorporated. Therefore, this review focuses on the most used methods for melanin extraction: alkali solubilization and acid precipitation, enzyme extraction, ultrasonic and microwave-assisted methods. Here, we organized the extraction methods of melanin on Table 3.

Table 3 Extraction method of melanin

Method	Pros (P) & Cons (C)	Application
Alkali-soluble acid precipitation extraction (AAE)	P: Convenience of operation;^38	Eumelanin;^98 pheomelanin;^106 pyomelanin;^14 allomelanin.^62
	C: Destroyed the structure of melanin,^98,100,102 and not conducive to green production in terms of expansion.	(Suitable for crude extraction)
Enzymic extraction (EE)	P: Isolated more uniform melanin particles, preserve the morphology of melanin;^98,102 digest protein tightly bound to melanin; higher purity.^53	Eumelanin;^53,99 pheomelanin;^99,104,105 melanin from iris.^103
	C: Long time and need heat.	(Suitable for promotion)
Ultrasonic-assisted extraction (UAE)	P: Reduce solvent use, shorter extraction times, and increased extraction efficiency.^107	Eumelanin;^104 DOPA-melanin;^6 Melanin in fungi^108 and edible mushroom.^109,110
	C: Alter melanin particles (deformation, bursting, etc.).^97,98	(Suitable for promotion)
Microwave-assisted extraction (MAE)	P: Accelerate the extraction process of adsorption and desorption of target compounds from the matrix.^111 Reduce extraction time, save energy, and improve yields^107	Melanin in fungi.^112
	C: Non-uniform heating.^111	(Suitable for promotion)

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4.1 Alkali-soluble acid precipitation extraction (AAE)

Due to the convenience of operation, alkali-soluble acid precipitation extraction is the most widely used.³⁸ However, melanin dissolved in alkaline solution and then precipitated was found to have the possibility of destroying the structure of melanin.^98,100 Moreover, due to the addition of alkaline and acidic solutions in the extraction process, it is not conducive to green production in terms of expansion. Kuttan et al.¹⁴ used acid–base extraction to extract pyomelanin from Shewanella sp. but the pigment was found to be irregular in shape size by SEM, and it is likely this was probably destroyed by repeated acid–base extraction. Similarly, Singla et al.⁶² observed that allomelanin extracted from pathogenic black knot fungus was disrupted and showed irregular morphology, and it was uncertain whether this structure was present in the pathogenic bacteria. The extraction method of neuromelanin is complicated because it requires the isolation of melanin granules from brain cells.^30,101

4.2 Enzymatic extraction (EE)

The enzymatic extraction method is an upgrade of the alkaline extraction and acid precipitation method. In a previous study using three different methods: two acid/base extractions and one enzyme extraction, it was found that the morphological and spectral properties of the isolated pigments differed significantly.¹⁰⁰ Both acid/base methods produced amorphous material, while the enzyme extraction produced ellipsoidal melanosomes. Acid or base treatment altered and destroyed the ultrastructure of the melanosomes, while enzymatic digestion isolated more uniform particles than acid and base extraction.^98,102 Amino acid analysis showed that even after alkaline solubilization and acid precipitation and enzymatic extraction methods, a significant amount of protein was still present in the melanin, accounting for 50 and 14% of the total mass, respectively. MALDI-MS structural analysis of eumelanin in the iris was only possible after enzymatic digestion, as eumelanin without digestion could only be observed in clusters of peaks around 7, 25, and 49 kDa.¹⁰³ Enzyme extraction of melanin technology has a well-developed history of research. Studies have tried enzymatic extraction methods for melanin in hair, where some studies started to use proteinase K, papain or trypsin to extract melanin by hydrolyzing keratin.^104,105 Eumelanin from squid ink was extracted by alkaline protease by Song et al.,⁵³ the extraction rate was found to be 88.3% for enzymatic extraction and 16.82% for the alkaline acid precipitation extraction.

The enzymatic extraction of melanin is gentle and does not require extreme pH adjustment, which is increasingly being widely used for melanin extraction and is a method worth advocating. Thus, melanin is a combination of macromolecular cross-linking and protein. Enzymatic digestion increases the purity, solubility, and stability of melanin in water by removing some of the proteins. The enzymatic extraction method helps to improve the stability and purity of melanin dispersion in solution by enzymatic digestion of the proteins tightly bound to melanin.

