The growing interest of the food, pharmaceutical and cosmetics industries in naturally occurring bioactive compounds or secondary plant metabolites also leads to a growing demand for the development of new and more effective analysis and isolation techniques. The extraction of bioactive compounds from plant material has always been a challenge, accompanied by increasingly strict control requirements for the final products and a growing interest in environmental protection. However, great efforts have been made in this direction and today a considerable number of innovative extraction techniques have been developed using green, environmentally friendly solvents. These solvents include the deep eutectic solvents (DES) and their natural equivalents, the natural deep eutectic solvents (NADES). Due to their adjustable physical-chemical properties and their green character, it is expected that DES/NADES could be the most widely used solvents in the future, not only in extraction processes but also in other research areas such as catalysis, electrochemistry or organic synthesis. Consequently, this review provided an up-to-date systematic overview of the use of DES/NADES in combination with innovative extraction techniques for the isolation of bioactive compounds from various plant materials. The topicality of the field was confirmed by a detailed search on the platform WoS (Web of Science), which resulted in more than 100 original research papers on DES/NADES for bioactive compounds in the last three years. Besides the isolation of bioactive compounds from plants, different analytical methods are presented and discussed.

The formation of metal complexes with phytic acid is a complex process that depends strongly on the metal-to-ligand molar ratio, pH value and consequent protonation level of the phytate ligand as well as accompanying side reactions, in particular metal hydrolysis and precipitation of the formed coordination compounds. In the present work, the potentiometric titration technique was used in combination with a detailed analysis of the equivalent point dependencies for selected biologically relevant monovalent and divalent cations from the groups of alkaline earths and transition metals, namely: Mg(II), Zn(II), Fe(II), Cu(I), and Cu(II) ions. The investigation of complex formation mechanism, the evaluation of the species formed, and the identification of other side reactions was based on the examination of three distinct equivalent points, which were detectable by alkalimetric titrations of phytic acid in the presence of selected metal ions. It has been demonstrated that alkaline earth metals interact with different binding site(s) than the transition metals, and experiments with both oxidation states of copper revealed similar complexing characteristics, which depend mainly on the ionic radius (and not on the ionic charge as initially expected). Quantitative data on phytate complexation, hydroxide formation and complex precipitation are presented herein for all metals studied, including Cu(I), which was investigated for the first time by means of alkalimetric titration.

From the early precipitation-based techniques, introduced more than a century ago, to the latest development of enzymatic bio- and nano-sensor applications, the analysis of phytic acid and/or other inositol phosphates has never been a straightforward analytical task. Due to the biomedical importance, such as antinutritional, antioxidant and anticancer effects, several types of methodologies were investigated over the years to develop a reliable determination of these intriguing analytes in many types of biological samples; from various foodstuffs to living cell organisms. The main aim of the present work was to critically overview the development of the most relevant analytical principles, separation and detection methods that have been applied in order to overcome the difficulties with specific chemical properties of inositol phosphates, their interferences, absence of characteristic signal (e.g., absorbance), and strong binding interactions with (multivalent) metals and other biological molecules present in the sample matrix. A systematical and chronological review of the applied methodology and the detection system is given, ranging from the very beginnings of the classical gravimetric and titrimetric analysis, through the potentiometric titrations, chromatographic and electrophoretic separation techniques, to the use of spectroscopic methods and of the recently reported fluorescence and voltammetric bio- and nano-sensors.

In this study, the extraction efficiency of natural deep eutectic solvents (NADES) based on choline chloride as a hydrogen bond acceptor (HBA) and five different hydrogen bond donors (HBD; lactic acid, 1,4-butanediol, 1,2-propanediol, fructose and urea) was evaluated for the first time for the isolation of valuable bioactive compounds from Achillea millefolium L. The phytochemical profiles of NADES extracts obtained after ultrasound-assisted extraction were evaluated both spectrophotometrically (total phenolic content (TPC) and antioxidant assays) and chromatographically (UHPLC-MS and HPLC-UV). The results were compared with those obtained with 80% ethanol, 80% methanol, and water. The highest TPC value was found in the lactic acid-based NADES (ChCl-LA), which correlated with the highest antioxidant activity determined by the FRAP analysis. On the other hand, the highest antiradical potential against ABTS^+• was determined for urea-based NADES. Phenolic acids (chlorogenic acid and dicaffeoylquinic acid isomers), flavones (luteolin and apigenin), and their corresponding glucosides were determined as the dominant individual phenolic compounds in all extracts. The antibacterial and antifungal properties of the extracts obtained against four bacterial cultures and two yeasts were evaluated using two methods: the agar dilution method to obtain the minimum inhibitory concentration (MIC) and the minimum bactericidal or fungicidal concentration (MBC or MFC), and the disc diffusion method. ChCl-LA had the lowest MIC and MBC/MFC with respect to all microorganisms, with an MIC ranging from 0.05 mg mL⁻¹ to 0.8 mg mL⁻¹, while the water extract had the weakest inhibitory activity with MIC and MBC/MFC higher than 3.2 mg mL⁻¹.

For the isolation of selected phenolic compounds from dried chokeberries, natural deep eutectic solvents (NADESs) were investigated as a green alternative to conventionally used extraction solvents. Four types of NADESs were synthesised, with choline chloride as the hydrogen bond acceptor in combination with different hydrogen bond donors (sugars, organic acid and urea). Ultrasound-assisted extraction was used to improve the extractability of the phenolic compounds and the results were compared to those obtained with 80% methanol as the extraction media. The highest values of total phenols and total flavonoids were found in the extract obtained with choline chloride–fructose NADES (36.15 ± 3.39 mg gallic acid g⁻¹ dry weight (DW) and 4.71 ± 0.33 mg rutin g⁻¹ DW, respectively). The extraction recoveries for the individual phenolic compounds depended strongly on the phenolic compound’s structure, with relative mean values between 70% and 97%.

When dealing with simple phenols such as caffeic acid (CA) and ferulic acid (FA), found in a variety of plants, it is very important to have control over the most important factors that accelerate their degradation reactions. This is the first report in which the stabilities of these two compounds have been systematically tested by exposure to various different factors. Forced degradation studies were performed on pure standards (trans-CA and trans-FA), dissolved in different solvents and exposed to different oxidative, photolytic and thermal stress conditions. Additionally, a rapid, sensitive, and selective stability-indicating gas chromatographic-mass spectrometric method was developed and validated for determination of trans-CA and trans-FA in the presence of their degradation products. Cis-CA and cis-FA were confirmed as the only degradation products in all the experiments performed. All the compounds were perfectly separated by gas chromatography (GC) and identified using mass spectrometry (MS), a method that additionally elucidated their structures. In general, more protic solvents, higher temperatures, UV radiation and longer storage times led to more significant degradation (isomerization) of both trans-isomers. The most progressive isomerization of both compounds (up to 43%) was observed when the polar solutions were exposed to daylight at room temperature for 1 month. The method was validated for linearity, precision as repeatability, limit of detection (LOD) and limit of quantitation (LOQ). The method was confirmed as linear over tested concentration ranges from 1−100 mg L⁻¹ (r²s were above 0.999). The LOD and LOQ for trans-FA were 0.15 mg L⁻¹ and 0.50 mg L⁻¹, respectively. The LOD and LOQ for trans-CA were 0.23 mg L⁻¹ and 0.77 mg L⁻¹, respectively.