**4. Conclusions**

This study characterized the phytochemical profile of hazelnut (*Corylus avellana* L.) involucre and described the optimal experimental conditions for maximizing the extraction efficiency of biologically active compounds from this tree nut by-product. We aimed to obtain hazelnut involucre extracts with high contents of phenolics, strong antioxidant capacity, and biological potential based on experimental designs. Besides, the phytochemical profile of the extracts using LC-MS methods was assayed. In order to determine the ideal extraction conditions, several parameters (stirring time, pH, solvent) were coupled with chemical analysis and statistical tools. From the eleven polyphenols (epicatechin, catechin, syringic acid, gallic acid, protocatechuic acid, vanillic acid, *p*-coumaric acid, ferulic acid, hyperoside, isoquercitrin, quercitrin) and three phytosterols (stigmasterol, campesterol, beta-sitosterol) determined and quantified, catechin (up to 0.243 mg/g dw HI), protocatechuic acid (up to 0.227 mg/g dw HI), and beta-sitosterol (up to 5.305 mg/g dw HI) were found in the highest amount. From the extraction conditions, the amount of water in the solvent proved to have a statistically significant influence upon all evaluated responses.

The best extraction conditions to attain the richest extract in total phenols were accomplished using turbo-extraction by Ultra-Turrax at stirring time 3 min, pH 3, and 50% acetone in water (*v*/*v*) as extraction solvent, while the best extraction conditions for sterols were 75% acetone in water (*v*/*v*), pH 5, and stirring time 2 or 3 min. The richest extract in polyphenols presented strong in vitro antioxidant activity in classical tests (TEAC, DPPH, FRAP) as well as in two cancer cell line assays (T47D-KBluc and A549). Cytotoxic and antioxidant effects of HI extract were more intense on these cancer cells than on a normal cell line (HGF). These results are promising and justify further research to characterize HI as a valuable source of bioactive compounds and to assay its anticancer potential. Moreover, the richest polyphenol HI extract demonstrated good enzyme inhibitory potential on tyrosinase (165.17 ± 1.88 mg KAE/g) and α-glucosidase (eight times stronger inhibition than acarbose), two key enzymes involved in age-related diseases. Phytosterols, compounds with proven cardioprotective effects and antioxidant capacity, are other valuable molecules found in HI.

The results of our study successfully accomplished the proposed objectives and justify future scientific investigations, including a comprehensive identification of individual polyphenols. Moreover, the content in bioactive compounds, correlated with good results for antioxidant and enzyme inhibitory activities, warrants more research in order to understand the bioavailability of specific molecules and to reveal their potential mechanisms of action for future use of hazelnut involucre in the cosmetic industry, in addition to food and pharmaceutical production.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/8/10/460/s1, Figure S1: The chromatograms for the individual polyphenolic compounds quantified by the LC-MS method II according to the retention time and m/z values in Supplementary Table S2, Figure S2: The chromatograms for the individual polyphenolic compounds quantified by an LC-MS method according to the retention time and m/z values in Supplementary Table S1, Figure S3: The chromatograms for the individual polyphenolic compounds quantified by an LC-MS method according to the retention time and m/z values in Supplementary Table S1, Figure S4: The chromatograms for phytosterols quantified by an LC-MS/MS method according to the retention time and m/z values in Supplementary Table S3; Table S1: Retention times and values of m/z for the individual polyphenolic compounds, Table S2: Retention times and values of m/z for the individual polyphenolic compounds quantified by LC-MS method II, Table S3: Retention times (RT) and specific ions of the phytosterols, Table S4: Regression equation coefficients for total bioactive compounds in hazelnut involucre extracts, Table S5: Quantitative evaluation of the recovery of main bioactive compounds in non-hydrolyzed and hydrolyzed samples of hazelnut involucre extracts, Table S6: Statistical parameters after data analyze and fit with factorial model for bioactive compounds in hazelnut involucre extracts, Table S7: Regression equation coefficients for individual bioactive compounds in hazelnut involucre extracts.

**Author Contributions:** Conceptualization, M.E.R., A.M., L.V., D.-S.P., and I.T.; methodology, M.E.R., A.M., D.-S.P., and I.T.; software, M.E.R., A.-M.G., I.F., A.P., and I.T.; formal analysis, M.E.R., A.-M.G., I.F., A.P., A.M., L.V., and D.-S.P.; investigation, M.E.R., I.F., A.P., A.M., L.V., and I.T.; supervision, L.V., G.C., F.L., D.-S.P., and I.T.; writing—original draft preparation, M.E.R., A.-M.G., I.F., A.P., and D.-S.P.; writing—review and editing, M.E.R., A.-M.G., I.F., A.P., A.M., G.C., L.V., F.L., D.-S.P., and I.T.

**Funding:** This research was funded in part by "Iuliu Hatieganu" University of Medicine and Pharmacy in Cluj-Napoca, Romania through a Ph.D. grant (PCD No. 1529/60/18.01.2019).

**Conflicts of Interest:** The authors declare no conflict of interest.
