Enhanced Recovery of Antioxidant Compounds from Hazelnut (Corylus avellana L.) Involucre Based on Extraction Optimization: Phytochemical Profile and Biological Activities

Abstract

Tree nut by-products could contain a wide range of phytochemicals, natural antioxidants, which might be used as a natural source for dietary supplements. The aim of the present study was to evaluate the phenolic and sterolic composition, as well as the antioxidant and other biological activities, of hazelnut involucre (HI) extracts. Experimental designs were developed in order to select the optimum extraction conditions (solvent, temperature, time) using turbo-extraction by Ultra-Turrax for obtaining extracts rich in bioactive compounds. Qualitative and quantitative analyses were performed by LC-MS and LC-MS/MS and they revealed important amounts of individual polyphenols and phytosterols, molecules with antioxidant potential. The richest polyphenolic HI extract with the highest antioxidant activity by TEAC assay was further evaluated by other in vitro antioxidant tests (DPPH, FRAP) and enzyme inhibitory assays. Additionally, the cytotoxic and antioxidant effects of this extract on two cancerous cell lines and on normal cells were tested. This is the first study to analyze the composition of both hydrophilic and lipophilic bioactive compounds in HI extracts. Our findings reveal that this plant by-product presents strong biological activities, justifying further research, and it could be considered an inexpensive source of natural antioxidants for food, pharmaceutical, or cosmetic industry.

Keywords: LC-MS; Ultra-Turrax; antioxidants; biological activity; enzymatic inhibition; experimental design; hazelnut involucre; phytosterols; polyphenols; turbo-extraction.

Figure 1

Influence of working conditions on total phenolic content (TPC), total flavonoid content (TFC), condensed tannin content (CTC), and antioxidant activity (AA) by TEAC assay of hazelnut involucre extracts, presented as scaled and centered coefficient plots. X₁, stirring time (min); X₂, pH; X₃, water in solvent (% v/v).

Figure 2

Response surface for predicting the recovery yield for total phenolic content (TPC), total flavonoid content (TFC), condensed tannin content (CTC,) and antioxidant activity (AA) by TEAC assay for hazelnut involucre extracts with regard to: X₁, stirring time (min); X₃, water in solvent (% v/v); X₂, pH = 5. The regions in red represent the domains of working conditions assuring the maximum extraction yield for the evaluated bioactive compounds.

Figure 3

Influence of working conditions on the bioactive compound recovery from hazelnut involucre extracts, depicted as scaled and centered coefficient plots. X₁, stirring time (min); X₂, pH; X₃, water in solvent (% v/v).

Figure 3

Influence of working conditions on the bioactive compound recovery from hazelnut involucre extracts, depicted as scaled and centered coefficient plots. X₁, stirring time (min); X₂, pH; X₃, water in solvent (% v/v).

Figure 4

Response surface for prediction of bioactive compound recovery from hazelnut involucre extracts with respect to: X₁, stirring time (min); X₂, pH; X₃, water in solvent (% v/v). The regions in red represent the domains of working conditions assuring the maximum extraction yield for the evaluated bioactive compounds.

Figure 5

Cytotoxic effect of the HI extract observed using Alamar Blue assay on T47D-KBluc (A), A549 (C), and HGF (E) and using Neutral Red assay on T47D-KBluc (B), A549 (D), and HGF (F). The results are expressed as relative means ± standard deviations (six technical replicates for each of the three biological replicates) where the negative control (DMSO 0.2%) is 100%. (*) indicates significant differences compared to negative control (ANOVA + Dunnett’s; p < 0.05).

Figure 6

Antioxidant effect of the HI extract evaluated using DCFH-DA assay on T47D-KBluc, A549, and HGF. The cellular model was pre-exposed to the extract (25, 50, and 75 μg/mL) or NAC (20 mM) for 24 h, and further incubated with 50 μM DCFH-DA. The antioxidant effect of the HI extract was assessed after 2 h from oxidative stress induced by exposure to 250 μM H₂O₂. The results are expressed as relative means ± standard deviations (six technical replicates for each of the three biological replicates) where the negative control (DMSO 0.2%) is 100%. (*) indicates significant differences compared to H₂O₂ exposure alone; (#) indicates significant differences compared to negative control (ANOVA + Dunnett’s; p < 0.05).