4.3 Ultrasonic-assisted extraction (UAE)

Ultrasound-assisted extraction methods have the advantage of reduced solvent use, shorter extraction times, and increased extraction efficiency.¹⁰⁷ However, ultrasound can alter melanin particles to some extent. This includes deformation, bursting, etc. It was found by SEM that after ultrasound-assisted extraction of eumelanin extracted from squid ink, some of the melanin particles were deformed and the physical structure of the round particles was disrupted to some extent.⁹⁷ Ultrasound disrupts the eumelanin, and the huge polymer is degraded into smaller soluble particles, exposing some of the hydrophilic groups, while showing the effect of increased solubility. Based on ultrasound-assisted extraction, some studies will combine some two methods, such as ultrasound-assisted extraction after alkali solubilization and acid deposition¹⁰⁸ and ultrasound-assisted extraction after enzymatic digestion.⁶ Hou et al.⁶ also used a response surface optimized ultrasound-assisted extraction process in dihydroxyphenylalanine (DOPA)-melanin from Inonotus hispidus mushroom. Ultrasound-assisted extraction is commonly used for melanin extraction from Auricularia.¹⁰⁹

Thus ultrasound-assisted extraction can be tried from plants and microorganisms. Ultrasound-assisted extraction of natural melanin from the dried seeds of A. auricula was carried out by Zou et al.¹¹⁰ Ultrasound-assisted extraction methods have gained popularity in melanin research due to their advantages of reduced solvent use, shorter extraction times, and increased efficiency. However, it is important to note that ultrasound can alter the physical structure of melanin particles, which may affect their characterization. Despite this limitation, future research could combine ultrasound-assisted extraction with other methods, such as enzymatic digestion or alkali solubilization, to improve the accuracy of analysis. Ultrasound-assisted extraction is commonly used for melanin extraction from various sources, including plants and microorganisms. With further optimization and refinement, ultrasound-assisted extraction holds promise as a valuable tool in melanin research.

4.4 Microwave-assisted extraction (MAE)

Both microwave-assisted extraction and ultrasound-assisted extraction can accelerate the extraction process of adsorption and desorption of target compounds from the matrix. Microwave-assisted extraction can effectively reduce extraction time, save energy, and improve yields, and there is an increased interest in applying acoustochemistry to natural product extraction.¹⁰⁷ However, the disadvantage of microwave-assisted extraction is the non-uniform heating.¹¹¹ Lu et al.¹¹² obtained the intracellular melanin YM296 (LIM) from Lachnum singerianum by microwave-assisted extraction.

Ultrasound-microwave-assisted extraction (UMAE) is a novel technology that combines the advantages of both ultrasound and microwave methods, offering improved extraction efficiency and selectivity. While microwave-assisted extraction can reduce extraction time, save energy, and improve yields, its non-uniform heating can be a drawback. To overcome this limitation, there is growing interest in applying cosmochemistry to natural product extraction. Ultrasound-microwave-assisted extraction (UMAE) is a novel technology that combines the advantages of both ultrasound and microwave methods, offering improved extraction efficiency and selectivity. With further optimization and development, these techniques have the potential to revolutionize the extraction of melanin and other natural products and become valuable tools for researchers in various fields.

5. Biological activities of melanin

5.1 Antimicrobial activity

Eumelanin derived from different sources, such as squid ink⁷ and animal hair (e.g., horsehair),¹¹³ has exhibited antibacterial properties against various biological pathogens, including Staphylococcus aureus and Escherichia coli. Barretto et al.¹¹⁴ found that yeast isolated from pyomelanin is also an antibacterial and antifungal agent against external microbial attack, reducing biological contamination.⁶⁸

Through the progress of science and technology, people are no longer satisfied with the antimicrobial effects of melanin itself. The study of combining melanin particles as carriers with other substances has been started. For example, Liu et al.¹¹⁵ combined eumelanin nanoparticles with silver nanoparticles for effective bactericidal activity through the fast photothermal effect of eumelanin. Liang et al.¹¹⁶ found that eumelanin nanoparticles could bind natural pectin to form a thin film and achieve a bactericidal rate of more than 90% in only 5 min against Listeria monocytogenes. The application in making antibacterial films nowadays is mainly in the food industry, but it can be extended and developed in medical care as new antibacterial dressings.

