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The main objective of this research was the extraction of bioactive compounds from different parts of two wild edible plants, Diplotaxis erucoides (pods, leaves, stems) and Oxalis pes- caprae (flowers, leaves, stems), by green strategies and encapsulation of the extracts. The flowers of Oxalis pes-caprae showed the highest content (about 750 mg GAE g-1) of total phenolics. The pods of Diplotaxis erucoides showed the highest total phenolic content (47 mg GAE g-1) compared to the leaves and stem. All encapsulated samples showed promising results regarding the release of bioactive compounds. Another important factor of the encapsulations for their practical application is the swelling capacity, and the pods of Diplotaxis erucoides samples showed the highest percentage of swelling (217%). However, for Oxalis pes-caprae, the results were very similar for flowers and stems (124% and 127%, respectively). In conclusion, these encapsulated plant extracts showed potential properties so they could be used as promising candidates for application in various fields such as food, cosmetics or agriculture.

Recently, a new window is opened with subclass of DESs, a hydrophobic DESs (hDESs), which application could be of great value for the pharmaceutical and cosmetic industry. It could be expected that hDESs can overpass the problems that occur with substances poorly soluble in water by increasing their solubility, permeability and stability. To be safely used in aforementioned industries it is of great importance to test hDESs cytotoxicity, solubility and permeability potential. Therefore, in the presented work we have prepared eight hDESs which are physico-chemically characterized and their cytotoxic profile was assessed on two human cell lines. The Parallel Artificial Membrane Permeability Assay (PAMPA) was applied as an in vitro model of passive permeation to determine potential of hDESs to be used as a solvent or a carrier for different compounds of interest. Furthermore, properties important for stability of formulation are also assessed i.e. antioxidant and antimicrobial potential of eight synthetized hDESs were evaluated based on ORAC method and disc diffusion assay.

Deep Eutectic Solvents (DESs) are in the focus of fundamental, but also applicative researches in the field of Green Chemistry. Their green character arises from the fact that DESs are non-toxic and non-flammable, as well as easily synthetized from cheap and renewable compounds. Recently, a new window is opened with subclass of DESs, hydrophobic DESs (hDESs), which could be of great value for the pharmaceutical and cosmetic industry. hDESs can overpass the problems that occur with the compounds poorly soluble in water by increasing their solubility, permeability and bioavailability. In order to safely use it in aforementioned industries it is of great importance to test their cytotoxicity and their own permeability potential. Therefore, in the presented work we have synthetized eight hydrophobic DESs and evaluated their cytotoxic profile in vitro on two human cell lines CaCo-2 and HaCaT. The Parallel Artificial Membrane Permeability Assay (PAMPA) was applied as an in vitro model of passive permeation to determine potential of hDESs to be used as a solvent or a carrier for different compounds of interest.

Agro-industrial sustainable economy indicates further exploitation of waste disposal because industry by-products are a source of esteemed bioactive compounds. Grape pomace is a major by-product coming from the winemaking industry and is generated in large quantities every year. To improve the environmental impact of the wine production process, grape pomace is used as a source for the isolation of phytochemicals such as phenolic acids, flavonoids, and tannins. Optimization of extraction techniques is an important step to achieving a maximum level of phenolic compounds from the source. Extraction processes favour the implementation of novel green technology methods as an alternative to energy-consuming and aggressive techniques that are still used. Solidliquid extraction is a classic method for extracting polyphenols made by agitation at a certain temperature using different solvents. Natural deep eutectic solvents (NADES) fulfl principles of green chemistry, making them a green alternative to conventional solvents, usually different organic chemicals. NADES are an easy-to-formulate mixture of naturally occurring primary metabolites with hydroxyl, carboxyl, or amino groups that provide hydrogen bond interactions, resulting in structured liquid solvents. NADES have adjustable viscosity, a broad range of polarity, and most importantly, the capacity to solubilize a wide variety of bioactive molecules. NADES increase extraction efciency due to the possibility of NADES being tailor-made depending on the physicochemical properties of desired extracts. The formation of additional hydrogen bonds between NADES and solutes increases the solubilization strength of NADES. While using NADES as a solvent, extraction can be further improved by using a microwave or ultrasound-assisted extraction process. NADES are not only used for extraction but also as a solvent for organic synthesis, biocatalysis, separation process, electrochemistry, etc. In this work, we used NADES as solvents of choice for the polyphenols extraction of Graševina white grape pomace. We also compared different NADES with conventional types of solvents and different extraction techniques. Using alternative green technologies for the extraction of bioactive molecules results in a higher yield in a short extraction period with minimized energy consumption.

