Rational design of deep eutectic solvents for the stabilization of dehydrogenases: an artificial neural network prediction approach.

Stabilized enzymes are crucial for the industrial application of biocatalysis due to their enhanced operational stability, which leads to prolonged enzyme activity, cost-efficiency and consequently scalability of biocatalytic processes. Over the past decade, numerous studies have demonstrated that deep eutectic solvents (DES) are excellent enzyme stabilizers. However, the search for an optimal DES has primarily relied on trial-and-error methods, lacking systematic exploration of DES structure-activity relationships. Therefore, this study aims to rationally design DES to stabilize various dehydrogenases through extensive experimental screening, followed by the development of a straightforward and reliable mathematical model to predict the efficacy of DES in enzyme stabilization. A total of 28 DES were tested for their ability to stabilize three dehydrogenases at 30°C: (S)-alcohol dehydrogenase from Rhodococcus ruber (ADH-A), (R)-alcohol dehydrogenase from Lactobacillus kefir (Lk-ADH) and glucose dehydrogenase from Bacillus megaterium (GDH). The residual activity of these enzymes in the presence of DES was quantified using first-order kinetic models. The screening revealed that DES based on polyols serve as promising stabilizing environments for the three tested dehydrogenases, particularly for the enzymes Lk-ADH and GDH, which are intrinsically unstable in aqueous environments. In glycerol-based DES, increases in enzyme half-life of up to 175-fold for Lk-ADH and 60-fold for GDH were observed compared to reference buffers. Furthermore, to establish the relationship between the enzyme inactivation rate constants and DES descriptors generated by the Conductor-like Screening Model for Real Solvents, artificial neural network models were developed. The models for ADH-A and GDH showed high efficiency and reliability (R2 > 0.75) for in silico screening of the enzyme inactivation rate constants based on DES descriptors. In conclusion, these results highlight the significant potential of the integrated experimental and in silico approach for the rational design of DES tailored to stabilize enzymes.

Effect of Pulsed Electric Field Pretreatment on the Concentration of Lipophilic and Hydrophilic Compounds in Cold-Pressed Grape Seed Oil Produced from Wine Waste.

Pretreatment of grape pomace seeds with a pulsed electric field (PEF) was applied to improve the extraction yield of cold-pressed grape seed oil. The effects of different PEF conditions, electric field intensities (12.5, 14.0 and 15.6 kV/cm), and durations (15 and 30 min) on the oil chemical composition were also studied. All PEF pretreatments significantly increased the oil yield, flow rate and concentration of total sterols (p < 0.05). In addition, similar trends were observed for total tocochromanols and phenolic compounds, except for PEF pretreatment under the mildest conditions (12.5 kV/cm, 15 min) (p < 0.05). Notably, the application of 15.6 kV/cm for 30 min resulted in the highest relative increase in oil yield and flow rate (29.6% and 56.5%, respectively) and in the concentrations of total tocochromanols, nonflavonoids, and flavonoids (22.1%, 60.2% and 81.5%, respectively). In addition, the highest relative increase in the concentration of total sterols (25.4%) was achieved by applying 12.5 kV/cm for 30 min. The fatty acid composition of the grape seed oil remained largely unaffected by the PEF pretreatments. These results show that PEF pretreatment effectively improves both the yield and the bioactive properties of cold-pressed grape seed oil.

Enhancing protein stability under stress: osmolyte-based deep eutectic solvents as a biocompatible and robust stabilizing medium for lysozyme under heat and cold shock.

