The melanin was isolated from mantle of P. penguin and oxidized as follows (15 (link)). 1 g mantle sample was finely homogenized on ice, mixed with 15 ml PBS (pH 7.4) with 2% (m/V) papain (J&K, China), and incubated at 55°C for 20 h. The precipitate was obtained by centrifuging at 12,000g for 10 min, and then was successively washed with 2 ml mineral ether, ethanol and water. After that, the obtained black precipitate was raw melanin production.
8.6 ml of 1 mol/L K2CO3 and 0.8 ml of 30% H2O2 were used to dissolve and oxidize the raw melanin. The mixture was heated at 100°C for 20 min and cooled down to room temperature. The residual H2O2 was decomposed by 0.4 ml of 10% Na2SO3, and 6 mol/L HCl was then added to adjust pH to 1.0. The mixture was centrifuged at 8,000g for 10 min to get the supernatant, which then was extracted using 70 ml of ether and dried to crystalline residue. Finally, crystalline residues were redissolved in mobile phase or water, and were filtered through 0.45 μm nylon membrane (Millipore, USA) before using.
8.6 ml of 1 mol/L K2CO3 and 0.8 ml of 30% H2O2 were used to dissolve and oxidize the raw melanin. The mixture was heated at 100°C for 20 min and cooled down to room temperature. The residual H2O2 was decomposed by 0.4 ml of 10% Na2SO3, and 6 mol/L HCl was then added to adjust pH to 1.0. The mixture was centrifuged at 8,000g for 10 min to get the supernatant, which then was extracted using 70 ml of ether and dried to crystalline residue. Finally, crystalline residues were redissolved in mobile phase or water, and were filtered through 0.45 μm nylon membrane (Millipore, USA) before using.
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