Natural deep eutectic solvents (NADES) and biocatalysis's synergistic use fit logically to the efficient and sustainable production of enantiomerically pure compounds. The biotechnological approach ensures catalysing otherwise difficult transformations in high regio-, chemo- and enantioselective, at mild and economic conditions. NADES can serve as the green support for modulating/directing the reaction route to obtain the desired product. Papers dealing with the use of NADES as a medium for enantiopure compounds preparation/recovery implied that the unique properties of these solvents make them a promising candidate for reactions catalysed either by isolated enzymes or whole-cells through improved substrate/product solubility ; enhanced enzyme activity, and stability ; enhanced reaction yield ; possibility to tailor reaction enantioselectivity and regioselectivity ; the possibility of solvent NADES recycle and reuse [1, 2] The abundance of possible NADES formulations, makes it impossible to prepare and characterize all of them, pointing out the need to develop predictive NADES structure-activity mathematical models. Approaches used so far for screening appropriate/ideal NADES (out of an enormous pool of structural possibilities) have been governed by time- consuming empirical methods. The systematic investigation in NADES structure-activity relationship, as a backbone for these solvents' rational design, is still lacking. Current literature suggests COSMOtherm, software that can predict thermodynamic properties and phase equilibrium, which would help design the best possible solvent for a specific application. Thus, the trial-and-error method of NADES preparation can be avoided. Based on those mentioned above, this work aims to apply COSMOtherm in NADES screening for application in Saccharomyces cerevisiae - catalysed asymmetric reduction of prochiral ketones for chiral building block synthesis. By using COSMOtherm, the most suitable NADES for biocatalytic reaction is selected among 100 different NADES from our database. Further steps in rational design include optimization of reaction conditions, development of downstream protocol and scale-up.

Coenzymes are an indispensable part of oxidoreductase-catalyzed reactions, with nicotinamide coenzymes NAD+ and NADH being most frequently present. They have found increasing application in biocatalysis, especially in the reduction of prochiral compounds. When designing such processes, the major drawbacks are the high coenzyme price, its instability in aqueous solution, pH dependence and recyclation.[1] Because coenzymes are known for their labile nature and short-term stability, new and fresh stock solutions need to be prepared prior to the reaction. Therefore, finding a solvent that could simultaneously stabilise both nicotinamide coenzymes in oxidoreductive biocatalytic reactions and minimize their degradation during storage could be of great significance. In the last decade, new natural deep eutectic solvents (NADES) have arisen as a promising tool to shape already green biocatalysis into an even more ecological and sustainable process. NADES are solvents with valuable characteristics such as reduced emissions and toxicity, low volatility, biodegradability, but most importantly easy tunability. Their structural flexibility offers a possibility for rational solvent design to fulfill specific purposes and industrial requests.[2] One of their applications has also been shown for solubilisation and stabilisation of a wide range of biomolecules.[3] In this work, NADES were investigated as a stabilisation medium for nicotinamide coenzymes, NAD+ as an oxidized and NADH as a reduced form. Results showed that coenzymes stabilisation strongly depends on the hydrogen bond donor nature and water content. Cholinium-based NADES containing amide urea as hydrogen bond donor, stabilise both coenzyme forms to a much greater extent than reference buffers. The observed stabilisation effect is significant considering that these two coenzyme forms prefer media with different pH values. Moreover, long term stability of the coenzymes within studied NADES up to 50 days was confirmed. Several approaches were investigated to better understand the coenzyme stabilisation phenomena in NADES by implementing UV/VIS spectroscopy, NMR analysis and molecular dynamics simulations. This discovery can open a new chapter in coenzyme recycling and reuse during continuous biocatalytic reactions and most importantly, it could solve coenzyme storage shortcomings.

