Eumelanin: A natural antioxidant isolated from squid ink by new enzymatic technique and prediction of its structural tetramer

Abstract

The investigation into natural eumelanin found in squid ink is an emerging research topic due to its antioxidative properties. However, the methodologies for acquiring pure eumelanin and establishing the intricate relationship between its structure and antioxidative efficacy remain limited. Novel enzyme extraction technique, termed response surface optimization, was employed to synthesize high-purity eumelanin, and its antioxidant activity was evaluated at free radical scavenging and cellular levels and correlated with detailed tetrameric structure prediction for the first time. Results demonstrated that enzymatic extraction technique yielded eumelanin (ANF) nanostructure with stability, boasting particle size of 112.35 nm, a maximum absorption wavelength of 224 nm, a ΔHpp value of 0.4802 mT, and a purity of 92%. Compared with melanin standard (MelS), ANF had superior free radical scavenging activity, eliminating 89.62% of DPPH radicals, 96.79% of superoxide ion radicals, and 93.20% of hydroxyl radicals, and better repair of cellular oxidative damage. Based on molecular energy, molecular electrostatic potential (MEP), and molecular orbitals by Gaussian calculations, the tetrameric structure (DHICA3-DHI1‴ and “DHICA3-DHI1”) with significant antioxidant activity contained inside eumelanin was anticipated. Our findings reveal its potential as a natural antioxidant, offering broad prospects for antioxidant food additives or therapeutic applications in nutritional and pharmaceutical ingredients.