Red-leaf hazelnut: Biotechnological approaches for secondary metabolite production and potential biological activities

This study provides the first evidence of anthocyanin and secondary metabolite production in the red-leaf hazelnut cultivar Corylus avellana L. cv. Fructo Rubro. Phenols, flavonoids and anthocyanins were analyzed in both field-grown leaves and in vitro callus cultures, assessing the effects of light quality, physical and chemical elicitors. Field-grown Fructo Rubro leaves exhibited higher content of anthocyanins early in the vegetative season, which then decreased as the chlorophyll content increased. In vitro cultures were successfully established from leaf explants, with callus cultures demonstrating significant anthocyanin biosynthesis under controlled conditions. To enhance metabolite production in callus culture, different LED spectra were used for physical elicitation, with NS1 (a spectrum close to natural sunlight) and G2 (rich in red and far-red light) being the most effective in stimulating anthocyanin biosynthesis. Chemical elicitation with jasmonic and abscisic acid, tested in combination with LED treatments, had a limited impact on anthocyanins. The HPLC analysis identified delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, and cyanidin 3-O-rutinoside as the predominant anthocyanins; furthermore, elicitation of callus culture demonstrated that the levels of these anthocyanins are comparable to or even exceeding those found in field-grown leaves. Additionally, polyphenols and flavonoids showed distinct accumulation patterns influenced by both physical and chemical elicitation. Furthermore, the effect of the extracts on the cell viability of human breast cells, both cancerous and non-cancerous, was preliminarily tested using the MTT assay. Extracts from field-grown leaves exhibited very low IC₅₀ values, indicating a strong ability to reduce the viability of cancerous cells. Notably, physical elicitation with LED light allowed in production of callus-derived extracts with comparable or even enhanced effects. Relevant, none of the extracts showed significant toxicity toward non-cancerous cells, highlighting their promising selectivity and potential safety for therapeutic applications. These findings highlight the potential of LED-enhanced in vitro cultures as a scalable and sustainable alternative for anthocyanin production, providing a controlled platform for optimizing bioactive compound synthesis. This research supports the development of biotechnological strategies for producing high-value phytochemicals that could be of interest for applications in health, food, and cosmetics.