5.2 Antioxidant activity

Many studies show melanin has superior antioxidant activities.^113,117 Eumelanin extracted from horse hair has excellent free radical scavenging properties.¹¹³ However, pheomelanin synthesis requires the consumption of glutathione and NADH (the main intracellular antioxidant) and therefore pheomelanin may have pro-oxidant properties.¹¹⁸ Allomelanin has a wide range of antioxidant activities because its monomer 1,8-DHN is a potent antioxidant with powerful hydrogen atom transfer properties.¹¹⁹ Allomelanin in fungi scavenges reactive oxygen and nitrogen species, such as hydrogen peroxide, hydroxyl and thereby promotes fungal virulence through its high antioxidant capacity.¹⁵ Ben Tahar et al.¹¹⁷ extracted pyomelanin produced from Yarrowia lipolytica W29 with a scavenging effect on DPPH. Hou et al.⁶ tested the stability and antioxidant activity of melanin extracted from Inonotus hispidus with good scavenging function of free radicals. The antioxidant capacity of melanin is caused by their paramagnetic center (PC), the quinone and hydroquinone radicals in dynamic equilibrium with the semi-quinone radicals in dynamic equilibrium.⁸¹

5.3 Hypoglycemic activity

Melanin has been found to have potential hypoglycemic activity, an activity that has been gradually discovered in recent years.¹²⁰ Eumelanin in squid ink can effectively inhibit the activity of α-glucosidase and α-amylase, and it can accelerate cellular intake of glucose and promote hepatic glycogen synthesis.⁵³ Lu et al.¹⁰⁸ found that melanin in begonia has effects that can inhibit α-glucosidase and PTP1B for the first time. Alam et al.⁸⁹ found that high levels of allomelanin in date palm fruits (Phoenix dactylifera L.) had good inhibitory effects on glycosidase and amylase activities.

5.4 Anti-radiation activity

Melanin polymers have a highly conjugated structure, therefore light absorption is their significant characteristic.¹²¹ In studies on the anti-radiation activity of melanin, fungi and plants were shown to be the most common sources of melanin, with allomelanin being the most common type. The exogenous melanin extracted from Lachnum YM404 has the ability to significantly increase the survival of Escherichia coli and Staphylococcus aureus under UV radiation, and after anti-radiation experiments in mice, it was shown that no edema, keratinization and brown pigmentation were observed in the skin of the experimental group of mice at low radiation doses.¹⁰⁶ Revskaya et al.¹²² found that mice consuming black mushrooms could avoid gastrointestinal syndrome caused by radiation and could be used as an oral antiradiation agent. Later, Kunwar et al.¹²³ further investigated the mechanism by which consumption of black mushrooms could be anti-radiation with the appropriate concentration, and found that mice given 50 mg kg⁻¹ of melanin content could avoid 100% of radiation.

5.5 Photothermal conversion effect

Melanin has a superior photothermal conversion effect, and there are materials inspired by the melanin photothermal effect to prepare sustainable photothermal materials.^116,124 As a natural nanoscale particle, melanin can be applied in the field of cancer diagnosis and therapy based on nanoplatform, such as cancer photothermal therapy.⁴ Zhang et al.¹²⁴ synthesized hematoporphyrin-eumelanin nanocouples, the hematoporphyrin fraction could be ultrasonically excited to produce cytotoxic singlet oxygen, while the melanin fraction was used for the photothermal effect and thus acted together on malignant tumors. Liang et al.¹¹⁶ obtained antimicrobial films with photothermal bactericidal effect by synthesizing eumelanin with pectin.

5.6 Anti-cancer activity

Melanin has also demonstrated potential for anticancer activity. By virtue of the photochemical properties of melanin, melanin-based nanoparticles can also exert immunomodulatory effects and promote anti-cancer responses in the backdrop of photothermal therapy.⁴ El-Naggar et al.¹²⁵ found eumelanin produced in Streptomyces glaucescens strain exhibited potent cytotoxic activity against HFB4 skin cancer cell line. Al-Obeed et al.¹²⁶ found melanin extracted from Nigella sativa can inhibit the proliferation of colorectal adenocarcinoma HT29 and mCRC SW620 cell lines. Fan et al.⁸ created melanin nanoparticles (MNP) with unique photoacoustic capabilities and the capacity to spontaneously attach to metal ions for usage as photoacoustic contrast agents as well as nanoplatforms for positron emission tomography (PET) and magnetic resonance imaging (MRI). Zhang et al.⁹ found that melanin, a biopolymer with strong biocompatibility and biodegradability, drug and ion binding ability, and inherent photoacoustic characteristics, may be used as an effective endogenous nanosystem for imaging-guided tumor treatment in living mice. The application of the functional biomarker melanin can ease the development of multimodal imaging probes, and MNPs have significant promise as nanoplatforms for molecular therapeutic diagnostics and clinical translation. Therefore, melanin has great potential for applications in photothermal therapy, anticancer drug delivery, preclinical and clinical imaging-guided chemotherapy.

