Potential application of green extracts rich in phenolics for innovative functional foods: natural deep eutectic solvents as media for isolation of biocompounds from berries.

The health-promoting effects of berries have attracted attention due to the possible application of their extracts as functional ingredients in food products. Natural deep eutectic solvents (NADESs) are a new generation of environmentally friendly solvents for the extraction of natural products, and they are green alternatives to organic solvents, and they can improve the solubility, stability, and bioavailability of isolated biocompounds. In this study, an efficient eco-friendly method was used for the extraction of phenolic compounds from different berries: chokeberries, blueberries, and black goji berries with a range of eutectic solvents consisting of hydrogen bond acceptors (HBAs) such as choline chloride, L-proline, L-glycine, and L-lysine and hydrogen bond donors (HBDs) such as malic, citric, tartaric, lactic and succinic acids, glucose and glycerol. The obtained results indicated the ability of NADESs towards selective extraction of phenolics; the eutectic system choline chloride : malic acid showed selective extraction of anthocyanins, while choline chloride : glycerol and choline chloride : urea showed selectivity towards flavonoids and phenolic acids. The methodology for screening of the NADES extraction performance, which included chromatographic profiling via high-performance thin layer chromatography combined with chemometrics and spectrophotometric essays, allowed effective assessment of optimal eutectic solvents for isolation of different groups of phenolics. Great antioxidant and antimicrobial activities of extracts, along with the green nature of eutectic solvents, enable NADES berry extracts to be used as "green-labelled" functional foods or ingredients.

Assessing radical scavenging capacity of Sempervivum tectorum L. leaf extracts: An integrated high-performance thin-layer chromatography/in silico/chemometrics approach.

High-Performance Thin-Layer Chromatography (HPTLC)-radical scavenging capacity (RSC) assays are standard techniques for the separation and identification of antioxidants from complex mixtures. HPTLC coupled with DPPH· visualization of chromatograms allows for the detection of individual antioxidants. However, other HPTLC-RSC assays that recognize compounds exhibiting different mechanisms of radical-scavenging activity are rarely reported. In this study, we developed an integrated approach that combines five HPTLC-RSC assays, principal component analysis (PCA) and quantum chemical calculations to assess the antioxidant capacity of Sempervivum tectorum L. leaf extracts. Two HPTLC assays - potassium hexacyanoferrate(III) total reducing power assay (TRP) and total antioxidant capacity by phosphomolybdenum method (TAC) - were developed for the first time. The method supports a more in-depth study of the RSC of natural products, as it compares the radical scavenging fingerprints of S. tectorum leaf extracts and recognizes differences in their individual bioactive constituents. Kaempferol, kaempferol 3-O-glucoside, quercetin 3-O-glucoside, caffeic acid, and gallic acid were identified as the compounds that discriminate HPTLC-RSC assays according to their mechanism of action and capture the similarities between 20 S. tectorum samples. Additionally, DFT calculations on M06-2X/6-31+G(d,p) level were applied to map thermodynamic feasibility of hydrogen atom transfer (HAT) and single electron transfer (SET) mechanisms of the identified compounds. Based on experimental and theoretical results, a combination of HPTLC-ABTS and HPTLC-TAC assays were proposed as the optimal method for mapping the antioxidants from S. tectorum. This study represents a step forward in identifying and quantifying individual antioxidants from complex food and natural product matrices in a more rational manner.

Protective Effects of Arbutus unedo L. Honey in the Alleviation of Irinotecan-Induced Cytogenetic Damage in Human Lymphocytes-An In Vitro Study.

Strawberry tree (Arbutus unedo L.) honey (STH) has been used since ancient times as a folk medicine remedy, especially in certain Mediterranean countries. This honey, rich in phenolic content, is well recognized for its antioxidant, anti-inflammatory, and antimicrobial activities, and is used for the treatment of skin lesions as well as gastrointestinal and respiratory disorders. This study investigated whether STH alleviates genome damage in human peripheral blood lymphocytes produced by the cytotoxic drug irinotecan. The phenolic profile of STH was previously estimated by ultra-high-performance liquid chromatography coupled to a linear ion trap-Orbitrap hybrid mass spectrometer. The effects of STH were evaluated at three concentrations (1×, 5×, and 10×), based on the daily consumption of the honey by an adult person. After 2 h of in vitro exposure, standard lymphocyte cultures for the analysis of chromosome aberrations and the cytokinesis-block micronucleus cytome assay were established. Our results demonstrate that STH offered remarkable geno- and cytoprotection when administered with irinotecan. These findings are relevant for drawing preliminary conclusions regarding the in vitro safety of the tested honey. However, further studies are needed with the application of more complex experimental models.

Quality Assessment of Bee Pollen-Honey Mixtures Using Thin-Layer Chromatography in Combination with Chemometrics.

