a b s t r a c tIn vitro culture emerges as a sustainable way to produce bioactives for further applicability in the food industry. Herein, vegetative parts of Fragaria vesca L. (wild strawberry) obtained by in vitro culture were analyzed regarding nutritional and phytochemical compounds, as well as antioxidant activity. These samples proved to have higher content of protein, polyunsaturated fatty acids, soluble sugars, organic acids (including ascorbic acid) and tocopherols (mainly a-tocopherol) than wild grown F. vesca, as well as containing additional phenolic compounds. The antioxidant activity of hydromethanolic extracts could be correlated with the content of different phenolic groups and other compounds (sugars and organic acids). It was demonstrated that in vitro culture could enhance nutritional and bioactive compounds of Fragaria vesca L. plants, providing a very interesting biotechnological tool for potential food applications.

A B S T R A C TThe extraction of phenolic compounds from walnut leaves (Juglans regia L.) was optimized using heat-assisted extraction and deep eutectic solvents based on choline chloride and carboxylic acids. A preliminary solvent screening was performed using a selected group of carboxylic acids as hydrogen bond donors, showing that the highest extraction yield of phenolic compounds was obtained using choline chloride mixtures with butyric or phenylpropionic acid at a mole ratio 1:2, with 20% of water (w/w). The extraction conditions (time, temperature and water proportion) were then optimized by an experimental design, assisted by response surface methodology. To evaluate the response, the three most abundant compounds identified by HPLC (neochlorogenic acid, quercetin 3-O-glucoside and quercetin O-pentoside) were quantified. Additionally, the solid/liquid ratio effect at the optimal conditions, in dose-response format, was studied in view of its upscale, not showing any significant decrease until 140 g/L. The results here presented provide valuable information towards the design of a process in a pre-industrial form for the extraction of phenolic compounds from J. regia leaves using deep eutectic solvents.

Juglans regia L. (walnut tree) is a recognized source of bioactive compounds with potential health benefits. In this work, hydroethanolic extracts of J. regia leaves were obtained by heat assisted extraction from different Portuguese samples in two phenological stages (green and yellow leaves) aiming to assess the impact of seasonal variations. The samples were compared regarding their phenolic composition and bioactivity. Seventeen phenolic compounds were identified by liquid chromatography combined with a diode array detector and electrospray ionization mass spectrometer (LC-DAD-ESI/MS n ): six phenolic acids, ten flavonoids and one tetralone derivative. The green leaves extracts presented a higher amount of total phenolic compounds (29.70 ± 0.03 mg/g extract) compared with the yellow leaves (23.26 ± 0.06 mg/g extract). In particular, yellow samples were richer in flavonoids (17.4 ± 0.2 mg/g extract; mainly quercetin-3-O-glucoside: 3.64 ± 0.01 mg/g extract), while the green ones presented higher phenolic acids content (16.7 ± 0.2 mg/g extract; mainly trans 3-p-coumaroylquinic acid: 6.9 ± 0.5 mg/g extract). Green leaves extract also presented higher antioxidant potential, achieving IC 50 values around 32 ± 2 μg/mL and 26.8 ± 0.2 μg/mL for the oxidative haemolysis inhibition and the thiobarbituric acid reactive substances assays, respectively. Furthermore, only green leaves samples showed anti-inflammatory potential. The cytotoxic evaluations revealed similar antiproliferative action of both extracts against the tumor cell lines tested. Also, an analogous anti-bacterial potential of the extracts was observed, with preferential action against Gram-positive clinical isolated bacteria, with lower minimum inhibitory concentration (MIC) values for Enterococcus faecalis and Listeria monocytogenes (MIC = 2.5 mg/mL). Therefore, the present study suggests the use of walnut leaves as a source of active ingredients without hepatotoxic effects to be used in different applications in the food or pharmaceutical areas.

Juglans regia L. (walnut) green husks are an important fraction of waste resulting from the walnut production, thus representing an interesting natural matrix to explore as a source of bioactive compounds. In this work, the hydroethanolic extract of walnut green husks was studied considering the phytochemical composition and the biological activity using different cell model assays, most of them evaluated for the first time for this matrix. From the HPLC-DAD-ESI/MS n analysis, sixteen compounds were identified, being the extract mostly composed of naphthalene derivatives (including tetralone derivatives) and less abundant in phenolic compounds (hydroxycinnamic acids and flavonols). The cytotoxic potential of the extract was assessed against tumour (MCF-7, NCI-H460, HeLa and HepG2) and non-tumour (PLP2) cell lines. Moreover, the antioxidant activity of the extract was evaluated by inhibition of the oxidative haemolysis (OxHLIA) and the formation of thiobarbituric acid reactive substances (TBARS), and the anti-inflammatory potential by the inhibition of the NO production by the RAW264.7 cell culture. The antibacterial effects of the extract were also evaluated against Gram-negative and Gram-positive bacteria. The results obtained represent a stepping stone for the development of future applications using walnut green husks as a source of added value compounds with bioactive potential.