In biomedical and biotechnological domains, liquid protein formulations are vital tools, offering versatility across various fields. However, maintaining protein stability in a liquid form presents challenges due to environmental factors, driving research to refine formulations for broader applications. In our recent study, we investigated the relationship between deep eutectic solvents (DESs) and the natural presence of osmolytes in specific combinations, showcasing the effectiveness of a bioinspired osmolyte-based DES in stabilizing a model protein. Recognizing the need for a more nuanced understanding of osmolyte-based DES stabilization capabilities under different storage conditions, here we broadened the scope of our osmolyte-based DES experimental screening, and delved deeper into structural changes in the enzyme under these conditions. We subjected lysozyme solutions in DESs based on various kosmotropic osmolytes (TMAO, betaine, sarcosine, DMSP, ectoine, GPC, proline, sorbitol and taurine) paired either with another kosmotropic (glycerol) or with chaotropic osmolyte urea to rigorous conditions: heat shock (at 80 °C) and repetitive freeze-thaw cycles (at -20 and -80 °C). Changes in enzyme activity, colloidal stability, and conformational alterations were then monitored using bioassays, aggregation tests, and spectroscopic techniques (FT-IR and CD). Our results demonstrate the remarkable effectiveness of osmolyte-based DES in stabilizing lysozyme under stress conditions, with sarcosine- and betaine-based DESs containing glycerol as a hydrogen bond donor showing the highest efficacy, even at high enzyme loadings up to 200 mg ml-1. Investigation of the individual and combined effects of the DES components on enzyme stability confirmed the synergistic behavior of the kosmotrope-urea mixtures and the cumulative effects in kosmotrope-glycerol mixtures. Additionally, we have shown that the interplay between the enzyme's active and stable (but inactive) states is highly influenced by the water content in DESs. Finally, toxicity assessments of osmolyte-based DESs using cell lines (Caco-2, HaCaT, and HeLa) revealed no risks to human health.

Application of Multivariate Regression and Artificial Neural Network Modelling for Prediction of Physicochemical Properties of Grape-Skin Compost.

The reusability of by-products in the food industry is consistent with sustainable and greener production; therefore, the aim of this paper was to evaluate the applicability of multiple linear regression (MLR), piecewise linear regression (PLR) and artificial neural network models (ANN) to the prediction of grape-skin compost's physicochemical properties (moisture, dry matter, organic matter, ash content, carbon content, nitrogen content, C/N ratio, total colour change of compost samples, pH, conductivity, total dissolved solids and total colour change of compost extract samples) during in-vessel composting based on the initial composting conditions (air-flow rate, moisture content and day of sampling). Based on the coefficient of determination for prediction, the adjusted coefficient of determination for calibration, the root-mean-square error of prediction (RMSEP), the standard error of prediction (SEP), the ratio of prediction to deviation (RPD) and the ratio of the error range (RER), it can be concluded that all developed MLR and PLR models are acceptable for process screening. Furthermore, the ANN model developed for predicting moisture and dry-matter content can be used for quality control (RER >11). The obtained results show the great potential of multivariate modelling for analysis of the physicochemical properties of compost during composting, confirming the high applicability of modelling in greener production processes.

Grape Skin Composting Process to Recycle Food Waste: Kinetics and Optimization.

Within the various approaches to organic waste handling, composting has been recognized as an acceptable method to valorize organic waste. Composting is an aerobic technique of microbial disruption of organic matter which results with compost as a final product. To guarantee the quality of the compost, key process factors (like the moisture content, temperature, pH, and carbon-to-nitrogen ratio) must be maintained. In order to optimize the process, nine composting trials using grape skins were conducted in the present study under various initial moisture content and air flow rate conditions over the course of 30 days. The processes were monitored through physicochemical variables and microbiological activity. Also, the kinetics of the organic matter degradation and microbial growth were investigated. Although the thermophile phase was only achieved in experiments 3 and 8, the important variables proved the efficiency of all nine composting processes. The organic carbon content and C/N ratio decreased after the 30 days of composting processes and a great color change was noticed too. The values for the germination index for all experiments were above 80%, which means that the final products are non-toxic for plants. Also, the greatest change in organic carbon content in was evident in experiment 3; it decreased from 71.57 to 57.31%. And consequently, the rate of degradation for that experiment was the highest, at 0.0093 1/day. Furthermore, the response surface methodology was used to identify optimal operating conditions for grape skin composting and the obtained conditions were 58.15% for the initial moisture content and 1.0625 L/min for the air flow rate.

Food-Waste Valorisation: Synergistic Effects of Enabling Technologies and Eutectic Solvents on the Recovery of Bioactives from Violet Potato Peels.

