Preparation of the Extracts

The involucre was ground in a coffee grinder (Argis, RC-21, Electroarges SA, Curtea de Arges, Romania) for 5 min and the powder was screened through a 200 μm Retsch sieve. HI was weighed (2 g) and mixed with the extraction solvent (20 mL) in Falcon tubes. TE was accomplished using an Ultra-Turrax homogenizer (T 18; IKA Labortechnik, Staufen, Germany) for 1 to 3 min (12,000 rpm) and a Vortex RX-3 (Velp Scientifica, Usmate, Italy) for 2 min. The homogenates were centrifuged (Hettich, Micro 22R, Andreas Hettich GmbH & Co., Tuttlingen, Germany) 15 min at 5000 rpm. The supernatant was carefully separated and, using a rotary evaporator (Hei-VAP, Heidolph Instruments GmbH & Co., Schwabach, Germany), the solvent was removed under vacuum at 45 ◦C. The dry residue was taken up in water, placed in amber glass vials, and lyophilized (Advantage 2.0, SP Scientific, Warminster, PA, USA). After lyophilization, the samples (weight between 6 and 520 mg) were stored at room temperature. For further analyses, the lyophilized extracts were dissolved in 70% EtOH (10 mg/mL), if not specified otherwise. All assays were performed in triplicate.

Two d-optimal experimental designs, implemented by Modde software, version 11.0 (Sartorius Stedim Data Analytics AB, Umeå, Sweden), were developed for the extraction process optimization. Three factors, stirring time, pH, and percentage of water in acetone, were the independent variables for both d-optimal experimental designs. The total phenolic content (TPC), total flavonoid content (TFC), condensed tannin content (CTC), and the AA-TEAC were the dependent variables in the first experimental design used in the screening step (Table 1). The extracts were prepared according to this first experimental design. The individual concentrations of the bioactive compounds quantified by LC/MS methods were the dependent variables in the second experimental design used in the optimization step (Table 2).


**Table 1.** Independent and dependent variables of the experimental design used in the screening step.

*Dependent variables (responses)*

Total phenolic content (TPC, mg GAE/g dw 1) (Y1)

Total flavonoid content (TFC, mg QE/g dw 2) (Y2)

Condensed tannin content (CTC, mg CE/g dw 3) (Y3)

Antioxidant activity (AA, mg TE/g dw 4) (Y4)

1—mg GAE/g dw = gallic acid equivalents per dry weight of hazelnut involucre, 2—mg QE/g dw = quercetin equivalents per dry weight of hazelnut involucre, 3—mg CE/g dw = catechin equivalents per dry weight of hazelnut involucre, 4—mg TE/g dw = Trolox equivalents per dry weight of hazelnut involucre.


**Table 2.** Independent and dependent variable of experimental design used in the optimization step.

dw—dry weight hazelnut involucre.