The pivotal role of acetylcholinesterase (AChE) in hydrolyzing the neurotransmitter acetylcholine can be disturbed by inhibitors like organophosphates. The formed adducts are not efficiently hydrolyzed by water and AChE loses its catalytic function. Consequent overstimulation of cholinergic receptors can lead to death. To overcome the life- threatening state, antimuscarinic atropine and an oxime reactivator must be administered promptly. Oximes, as nucleophiles stronger than water, restore phosphylated AChE activity, but due to their positive charge, standard oximes poorly pass the blood-brain barrier (BBB) and fail to reactivate synaptic AChE. Lately, zwitterionic molecules with a sufficient neutral species at physiologic pH to cross the BBB have been investigated. AChE is then reactivated by cationic species formed upon establishing pH equilibrium in the brain. Such oxime is RS- 194B, but it has low solubility in water. Natural deep eutectic solvents (NADES) can dissolve compounds of low water solubility, and besides being considered as safe for human use, often have beneficial features like antioxidative activity. With the aim to test the suitability of such solvents for oxime formulation, RS-194B was dissolved in NADES composed of glucose and citric acid with 30% water. The oxime was simultaneously dissolved in water, and the effect of such prepared formulations on AChE kinetics was compared. The results indicate that NADES induces conformational changes of the AChE enabling easier embedding of the oxime in the active site resulting in higher AChE affinity for the RS-194B/NADES than for RS- 194B/H2O. Yet, it seems that RS-194B/NADES orientation/vicinity to the phosphylated AChE’s catalytic serine was unfavorable, and the reactivation rate was higher with RS-194B/H2O. This NADES has been shown to inhibit AChE in fractions greater than 0.2%, implying its limited benefit ; however, further studies on other NADES are planned. We thank Prof P. Taylor for AChE and RS-194B 1Radić Z, et al. Refinement of structural leads for centrally acting oximes reactivators of phosphylated cholinesterases. J Biol Chem 2012 ; 287:11798-11809. 2Rosenberg Y, et al. Post- exposure treatment with the oxime RS194B rapidly reverses early and advanced symptoms in macaques exposed to sarin vapor. Chem-Biol Interact 2017 ; 274:50–7. 3Sit RK, et al. Pharmacology, pharmacokinetics, and tissue disposition of zwitterionic hydroxyiminoacetamido alkylamines as reactivating antidotes for organophosphate exposure. J Pharmacol Exp Ther 2018 ; 367:363– 72. 4Mitar A, et al. Physicochemical properties, cytotoxicity, and antioxidative activity of natural deep eutectic solvents containing organic acid. CYBEQ 2019 ; 33, 1-18.

In this dissertation, 15 natural deep eutectic solvents were successfully prepared and applied for developing an eco-friendly method for extraction of anthocyanins and lipase-catalyzed (R)-1-phenylethanol synthesis. Also, the biological activity of prepared grape pomace extracts was evaluated. With natural deep eutectic solvents efficiency of the process was increased in comparison with conventional processes. Extraction and biocatalytic process were optimised using the response surface methodology afterward laboratory-developed methods were scaled up. In both processes, the recyclation of used natural deep eutectic solvents was performed, as well as isolation and purification of the final product (anthocyanins, (R)-1-phenylethanol). U sklopu doktorske disertacije uspješno je pripremljeno i okarakterizirano 15 prirodnih eutektičkih otapala i primijenjeno za razvoj ekološki prihvatljivih metoda ekstrakcije antocijana iz komine grožđa te biokatalitičkog procesa priprave (R)-1-feniletanola pomoću lipaze. Također, evaluirana je biološka aktivnost pripremljenih polifenolnih ekstrakata u prirodnim eutektičkim otapalima. Primjenom prirodnih eutektičkih otapala u ekstrakciji polifenolnih spojeva i biokatalizi ostvarena je veća učinkovitost procesa u odnosu na konvencionalne postupke. Ekstrakcija i biokataliza su optimirani primjenom metode odzivnih površina te su laboratorijski razvijene metoda prenijete u veće mjerilo. U oba procesa razvijena je i metoda reciklacije korištenog prirodnog eutektičkog otapala, kao i izolacije i pročišćavanja produkta (antocijani, (R)-1-feniletanol).

