Study on the Preparation and Chemical Structure Characterization of Melanin from Boletus griseus

Abstract

In this study, melanin (BgM) was obtained from Boletus griseus. The chemical composition and structure of BgM were characterized by UV-visible absorption spectrum, Fourier transform infrared spectrum, elemental analysis, nuclear magnetic resonance, pyrolysis gas chromatography mass spectrometry, and ultra-performance liquid chromatography–high resolution mass spectrometry. The percentage contents of C, H, N, S and O elements were 56.38%, 5.86%, 6.17%, 2.44%, and 28.04%, and the S/N and C/N ratios were 0.17 and 10.66, respectively. The UV-vis spectrum of BgM showed a maximum absorption peak at 214 nm. Characteristic absorption peaks were observed at 3426, 1600 and 1105 cm−1, and BgM contained phenolic hydroxyl, amidogen, carbonyl, methylene, and methyl groups. Moreover, BgM is an eumelanin, and its main skeleton has both a benzene ring and an indole, and the branched chain mainly consists of alkanes, alcohols, and fatty acids. BgM was hydrolyzed by H2O2 and four compounds were tentatively analyzed from the UPLC-MS/MS profile. The chemical structure of BgM was characterized as 5,6-dihydroxyindole eumelanin, and the condensed molecular formula is [C28(OR1)4(OR2)3H11O6N4]n.

From Extraction to Advanced Analytical Methods: The Challenges of Melanin Analysis

“…In the study of Boletus griseus ’s melanin pigment [22], the most abundant products were benzene and indole derivatives, the characteristic thermal degradation products of eumelanin [136]. Also, few quinoline and isoquinoline molecules were detected, confirming a mixed eumelanin-pheomelanin structure of the analyzed pigment.…”

“…These slopes of linear plots are often used as an important criteria for the identification and characterization of melanins [8,9,23]. In addition, the lack of any absorption peaks at 260 nm and 280 nm suggests the absence of any nucleic acid, lipid or protein impurity [11,22,81].…”

“…In order to further confirm the molecular structure of extracted melanin, proton and carbon nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR) analysis can be carried out. First, the sample is dissolved in a deuterated solvent such as deuterium oxide/sodium deuteroxide (D 2 O/NaOD) [11,22] or dimethylsulfoxide-D6 (DMSO-D6) [15,86]. The sample is then subjected to NMR analysis using various resonant frequencies and usually referenced to tetramethylsilane [14,15,86].…”

“…In the aromatic regions, the chemical shifts between 6–8 ppm were attributed to the aromatic hydrogens of indole and/or pyrrole rings of the melanin polymer chain [22,47,84,85,86]. Between 3.2–4.2 ppm, the observed peaks were caused by the methyl or methylene groups attached to nitrogen and/or oxygen atoms [22,84,85,86]. The presence of a NH-group linked to indole was attributed to the signals within the range of 1.3–2.5 ppm [84,85].…”

“…In the 13 C-NMR spectra, the peaks between 160–185 ppm were typically ascribed to the carbon atoms in carboxyl and amide groups or the carbonyl groups of peptidic bonds [15,22]. Next, the peaks identified between 120–140 ppm could be generated by aromatic carbons, probably involved in indole or pyrrole systems.…”