The aim of this study is to develop a rapid, effect-directed screening method for quality assessment of bee pollen-honey mixtures. The comparative antioxidant potential and phenolic content of honey, bee pollen, and the bee pollen-honey mixtures, was performed using spectrophotometry. The total phenolic content and antioxidative activity of bee pollen-honey mixtures with 20 % bee pollen share were in the range 3.03-3.11 mg GAE/g, and 6.02-6.96 mmol TE/kg, respectively, while mixtures with 30 % bee pollen share contained 3.92-4.18 mg GAE/g, and 9.69-10.11 mmol TE/kg. Chromatographic fingerprint of bee pollen-honey mixtures was performed by high-performance thin-layer chromatography with conditions developed by authors and reported for the first time. Fingerprint analysis hyphenated with chemometrics enabled authenticity assessments of honey in mixtures. Results indicate that bee pollen-honey mixtures represent a food with highly, both, nutritious characteristics and health-promoting effect.

Authenticity assessment of cultivated berries via phenolic profiles of seeds.

Considering the health-benefits of berry fruits consumption and increased market demands for food authenticity as one of the most important quality assurances, phenolic profiling by high-performance thin layer chromatography and ultra-high-performance liquid chromatography hyphenated with mass spectrometry was combined with multivariate analysis for phytochemical characterization and intercultivar discrimination of cultivated berry seeds. The phenolic profiles of 45 berry seeds from nine genuine Serbian cultivated fruit species (strawberry, raspberry, blackberry, black currant, blueberry, gooseberry, cape gooseberry, chokeberry, and goji berry) revealed a good differentiation according to botanical origin. In order to determine biomarkers responsible for the classification, a total of 103 phenolic compounds were identified, including 53 phenolic acids and their derivatives, 26 flavonoids and 24 glycosides. Biomarkers derived from the phenolic profile of berry seeds proved to be a powerful tool in the authentication of botanical origin, and may be useful in detection of frauds in berry-based seed-containing product.

Antibacterial Fractions from Erodium cicutarium Exposed-Clinical Strains of Staphylococcus aureus in Focus.

Followed by a buildup of its phytochemical profile, Erodium cicutarium is being subjected to antimicrobial investigation guided with its ethnobotanical use. The results of performed in vitro screening on Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans strains, show that E. cicutarium has antimicrobial activity, with a particular emphasis on clinical S. aureus strains-both the methicillin sensitive (MSSA) and the methicillin resistant (MRSA) S. aureus. Experimental design consisted of general methods (the serial microdilution broth assay and the agar well diffusion assay), as well as observing bactericidal/bacteriostatic activity through time (the "time-kill" assay), investigating the effect on cell wall integrity and biofilm formation, and modulation of bacterial hemolysis. Observed antibacterial activity from above-described methods led to further activity-guided fractionation of water and methanol extracts using bioautography coupled with UHPLC-LTQ OrbiTrap MS4. It was determined that active fractions are predominantly formed by gallic acid derivatives and flavonol glycosides. Among the most active phytochemicals, galloyl-shikimic acid was identified as the most abundant compound. These results point to a direct connection between galloyl-shikimic acid and the observed E. cicutarium antibacterial activity, and open several new research approaches for future investigation.

Thin-layer chromatography in the authenticity testing of bee-products.

Quality control, nutritional value and the monitoring of hazardous residues in honey bee- products have become major topics for both producers and consumers. Due to its potential role in human health, bee-products rich in bioactive compounds are becoming increasingly popular. This review aims to provide an overview of thin-layer chromatography methods used in quality control,authenticity testing and chemical profiling of bee-products in order to help scientists engaged in the field of bee-products chemistry to utilize the advantages of this technique in the detection and elimination of fraudulent practices in bee-product manufacturing. Recently, hyphenation of thin-layer chromatography, image analysis and chemometrics support bee-products analysisbysimultaneousdeterminationofanalytes with different detection principles, identification of individual bioactive compounds as well as structure elucidation of compounds. Highlighted opportunities of thin-layer chromatography could encourage further investigations that would lead to improvements in the detection and elimination of marketing fraudulent practices.

Content and distribution of major and trace elements as a tool to assess the genotypes, harvesting time, and cultivation systems of potato.

The effects of organic versus integral and conventional crop management on content and distribution of major and trace elements in different varieties of potato were investigated in a three year field trial. A set of 48 potato tubers of four varieties with different harvesting times, cultivated in three types of agricultural systems: conventional (C), integral (I), and organic (O) were characterized based on the composition of the elements of their bulk and peel. A total of 16 elements were quantified. In order to determine the source of variation among the types of production, parts of potato and varieties, multivariate analysis of variance (MANOVA) was conducted. The results indicate that Ca, Mg, and K may be considered as important indicators of the type of production, genotypes of potato, and harvesting time. Additionally, the analyses show that ten microelements are able to distinguish between production types and genotypes of potato.

