**1. Introduction**

The interest in traditional bioactive herbal compounds with less detrimental effects on human body than their synthetic counterparts is growing. Many clinical trials and cohort studies showed that consumption of foods (vegetables, fruits, nuts) rich in biologically active molecules with demonstrated antioxidant capacity, through different mechanisms of actions, has the potential to protect against cardiometabolic diseases, neurodegenerative disorders, cancer, or age-related sarcopenia and

frailty [1–5]. Increased intake of antioxidants from tree nuts and peanuts influences risk factors associated with aging and can extend health span and lifespan [6].

Hazelnut (*Corylus avellana* L.) is known for the nutritional and therapeutic properties of its fruits, rich sources of healthy fatty acids, vitamins, minerals, and polyphenols [7,8]. Polyphenols, common secondary plant metabolites characterized by several hydroxyl groups linked to a phenol ring, can act synergistically with other phytochemicals to lower the oxidation and inflammation processes which may trigger many pathological conditions or age-related chronic diseases [9–11]. These biologically active molecules donate electrons or hydrogen atoms to reactive free radicals preventing lipid oxidation or cellular damage and acting as natural antioxidants with many health benefits [12]. In addition, plant polyphenols act as signaling molecules and can participate in many enzymatic pathways [13].

Recently, special interest was given to tree nut by-products, waste plant matrices, with great potential as sources of biologically active compounds. As little is known about the biochemical profile of hazelnut involucre (HI) [14], we intended to extend the knowledge about this promising by-product. In a previous study, we successfully applied a d-optimal experimental design to optimize the bioactive compound extraction from walnut septum, another tree nut by-product [15]. In that experiment, acetone was more efficient than ethanol to extract both polyphenols and phytosterols from the plant matrix. The objectives of this study were the optimization of the extraction process in order to identify the optimal extraction conditions for attaining rich bioactive compound extracts from HI and the emphasizing of key biological activities for the optimally selected extract, which would justify further research on this plant matrix. The first aim of our study was the analysis of phenolic and phytosterol compounds from HI based on experimental designs. Turbo-extraction (TE) by Ultra-Turrax, an efficient and economical extraction method, with higher outcomes compared to other methods, was chosen [15]. Three extraction variables, stirring time, pH, and percentage of water in acetone, were selected to define the optimal extraction conditions. LC-MS analysis was chosen for the identification and quantification of several individual polyphenols and phytosterols from involucre. Based on the obtained results, optimal conditions were selected to achieve the HI extract with the highest content of polyphenolic compounds and the highest antioxidant activity (AA) determined by Trolox equivalent antioxidant capacity (TEAC) assay. The biological activities of this optimal extract were further examined by other in vitro antioxidant assays (DPPH and FRAP), for enzymatic inhibitory capacity (tyrosinase and α-glucosidase), and a potential cytotoxic effect on lung and breast cancer cell lines.