The recovery of valuable bioactive compounds from the main underutilised by-products of the food industry is one of the greatest challenges to be addressed in circular economy. Potato peels are the largest waste generated during potato processing. However, they could be a potential source of valuable bioactive compounds, such as polyphenols, that can be reused as natural antioxidants. Currently, environmentally benign enabling technologies and new types of non-toxic organic solvents for the extraction of bioactive compounds may dramatically improve the sustainability of these processes. This paper focuses on the potential inherent in the valorisation of violet potato peels (VPPs) by recovering antioxidants using natural deep eutectic solvents (NaDES) under ultrasound (US)- and microwave (MW)-assisted extraction. Both the enabling technologies provided performances that were superior to those of conventional extractions in terms of antioxidant activity determined by the DPPH· (2,2-diphenyl-1-picrylhydrazyl) assay. In particular, the most promising approach using NaDES is proven to be the acoustic cavitation with a Trolox eq. of 1874.0 mmolTE/gExtr (40 °C, 500 W, 30 min), vs. the 510.1 mmolTE/gExtr of hydroalcoholic extraction (80 °C, 4 h). The shelf-life of both hydroalcoholic and NaDES-VPPs extracts have been assessed over a period of 24 months, and found that NaDES granted a 5.6-fold shelf-life extension. Finally, the antiproliferative activity of both hydroalcoholic and NaDES-VPPs extracts was evaluated in vitro using the MTS assay on human tumour Caco-2 cells and normal human keratinocyte cells (HaCaT). In particular, NaDES-VPPs extracts exhibited a significantly more pronounced antiproliferative activity compared to the ethanolic extracts without a noteworthy difference between effects on the two cell lines.

Are Foliar Nutrition Status and Indicators of Oxidative Stress Associated with Tree Defoliation of Four Mediterranean Forest Species?

Mediterranean forest ecosystems in Croatia are of very high significance because of the ecological functions they provide. This region is highly sensitive to abiotic stresses such as air pollution, high sunlight, and high temperatures alongside dry periods; therefore, it is important to monitor the state of these forest ecosystems and how they respond to these stresses. This study was conducted on trees in situ and focused on the four most important forest species in the Mediterranean region in Croatia: pubescent oak (Quercus pubescens Willd.), holm oak (Quercus ilex L.), Aleppo pine (Pinus halepensis Mill.) and black pine (Pinus nigra J. F. Arnold.). Trees were selected and divided into two groups: trees with defoliation of >25% (defoliated) and trees with defoliation of ≤25% (undefoliated). Leaves and needles were collected from selected trees. Differences in chlorophyll content, hydrogen peroxide content, lipid peroxidation and enzyme activity (superoxide dismutase, catalase, ascorbate peroxidase, non-specific peroxidase), and nutrient content between the defoliated and undefoliated trees of the examined species were determined. The results showed that there were significant differences for all species between the defoliated and undefoliated trees for at least one of the examined parameters. A principal component analysis showed that the enzyme ascorbate peroxidase can be an indicator of oxidative stress caused by ozone. By using oxidative stress indicators, it is possible to determine whether the trees are under stress even before visual damage occurs.

Prediction of pH Value of Aqueous Acidic and Basic Deep Eutectic Solvent Using COSMO-RS σ Profiles' Molecular Descriptors.

The aim of this work was to develop a simple and easy-to-apply model to predict the pH values of deep eutectic solvents (DESs) over a wide range of pH values that can be used in daily work. For this purpose, the pH values of 38 different DESs were measured (ranging from 0.36 to 9.31) and mathematically interpreted. To develop mathematical models, DESs were first numerically described using σ profiles generated with the COSMOtherm software. After the DESs' description, the following models were used: (i) multiple linear regression (MLR), (ii) piecewise linear regression (PLR), and (iii) artificial neural networks (ANNs) to link the experimental values with the descriptors. Both PLR and ANN were found to be applicable to predict the pH values of DESs with a very high goodness of fit (R2independent validation > 0.8600). Due to the good mathematical correlation of the experimental and predicted values, the σ profile generated with COSMOtherm could be used as a DES molecular descriptor for the prediction of their pH values.

