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 anti-proliferative 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
The authors thank the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), L. Barros, R. Calhelha and Carla Pereira contracts and V. Vieira (SFRH/BD/108487/2015) and T. Pires (SFRH/BD/129551/2017) grants. To UID/CTM/50011/2019 (CICECO), financed by national funds through the FCT/MCTES . To POCI-01-0145-FEDER-006984 (LA LSRE-LCM), funded by ERDF , through POCI-COMPETE2020 and FCT . To project AllNat - POCI-01-0145-FEDER-030463, financed by national funds through the FCT, and by COMPETE and Portugal2020, and to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E .
