Dual Antioxidant Activity: Preventive and Scavenger Effects of Wild and Cultivated P. nebrodensis Extracts Against ROS and SOX in Human Keratinocytes

<i>Pleurotus nebrodensis</i>, a rare endemic Sicilian mushroom with notable gastronomic and medicinal value, attracts interest for its potential antioxidant properties, though data on its biological effects in skin models are lacking. This study evaluated the antioxidant activities of several aqueous extracts from wild (1a, 2a, 3a, 1b, 2b, and 3b) and cultivated (CAN1 °F, 3A, 2B(II), and CAN2 °F) <i>P. nebrodensis</i> basidiomes in human keratinocytes (HaCaT cells). Extracts were characterized through DPPH radical scavenging assay, MTT viability assay, and intracellular ROS and mitochondrial SOX quantification by DCFH-DA and MitoSOX Red fluorescence analyses. The methodology specifically included two approaches in keratinocytes: co-treatment of extracts and H₂O₂ to investigate direct scavenger activity, and pre-treatment to assess the preventive activity on oxidative stress modulation. This analysis demonstrated that selected extracts (1b and CAN2 °F) exert a dual action, combining anti-intracellular ROS and anti-mitochondrial SOX preventive effect with a direct free radical scavenging activity in human keratinocytes. In particular, CAN2 °F exerts its activity predominantly through prevention (modulation of cellular defenses), while 1b primarily functions as a direct intracellular ROS and mitochondrial SOX scavenger. Notably, glucan quantification revealed a correlation between β-glucan content and the overall antioxidant activity. These findings provide the first evidence of <i>P. nebrodensis</i>'s anti-ROS and anti-SOX efficacy in human keratinocytes, highlighting its potential as a source of natural bioactives for cosmeceutical and dermatological applications.