The analysis of acidic and basic non-steroidal anti-inflammatory drugs in milk and muscle samples: a comprehensive analytical approach using UHPLC-MS/MS.

In animal husbandry for food production, the administration of non-steroidal anti-inflammatory drugs (NSAIDs) as anti-inflammatory, analgesic, and antipyretic agents, has seen an increase due to raised awareness of animal welfare issues. Residues of NSAIDs may be present in animal products that are intended for human consumption, and since some may pose a certain risk to human health, there is a need to monitor NSAID residues at low levels via routine and targeted surveillance. In analytical chemistry, NSAIDs are usually differentiated using their acid-base properties. Within this study, a method for simultaneous analysis of 27 NSAIDs, including both groups, in milk and muscle samples in 12.5 min is described. Sample processing consisted of enzymatic hydrolysis and acetonitrile extraction, followed by a clean-up of the extract by SPE, and measurement by UHPLC-MS/MS. The in-house validation study (alternative approach), covering trueness, precision, sensitivity, decision limit (CCα), detection capability (CCß) and matrix effect, was designed and evaluated with the help of validation software to meet the demands of regulatory compliance. The method recovery for milk and muscle matrix was in the range of 98.1% to 106.5% and 98.8% to 102.7%, whereas the CCß as the parameter for screening analysis, ranged from 0.07 to 46.7 µg/kg and 1.19 to 69.7 µg/kg, and the CCα, as the parameter for confirmatory analysis, from 0.11 to 56.7 µg/kg and 1.12 to 518.6 µg/kg, respectively. The occurrence of NSAID residues in milk and muscle samples was assessed using the developed method within the Croatian National Residue Control Plan, revealing quantifiable residues for diclofenac, ketoprofen, and salicylic acid mostly in milk samples. The most abundant NSAID in analysed samples was salicylic acid, which may be introduced into the food chain and be present in various types of matrices due to its natural occurrence in plants as a phytohormone.

Low-Phytotoxic Deep Eutectic Systems as Alternative Extraction Media for the Recovery of Chitin from Brown Crab Shells.

The versatility of chitin and its derivatives has allowed their utilization in a wide range of applications, from wastewater treatment to pharmaceutical or biomedical industries. However, even though the extraction method used industrially is extremely efficient, it involves the use of strong acids and bases and results in the disposal of large quantities of toxic effluents. Deep eutectic systems (DESs) have emerged as a promising new class of alternative solvents, including for chitin recovery. Yet, the assessment of their toxicity has often been neglected. Therefore, in this work, the phytotoxicity of choline chloride (ChCl)/organic acid-based DESs toward wheat seeds was evaluated by measuring different growth parameters and stress biomarkers. DESs were then explored for the efficient recovery of chitin contained in brown crab shell residues at varying conditions of temperature and processing time as well as with and without water addition. The obtained chitin was then characterized through different analytical techniques and compared to a standard as well as to chitin obtained by a conventional acid/alkaline hydrolysis. Results have shown that by applying a ChCl/lactic acid-based DES (which was the system that showed the least phytotoxic effects on wheat; EC50 ≥ 1.6 mg/mL) at 130 °C, it was possible to obtain pure chitin (up to 98%) with characteristics similar to those presented by commercial chitin or chitin recovered by conventional hydrolysis in a shorter time (more than 8-fold faster), thus suggesting that ChCl/organic acid-based DESs can truly represent a low-phytotoxic alternative extraction media for the recovery of chitin from the crab shell biomass.

Chilling and Freezing Temperature Stress Differently Influence Glucosinolates Content in Brassica oleracea var. acephala.