Over the past decade, natural deep eutectic solvents (NADES) have become promising green solvent from both environmental and technological perspectives. The number of structural combinations encompassed by NADES is tremendous ; thus, NADES with unique physicochemical properties can be designed for a particular purpose, such as for efficient biocatalytic processes. The aim of this work was to prepare several NADES based on renewable sources (biomass energy) and to apply these solvents in the industrially interesting process of lipase-catalysed (R)-1-phenylethanol synthesis. Based on experimental data on lipase behaviour in the tested NADES, choline chloride:glycerol (ChGly) was selected as the most promising solvent for the given reaction. The resulting (R)-1- phenylethanol was also successfully isolated and purified from the reaction mixture using liquid-liquid extraction with ethyl acetate, whilst ChGly was recycled and reused. (R)-1-phenylethanol and unreacted (S)-1- phenylethyl acetate were purified on silica gel columns with yields of 81.91 %. The biocatalyst was also successfully recycled and reused in 4 cycles. Based on laboratory- scale optimum conditions, (R)-1-phenylethanol synthesis performed in a 500 mL batch reactor achieved reaction conversions comparable to those obtained with millilitre-scale biocatalytic reactions. The downstream process was also performed on a preparative scale with a yield of 49.48 %.

Novel approach in food fortification by using natural deep eutectic solvent (NADES) was studied. Cocoa by-products were chosen as sustainable source of polyphenols and extracts were prepared following the green extraction principles by using NADES. Antioxidative and biological activities of prepared extracts rich in polyphenols were determined and they were used for fortification of chocolate milk. Furthermore, electronic tongue analysis combined with SIMCA multivariate data analysis was used for the first time, to evaluate the sensory acceptability of chocolate/cocoa drinks with addition of NADES extracts, which is after safety issues, surely one of the most important features for possible application of those extracts in food industry. Based on presented results it is evident that carefully selected NADES could be used for efficient extraction of polyphenols from cocoa by-products and that obtained NADES extracts could be used for fortification in food industry, without removal of extraction solvent, since they are proven safe and estimated as sensory acceptable.

Green chemistry is a prevention approach to designing products and processes that minimize the use of materials hazardous to human health and environment. Biocatalysis, green solvents, reducing energy consumption by using alternative energy sources (e.g. microwave, ultrasound) are most important tools of green chemistry. The biocatalysis (whole cells, cell extracts, or purified enzymes) has the ability to catalyse organic reactions in the high chemo-, regio-, and stereoselectivity under mild reaction conditions and therefore represents an environment-friendly method of performing reactions that are known to require highly acidic or alkaline levels, high energy consumption for heating or even highly toxic metal catalysts. During the last 10 years, in order to find an alternative to conventional organic solvents, natural deep eutectic solvents (NADES) have started to be assessed as tools for biocatalysis, either as solvents or as separative agents, to overcome challenging workup procedures. A number of enzymes have shown enhanced activity and stability in the presence of cholinium-based NADES. The possibility to design an optimal NADES for each specific biocatalytic reaction system allows to improve substrate/product solubility ; enhance enzyme activity, enantioselectivity, regioselectivity and stability ; or to tailor the reaction rate. Herein, the behavior of industrial biocatalyst, Candida antarctica lipase B, carrot root and yeast Saccharomyces cerevisiae in the asymmetric hydrolysis of prochiral compounds and asymmetric reduction of prochiral ketones in natural deep eutectic solvents was studied. The effects of NADES on the synthetic activity and enantioselectivity of selected biocatalysts (as solvents, co-solvents or immiscible second phase), as well as some key parameters of processes involving them, will be presented.

Olive pomace, a by-product of the olive oil production, represents a serious ecological problem in Mediterranean region. Olive pomace is a dark-coloured waste that contains high amounts of organic materials that are not easily degradable or can even be toxic to plant life. But some of these compounds, like phenolic compounds, can act as potent natural antioxidants with bioactive activities. Olive pomace is a great source of phenolic alcohols, phenolic acids, flavonoids, lignans and especially secoiridoids, specific phenolic compounds of the Oleaceae family plants. The removal of the phenolics can facilitate further processing of the pomace and extracted compounds could be used as antioxidants in different food systems or as nutraceuticals. Nowadays new technologies and new generation of solvents are investigated in order to reduce environmental impact by increasing efficiency and sustainability of the extraction process. That is why the aim of this research was optimization of the method for the extraction of phenolic compounds from olive pomace using natural deep eutectic solvents and microwaves and/or ultrasound. We tested several green solvents with choline chloride or betaine chloride as hydrogen acceptor and various hydrogen-bond donors (sugars, alcohols ad carboxylic acids). Of eighth different natural deep eutectic solvents tested in our research, choline citrate was found to be most effective. Selected solvent was further used for the optimization of extraction process using and an ultrasonic-microwave cooperative reactor. Results have showed that microwave assisted extraction is the method of choice for the isolation of phenolic compounds from olive pomace. Longer extraction time and higher power used have positive impact on the extractability. Optimal extraction parameters are 30% water in the solvent, 12 minutes extraction time, and microwave power 300 W. Using microwave assisted extraction of phenolic compounds with selected parameters resulted in reduction of extraction time by 92% and increase of phenolic yield for 28% when compared to conventional methanol extraction method.

