Using natural deep eutectic solvents for the extraction of bioactive compounds from plant material: valorizing walnut residues through the development of bio-based semi-solid formulas for topical use

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Phenolic profile, antioxidant and antibacterial properties of Juglans regia L. (walnut) leaves from the Northeast of Portugal

Publication . Vieira, Vanessa; Pereira, Carla; Pires, Tânia C.S.P.; Calhelha, Ricardo C.; Alves, Maria José; Ferreira, Olga; Barros, Lillian; Ferreira, Isabel C.F.R.

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

2019Journal article

Open access

Enhanced extraction of phenolic compounds using choline chloride based deep eutectic solvents from Juglans regia L.

Publication . Vieira, Vanessa; Prieto Lage, Miguel A.; Barros, Lillian; Coutinho, João A.P.; Ferreira, Isabel C.F.R.; Ferreira, Olga

The 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.

2018Journal article

Open access

Optimization of the extraction of phenolic compounds from walnut leaves using DES.

Publication . Vieira, Vanessa; Prieto Lage, Miguel A.; Barros, Lillian; Coutinho, João A.P.; Ferreira, Olga; Ferreira, Isabel C.F.R.

Deep eutectic solvents (DES) are a new generation of alternative solvents resulting from the mixture of two (or more) starting materials where the eutectic temperature of the mixture is considerably lower than the melting point of the individual components [1]. DES can be considered “designer solvents” due to the possibility of combining different hydrogen bond acceptors and donors, to obtain solvents with specific affinity to the target molecules. Among their many applications is the potential use of DES as both solvents and formulation media of extracts rich in phenolic compounds [2]. In this work, the heat assisted extraction of phenolic compounds from walnut leaves was optimized, using DES based on choline chloride (CC) and carboxylic acids. To evaluate the response, the main phenolic compounds present in the extract (acid 3-O-caffeyloquinic acid, quercetin 3-O-glucoside and quercetin O-pentoside) were determined by HPLC-DAD. From a preliminary solvent screening, butyric acid (BA) and phenylpropionic acid (PPA) were selected as hydrogen bond donors. The extraction conditions (time, temperature and water content) were then optimized by an experimental design, assisted by response surface methodology. Water content was the most relevant extraction variable, followed by temperature and, lastly, extraction time. Under the optimized conditions, it was possible to obtain a response of 37.9±4.0 mg/g dw for CC:BA and 31.7±4.2 mg/g dw, for CC:PPA. Compared to the traditional water + ethanol reference solvent, similar or higher extraction yields were obtained using the selected DES.

2017Conference object

Open access

Extraction of phenolic compounds from Juglans regia L. optimized by response surface methodology

Publication . Vieira, Vanessa; Ferreira, Olga; Barros, Lillian; Prieto Lage, Miguel A.; Coutinho, João A.P.; Ferreira, Isabel C.F.R.

The studies on plant secondary metabolites extraction, namely phenolic compounds, are increasing due to their in vitro bioactivity and marked effects on oxidative process related to several health diseases. Moreover, they have been widely used in pharmaceuticals, functional foods and natural cosmetics [1]. The solid-liquid extraction of these compounds from biomass is usually carried out using organic solvents such as methanol, ethanol, acetone and ethyl acetate. In this context, microwave assisted extraction (MAE) is one of the most employed alternative extraction methods commonly using methanol or ethanol in different concentrations by dilution with water. Ethanol will be selected in this work due to its lower toxicity. In addition, this alcohol can be obtained from renewable sources and is safe for human consumption. Among the advantages reported for MAE are its lower extraction times and solvent consumption, when compared to conventional methods [2-4]. Our research group is interested in the green extraction of valuable compounds from biomass and there are previous evidences that walnut leaves are rich in phenolic acids and flavonoids [5]. In the present work, a 5-level full factorial Box–Behnken design was successfully implemented for maceration and MAE optimization, in which the ethanol concentration (Et), processing time (t), temperature (T), and solid/liquid ratio (S/L) were relevant independent variables. HPLC-DAD results showed that MAE is more effective extracting higher contents of phenolic compounds than conventional maceration in a lower processing time.

2016Conference object

Open access

Tailloring deep eutectic solvents for the extraction of valuable compounds from natural sources using choline chloride and carboxylic acids mixtures: optimization of the extraction of phenolic compounds from Juglans regia L. leaves

Publication . Vieira, Vanessa; Prieto Lage, Miguel A.; Barros, Lillian; Ferreira, Isabel C.F.R.; Ferreira, Olga; Coutinho, João A.P.

Phenolic compounds are a wide group of plant secondary metabolites with increasing interest due to their antioxidant ability and marked effects on oxidative processes related to several chronic diseases. Nowadays, they find applications in pharmaceutical products, functional foods and natural-based cosmetics. Therefore, the extraction and identification of these valuable compounds from different plants have become a major research area. Nevertheless, conventional solid-liquid extraction of phenolics usually involves the use of flammable, toxic and volatile organic solvents. Deep eutectic solvents (DES) are gaining much interest as alternative solvents to extract valuable compounds from natural matrices. DES can be considered “designer solvents” due to the possibility of combining different HBA (hydrogen bond acceptor) and HBD (hydrogen bond donor) to obtain solvents with specific properties to the target application. In this study, DES composed of choline chloride (CC) and different groups of organic acids were prepared, in order to extract phenolic compounds from leaves of Juglans regia L. (walnut leaves). The initial screening involved monocarboxylic (acetic, propionic, butyric, valeric, lactic and glycolic acids), dicarboxylic (malonic, glutaric and malic acids), tricarboxylic (citric) and aromatic acids (phenylacetic acid, 3-phenylpropanoic acid). The initial extraction conditions were: 50 ºC, 60 min extraction time and 20% water content. The main phenolic compounds (neochlorogenic acid, quercetin 3-O-glucoside and quercetin O-pentoside) were quantified by high performance liquid chromatography coupled to a diode de array detector (HPLC-DAD at 280 and 370 nm). Higher extraction yields were obtained using CC:butyric acid and CC:3-phenylpropanoic acid. For these systems, the stoichiometric ratio of HBA and HBD as well as the water content in the DES solutions were further evaluated. Better results were obtained using DES compared to the conventional water + ethanol mixed solvent. The present work contributes to the valorization of walnut leaves extracts using alternative solvents that could be tailored for potential applications in the food and pharmaceutical areas.

2017Conference object

Open access