Brassica oleracea var. acephala is known to have a strong tolerance to low temperatures, but the protective mechanisms enabling this tolerance are unknown. Simultaneously, this species is rich in health-promoting compounds such as polyphenols, carotenoids, and glucosinolates. We hypothesize that these metabolites play an important role in the ability to adapt to low temperature stress. To test this hypothesis, we exposed plants to chilling (8 °C) and additional freezing (-8 °C) temperatures under controlled laboratory conditions and determined the levels of proline, chlorophylls, carotenoids, polyphenols, and glucosinolates. Compared with that of the control (21 °C), the chilling and freezing temperatures increased the contents of proline, phenolic acids, and flavonoids. Detailed analysis of individual glucosinolates showed that chilling increased the total amount of aliphatic glucosinolates, while freezing increased the total amount of indolic glucosinolates, including the most abundant indolic glucosinolate glucobrassicin. Our data suggest that glucosinolates are involved in protection against low temperature stress. Individual glucosinolate species are likely to be involved in different protective mechanisms because they show different accumulation trends at chilling and freezing temperatures.

Natural deep eutectic solvent as a unique solvent for valorisation of orange peel waste by the integrated biorefinery approach.

This research investigates the use of seven natural deep eutectic solvents (NADESs) for valorisation of orange peel waste, with the final goal to propose a unique NADES for integrated biorefinery. Initial screening of NADESs revealed the excellent ability of cholinium-based NADES with ethylene glycol as hydrogen bond donor (ChEg50) to serve as a medium for orange peel-catalysed kinetic resolution (hydrolysis) of (R,S)-1-phenylethyl acetate with high enantioselectivity (ee = 83.2%, X  = 35%), as well as it's stabilizing effect on the hydrolytic enzymes (hydrolytic enzymes within ChEg50 peel extract were stabile during 20 days at 4 °C). The ChEg50 also showed a satisfactory capacity to extract D-limonene (0.5 mg gFW-1), and excellent capacity to extract polyphenols (45.7 mg gFW-1), and proteins (7.7 mg gFW-1) from the peel. Based on the obtained results, the integrated biorefinery of orange peel waste using ChEg50 in a multistep process was performed. Firstly, enantioselective kinetic resolution was performed (step I; ee = 83.2%, X  = 35%), followed by isolation of the product 1-phenylethanol (step II; h = 82.2%) and extraction of polyphenols (step III; h = 86.8%) from impoverished medium. Finally, the residual orange peel was analysed for sugar and lignin content, and results revealed the potential of waste peel for the anaerobic co-digestion process. The main bottlenecks and futures perspective of NADES-assisted integrated biorefinery of orange peel waste were outlined through SWOT analysis.

Development, Optimization, and Comparison of Different Sample Pre-Treatments for Simultaneous Determination of Vitamin E and Vitamin K in Vegetables.

The absence of vitamin E from the diet can lead to cardiovascular disease, cancer, cataracts, and premature aging. Vitamin K deficiency can lead to bleeding disorders. These fat-soluble vitamins are important nutritional factors that can be determined in different methods in vegetables. In this work, the simultaneous determination of α-tocopherol, α-tocopheryl acetate, phylloquinone, and menaquinone-4 by gas chromatography-mass spectrometry (GC-MS) has been optimized using both direct injection and solid phase microextraction (SPME). Three different sample pre-treatment approaches based on: (A) solid-liquid-liquid-liquid extraction (SLE-LLE), (B) SLE, and (C) SPME were then applied to extract the target analytes from vegetables samples using menaquinone as internal standard. All the procedures allowed the determination of the target analytes in onion, carrot, celery, and curly kale samples. Similar results were obtained with the three different approaches, even if the one based on SPME offers the best performance, together with a reduced use of solvent, time consumption, and experimental complexity, which makes it the preferable option for industrial applications.

Enabling technologies for the extraction of grape-pomace anthocyanins using natural deep eutectic solvents in up-to-half-litre batches extraction of grape-pomace anthocyanins using NADES.

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.

Ready-to-use green polyphenolic extracts from food by-products.