Nutritional value of flaxseed could be singled out in three major compounds: oil with dominant alpha linolenic fatty acid, fibers and polyphenols in form of glucosides, mostly lignans. Quantity of lignans in flaxseed is far greater than any other natural source but during oil extraction process polyphenols are left in press cake which is rarely used for human consumption. Lignans show significant biological activity through their derivates formed in our gut by intestinal bacteria. Apart from lignans, other detected phenols in flaxseed include ferulic and p-coumaric acid glucosides. Current methods of polyphenol extraction from flaxseed are time consuming (6h or more) and use organic solvents which mixed with NaOH which have negative impact on an environment. Deep eutectic solvents (DES) present inexpensive and ecological alternative to organic solvents while their role will be attractive not only for academic and medicinal research but also for industrial research. Combination of microwave and ultrasound extraction with deep eutectic solvents replacing organic solvents opens new possibilities in significantly improving this process. In this research, we tried to replace organic solvents with different DES and compare the results with some standard methods. Four different DES mixtures with alkaline properties were used for extraction: choline chloride:urea:glycerol, choline chloride:ethylene glycol, choline chloride:urea:ethylene glycol and choline chloride:urea with different 0.1 M NaOH ratios. Combination of microwave and ultrasound extraction showed better results compared just with microwaves while higher NaOH ratios (from 30 up to 50 %) had significant influence on quantity of extracted polyphenols. Much better results were obtained with ferulic and p- coumaric acid glucosides then with lignans. Up to 50 % of them were extracted compared with standard methods while lignan recovery was closer to 20 %. While this method needs further improvement and optimization the most promising solvents are choline chloride:urea:glycerol and choline chloride:ethylene glycol with 40 % of 0.1 M NaOH, extraction time of 6 minutes and microwave power 200W for choline chloride:urea:glycerol and extraction time of 9 minutes and microwave power 300W for choline chloride:ethylene glycol.

Environmental profiling of newly synthetized chemicals, including alternative solvents such as ionic liquids (ILs) and deep eutectic solvents (DESs), could be done in vivo or in vitro. Using an (eco)toxicological test battery comprising different levels of biological complexity (from enzymes, cells, microorganisms up to organisms and multi-species-systems) is a good approach to assess solvent of the interest, but the most often acute in vivo toxicity and in vitro tests are employed to determine are those novel solvents toxic or green. Until now, toxicity studies on ILs and DESs were carried out on bacteria, yeast, algae, multicellular organisms, soil invertebrates, mammals, as well as in different mammalian and fish cell lines. In general, depending on the model used, good correlations between in vivo and in vitro results are observed. Also, results are dependent on chemical structure (e.g. type of forming compounds used for ILs and DESs synthesis). Furthermore, degradation of solvent is also under the scope of ecotocixological field since the whole “life-cycle” should be investigated, as defined by the twelve principles of Green Chemistry. Therefore, several biodegradation tests were performed to determine the biodegradability of those solvents. Based on many researches in this field, as well as our own knowledge gain through “Green Solvents for Green Technology” project (HRZZ Grant No. 9550) some general guidelines for the synthesis of greener solvents could be given. Also, it can be concluded that cholinium-based ILs and DESs, which are newer class of alternative solvents, have better ecotoxicological profile than classical imidazolium-based ILs. Therefore, they would surely be the further direction in development of alternative solvents. A few types of solvents, including DESs, have been suggested as promising candidates for green extraction. Several studies have reported successful application of natural deep eutectic solvents (NADESs) in the extraction of phenolic compounds, indicating its great potential in plant-extract production. But, currently a lack of data on toxicology and the biological activity of those extract limits their commercial and industrial applications. Therefore, in the present study we prepared various phenolic extracts in NADESs and valorised their biological activity in cell lines. Obtained results showed that NADESs components could be used to enhance the biological activities of plant extract. Also, considering excellent extractability and stability of phenolic compounds in NADESs, these solvents will certainly be used for sustainable and green extraction from plant material.