5.7 Heavy metal ion absorbent

Metal chelation is one of the most important biological functions of melanin.^10,120,127 Gao et al.¹⁰ found that polyethylene glycol synthesized with almond melanin (not a single species) nanoparticles can effectively bind Fe³⁺, Cu²⁺, and Zn²⁺, which may be used for the treatment of patients with metal poisoning or for loading ions for imaging. Manirethan et al.¹²⁷ used pyomelanin-coated PVDF membranes to remove heavy metal ions such as Hg²⁺, Cr²⁺, and others from water. Melanin combines with the strong interfacial hydrophobic interaction of PVDF membranes, such as hydrogen bonding between melanin and donor–acceptor reactions. The study also synthesized melanin as a new iron supplement, and the carboxyl and hydroxyl groups of melanin in Nigella sativa were involved in the chelation of iron ions, which could be developed as a comprehensive multifunctional iron supplement in the future.¹²⁸

5.8 Other activities and applications

Melanin is a natural active product with potential for anti-inflammatory activity.¹²⁰ Barretto et al.¹¹⁴ described the anti-inflammatory activity of melanin produced from microbial populations extracted from insects. Wang et al.¹²⁹ prepared ultrafast melanin-like nanoparticles with adjustable size and monodispersity, and melanin had the ability to phenotypically convert inflammatory macrophages.

The anti-hemolytic activity of melanin is an activity that has not been widely explored and developed. El-Naggar et al.¹²⁵ found that eumelanin produced in the Streptomyces glaucescens strain has superior potent anti-hemolytic activity due to the ability of phenolic compounds in melanin and free radicals, thus protecting the red blood cell membrane from damage and lysis.

Melanin acts by inhibiting the pathway (intracellular) activated by the virus. Montefiori et al.¹³⁰ synthesized soluble melanin and found that synthetic melanin effectively impeded the replication of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) in human lymphoblastoid cell lines and T-cell lines.

Gao et al. explored the role of melanin complexes in medicine (site-specific diagnosis and treatment of diseases) and in extending the shelf life of food products.¹⁰ As human activity in space continues to increase, it is becoming increasingly important to understand how biological assets respond to spaceflight conditions. A study has shown that melanin-producing fungi are able to survive in the vacuum of space and low Earth orbit under Mars-like conditions, thereby extending the life of biological assets in space.¹³¹

6. Conclusions and future perspectives

Melanin, a natural pigment, shows great potential for biological activity, which could be found in animals, plants, and microorganisms in nature. Melanin has diverse activities and functions, such as metal chelation, thermoregulation, drying, antioxidants, UV absorption and protection, free radical scavenging, electron transport, etc. Poor solubility is one of the major factors that limit the application of melanin. Therefore, the improvement of melanin solubility and the synthesis of new melanin complexes are now a popular research direction. In addition, melanin demonstrates potential applications in the fields of drug delivery, optoelectronics, and materials science. Thus, the full picture of melanin research involves a broader range of fields and aspects. And there is also a need to further understand the relationship between the structure of melanin and the specific functions it exhibits, such as light absorption, electrical conductivity, catalytic activity, etc. By resolving the structure of melanin molecules, their activity and application potential can be better understood.

Author contributions

W. S.: conceptualization, methodology, investigation, writing – original draft, and writing – review and editing. H. Y.: writing – review and editing and funding. S. L.: supervision. H. Y.: supervision. D. L.: supervision. P. L.: supervision. R. X.: conceptualization, writing-review and editing, supervision and funding.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by the 14th Five-Year Plan of Qingdao Pilot National Laboratory of Marine Science and Technology [2022QNLM030003-2], Shandong Key R&D Plan, Major Scientific and Technological Innovation Project [2022CXGC020413], and Fujian Science and Technology Planning Project-STS Program [2021T3013]. The authors would like to acknowledge the financial support provided by the the China Scholarship Council [202204910355].

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