To establish environmentally friendly polyphenolic extracts from grape and olive pomace, natural deep eutectic solvents (NADES) were used coupled with alternative energy sources - ultrasound and microwave irradiation. Obtained extracts were characterized by HPLC analysis, while antioxidant capacity was determined by ORAC method. Furthermore, in vitro cytotoxicity of prepared extracts was assessed by antiproliferation assay on two tumour cell lines, whereas for investigation of type of cell death or cell cycle arrest a flow cytometric analysis was applied. In addition, a detection of compounds with DNA/RNA-bindingaffinity in extracts was investigated by UV/Vis and circular dichroism spectroscopy. Grape pomace extract in NADES showed to be the best of all extracts tested, with regard to extraction of total polyphenolic compounds (p < 0.05) and related biological activities such as antioxidant and antiproliferative activity. Prepared polyphenolic extracts in NADES could be considered as ready-to-use in food and pharmaceutical industry without demanding and expensive downstream purification steps.

Green asymmetric reduction of acetophenone derivatives: Saccharomyces cerevisiae and aqueous natural deep eutectic solvent.

OBJECTIVE: Chiral building blocks [(S)-1-(3-methylphenyl)ethanol, (S)-1-(3,4-dimethylphenyl)ethanol and (S)-1-(2,4,6-trimethylphenyl)ethanol] for drug synthesis were prepared using two green approaches: (1) the yeast Saccharomyces cerevisiae as the biocatalyst and (2) the natural deep eutectic solvents (NADES) as the alternative solvents. Three different NADES with different water contents were prepared and screened for the highest conversion and enantiomeric excess of reduction of 1-(3-methylphenyl)ethanone, 1-(3,4-dimethylphenyl)ethanone (DMPA) and 1-(2,4,6-trimethyphenyl)ethanone by S. cerevisiae. The results were used in the development of eco-friendly procedures on a preparative scale. RESULTS: The highest enantioselectivity of baker´s yeast was for the bioconversion of DMPA in choline chloride:glycerol with 30% (v/v) of water (ChGly30). This reaction was used for further studies. Parameters such as pre-treatment of biocatalysts and recyclation of solvent were tested for a possible scale-up of this reaction system. Conversion was improved with the ultrasound pre-treatment of the biocatalysts in ChGly30. Moreover, the biocatalytic asymmetric reduction of DMPA in ChGly30 was successfully performed on a preparative scale with the efficient recyclation of NADES in two cycles, in which the reduction of DMPA was also successfully performed. CONCLUSION: Three enantioselective reductions in NADES with baker's yeast were successfully conducted. According to the highest enantioselectivity of the biocatalyst, the asymmetric reduction of 1-(3,4-dimethylphenyl)ethanone in ChGly30 was also performed on a preparative scale with efficient recyclation and reuse of NADES as a first step towards the implementation of this method on the industrial scale.

Comparative analysis of phytochemicals and activity of endogenous enzymes associated with their stability, bioavailability and food quality in five Brassicaceae sprouts.

Five Brassicaceae sprouts (white cabbage, kale, broccoli, Chinese cabbage, arugula) were comparatively analyzed based on phytochemicals (polyphenols, glucosinolates, carotenoids, chlorophylls, ascorbic acid) content and accompanying enzymes associated with phytochemical stability and bioavailability (peroxidases, myrosinase, and polyphenol-oxidase) that consequently impact food quality. Significantly high content of polyphenols and glucosinolates, as well as a high antioxidant activity were found in white cabbage, followed by kale sprouts. In addition, white cabbage contained higher amount of fibers and lower polyphenol-oxidase activity which potentially indicates prevention of browning and consequently better sprout quality. Arugula and broccoli showed higher activity of myrosinase that may result in higher bioavailability of active glucosinolates forms. According to our data, sprouts are cheap, easy- and fast-growing source of phytochemicals but also they are characterized by different endogenous enzymes activity. Consequently, this parameter should also be taken into consideration in the studies related to the health benefits of the plant-based food.

Extraction of Proanthocyanidins and Anthocyanins from Grape Skin by Using Ionic Liquids.