Over the last few years, natural deep eutectic solvents (NADES) have been dramatically expanding in popularity as promising alternatives to traditional organic solvents. NADES are based on compounds that are safe for human consumption which opens numerous possibilities for their applications in the field of life sciences extraction of biologically active compounds from plant material. Possibility to use NADES as an extraction solvent for various metabolites, DNA, proteins, cellulose, lignin and many other natural compounds has been demonstrated. Solubility for some of those molecules increased for several orders of magnitude in NADES when compared to organic solvents, proving that extraction of natural molecules with NADES has a lot of potential. Main benefits of their application as a solvent for extraction of natural compounds are: NADES are highly selective and could extract both polar and nonpolar compounds ; NADES probably occur in living cells and are involved in the biosynthesis, solubilisation and storage of various metabolites ; stability of target compounds in NADES is better than in conventional solvents ; extracts made with NADES could be directly used in food, pharmaceutical, cosmetical and agrochemical applications without the need for expensive downstream purification steps. To design efficient extraction methods on industrial scale independently of target compound by using NADES the following steps should be included: (1) selection and fine- tuning of physicochemical characteristics of NADES (2) selection and optimization of the extraction method, (3) recovery of the target compounds form NADES, if it is necessary and (4) scale-up. However, all published research data refer to milliliter scale, and according to our knowledge there are no NADES-based commercial process. NADES have a great potential for industrial application owing to the acceptable costs, the versatility of physicochemical properties, the simple and inexpensive preparation and the low cytotoxicity. Herein, development of green extraction method using NADES on case study grape pomace anthocyanins will be presented and possibility of process scale-up will be discussed.

A lot of industries are looking for efficient exploitation of organic residues from various domains of agriculture and food manufacturing. Oil cake, a substance that remains after oil extraction from oil plant seeds, is one of these organic residues with great potential of application. Because of their protein content, the flaxseed and hempseed oil cakes can potentially be used in animal cell technology. Animal cells are complex expression system applied in wide range of biotech and biomedical techniques. Cell growth supporting microenvironment is largely defined by nutrient composition of culture media. Content of the proteins as much as their origin have a key role in culture media preparation. Fetal bovine serum (FBS) is still used as a major protein source, despite its compositional inconsistency and safety related risks. To overcome these problems, cell culture experts developed media where serum proteins can be partially or fully replaced with non-animal origin proteins. In this work we used plant protein hydrolysates derived from flaxseed and hempseed oil cakes. They both contain about 20% proteins, and as such can be processed into nutrient media supplement for animal cell cultivation. The main objective of this study is to determine effects of flaxseed and hempseed oil cake protein hydrolysates on growth and productivity of recombinant protein producing CHO cell line. To obtain the hydrolysates, proteins were isolated from hempseed and flaxseed oil cake by extraction and precipitation steps. The hydrolysis of protein isolates was carried out with commercial microbial proteases. CHO cell cultivation was performed in standard growth media containing FBS partially replaced with total protein hydrolysates. Protein content of the culture media was determined using SDS-PAGE and colorimetric techniques. CHO cell growth was monitored with hemocytometer and recombinant protein production by immunodetection. Our preliminary results indicate that oil cake protein hydrolysates show potential of being a beneficial culture media supplement. Both hempseed and flaxseed protein hydrolysates have positive effects on cell growth and volumetric production when compared to medium without hydrolysates added.

Bioactive compounds should be extracted using alternative solvents and enabling technologies, in accordance with green extraction principles. The aim of this study is to develop an eco- friendly extraction method for grape-pomace anthocyanins on a larger scale. From a preliminary screening of 8 different natural deep eutectic solvents (NADES), a combination of choline chloride:citric acid was selected because of its price, physicochemical properties, and anthocyanin recovery and stability. The effects of multimode-microwave (MW), and low-frequency-ultrasound (US) irradiation (used alone or simultaneously), as well as that of process parameters on extraction efficiency have been investigated in order to maximise anthocyanin extraction yield. The best conditions were found to be: simultaneous ultrasound/microwave-assisted extraction (UMAE) (MW power at 300 W, US power 50 W), for 10 min with 30% (v/v) of water. This gave 1.77 mg gdw-1 of anthocyanins. Anthocyanins were efficiently recovered from NADES, which were recycled. The optimised procedure was scaled up to a half-litre batch.