In this study, eight different types of imidazolium-based ionic liquids (ILs) were applied as new solvents in the extraction of flavonoids from grape skin, and compared to the conventional organic solvent extraction that was not reported earlier. The structure of anions, cations and concentration of ILs significantly affected extraction yields. The highest mass fractions of proanthocyanidins and anthocyanins were obtained with 2.5 mol/L of 1-butyl-3-methylimidazolium bromide [C4mim][Br] and 2.5 mol/L of 1-ethyl-3-methylimidazolium bromide [C2mim][Br], respectively. The studied ILs provided an excellent preliminary result in the extraction of anthocyanins. Significantly higher mass fractions of total and all free anthocyanins were extracted with 2.5 mol/L of [C2mim][Br] and 2.5 mol/L of 1-methylimidazolium hydrogen sulfate [mim][HSO4] than with conventional solvent with the exception of anthocyanin-3-O-acetylmonoglucosides in the latter. On the other hand, 2.5 mol/L of [C4mim][Br] and 2.5 mol/L of 1-(4-sulfobutyl)-3-methylimidazolium hydrogen sulfate [sC4mim][HSO4] showed significantly higher selectivity towards anthocyanin-3-O-acetylmonoglucosides and anthocyanin-3-(6-O-p-coumaroyl)monoglucosides.

Phytochemical and Bioactive Potential of in vivo and in vitro Grown Plants of Centaurea ragusina L. - Detection of DNA/RNA Active Compounds in Plant Extracts via Thermal Denaturation and Circular Dichroism.

INTRODUCTION: The phytochemical composition and biological activity of non-volatile components of Centaurea ragusina L. has not been studied previously. OBJECTIVES: Our aim was to evaluate the phytochemical and bioactive potential (including interactions with polynucleotides) of C. ragusina L. depending on the origin of plant material (in vivo - leaves from natural habitats, ex vitro - leaves from plants acclimated from culture media, in vitro - leaves and calli from plants grown in culture media) and polarity of solvents used in extract preparation (80 and 96% ethanol and water combinations or single solvents). METHODOLOGY: The polyphenol composition was determined by spectrophotometric and HPLC analysis. Biological activity of extracts was evaluated by following methods: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods for antioxidative activity, 2,3,5-triphenyl tetrazolium chloride (TTC) microdilution method for antibacterial activity, crystal-violet test for cytotoxic activity and thermal denaturation (TD) and circular dichroism (CD) for DNA/RNA interactions. RESULTS: Conditions for the most efficient polyphenol extraction were determined: the 80% ethanol/water solvent system was the most suitable for callus and leaf ex vitro samples and 80 or 96% ethanol for leaf in vivo samples. Significantly higher levels of chlorogenic acid and naringenin were detected in callus tissue than in vivo plant. Ethanolic extracts exhibited the significant antibacterial activity against Staphylococcus aureus ATCC 25923. DNA/RNA active compounds in plant extracts were detected by TD and CD methods. CONCLUSIONS: Callus tissue and ex vitro leaves represent a valuable source of polyphenols as in vivo leaves. TD and CD can be applied for detection of DNA/RNA active compounds in extracts from natural resources. Copyright © 2017 John Wiley & Sons, Ltd.

Poplar response to cadmium and lead soil contamination.

An outdoor pot experiment was designed to study the potential of poplar (Populus nigra 'Italica') in phytoremediation of cadmium (Cd) and lead (Pb). Poplar was treated with a combination of different concentrations of Cd (w = 10, 25, 50mgkg-1 soil) and Pb (400, 800, 1200mgkg-1 soil) and several physiological and biochemical parameters were monitored including the accumulation and distribution of metals in different plant parts (leaf, stem, root). Simultaneously, the changes in the antioxidant system in roots and leaves were monitored to be able to follow synergistic effects of both heavy metals. Moreover, a statistical analysis based on the Random Forests Analysis (RFA) was performed in order to determine the most important predictors affecting growth and antioxidative machinery activities of poplar under heavy metal stress. The study demonstrated that tested poplar could be a good candidate for phytoextraction processes of Cd in moderately contaminated soils, while in heavily contaminated soil it could be only considered as a phytostabilisator. For Pb remediation only phytostabilisation process could be considered. By using RFA we pointed out that it is important to conduct the experiments in an outdoor space and include environmental conditions in order to study more realistic changes of growth parameters and accumulation and distribution of heavy metals. Also, to be able to better understand the interactions among previously mentioned parameters, it is important to conduct the experiments during prolonged time exposure., This is especially important for the long life cycle woody species.