Comparative Study on the Phenolic Fingerprint and Antioxidant Activity of Strawberry Tree (Arbutus unedo L.) Leaves and Fruits.

The strawberry tree (Arbutus unedo L., Ericaceae family) is an evergreen Mediterranean shrub whose leaves and fruits are used in traditional medicine due to their antioxidant, antimicrobial, antidiabetic, diuretic, and antiproliferative properties. The health benefits are mainly attributed to the presence of phenolic compounds. The aim of this study was to compare the phenolic profiles, total phenolic content (TPC), and radical scavenging activity (RSA) of A. unedo leaves and fruits collected at two locations in Croatia. Phenolic profiles were identified using an ultra-high-performance liquid chromatograph (UHPLC) coupled with a hybrid mass spectrometer (LTQ Orbitrap MS). TPC was determined by Folin-Ciocalteu's assay, while RSA was investigated using DPPH reagent. A total of 64 phenolics (60 and 42 compounds in leaves and fruits, respectively) were identified. Hyperoside and flavan-3-ols were predominant compounds in leaves, while gallocatechin and catechin were the major compounds found in fruits. To the authors' knowledge, 16 and 5 phenolics in leaves and fruits, respectively, were reported for the first time. Principal component analysis (PCA) showed that UHPLC-LTQ Orbitrap MS could be used to identify which phenolics were able to discriminate samples regarding plant tissue and geographical origin. TPC in leaves and fruits were in the ranges of 67.07-104.74 and 16.78-25.86 mg gallic acid equivalents (GAE)/g dried weight (dw), respectively. RSA for leaves and fruits were in the ranges of 408.92-430.98 and 74.30-104.04 µmol Trolox equivalents (TE)/g dw, respectively. The number of identified phenolics was lower in fruits compared to leaves. Such a large number of bioactive phenolics identified and the strong antioxidant activity pointed to A. unedo as a promising health-promoting plant and natural food preservative.

First Extensive Polyphenolic Profile of Erodium cicutarium with Novel Insights to Elemental Composition and Antioxidant Activity.

Erodium cicutarium is known for its total polyphenolic content, but this work reveals the first highly detailed profile of E. cicutarium, obtained with UHPLC-LTQ OrbiTrap MS4 and UHPLC-QqQ-MS/MS techniques. A total of 85 phenolic compounds were identified and 17 constituents were quantified. Overall, 25 new compounds were found, which have not yet been reported for the Erodium genera, or the family Geraniaceae. Along with methanolic extracts, the so far poorly investigated water extracts exhibited in vitro antioxidant activity according to all performed assays, including the ferric reducing/antioxidant power assay (FRAP), 2,2-diphenyl-1-picrylhydrazyl assay (DPPH), 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) assay (ABTS) and cupric ion reducing antioxidant capacity assay (CUPRAC). Elemental composition analysis performed with inductively coupled plasma atomic emission spectrometry (ICP-AES) and, additionally, hydride generation atomic absorption spectrometry (HydrEA-ETAAS) showed six most abundant elements to be decreasing as follows: Mg>Ca>K>S>P>Na, and gave first data regarding inorganic arsenic content (109.3-248.4 ng g-1 ). These results suggest E. cicutarium to be a valuable source of various phenolic compounds with substantial potential for further bioactivity testing.

Extraction as a Critical Step in Phytochemical Analysis.

Phytochemical analysis, the chemical investigation of plants, is a rapidly developing chemical discipline with various goals such as the determination of the substance classes and the isolation and qualitative and/or quantitative analyses of bioactive compounds or their structural analysis. The analysis of plants begins with optimizing extraction procedures because they play a key role in extraction outcomes, i.e., the isolation of bioactive compounds from natural products and the selection of the subsequent tests used in the analyses. The extraction is a critical step because it is necessary to extract the desired components of the complex natural matrix without destroying them. A wide range of techniques for the extraction of bioactive compounds from natural matrixes are currently available. This review aims to describe and compare the most commonly used methods, traditional/conventional and innovative/green. It will focus on the principles behind each method and on their strengths and limitations in order to help evaluate their suitability and economic feasibility, highlighting the advantages of new innovative techniques over conventional extraction procedures. Finally, the recent application of deep eutectic solvents as a new, eco-friendly method for the extraction of bioactive compounds from natural matrixes is also discussed.

Phenolic Composition Influences the Health-Promoting Potential of Bee-Pollen.

Information on compositional, nutritional and functional properties of bee-pollen, as a health-promoting food, is essential for defining its quality. Concerning the nutritional importance of phenolic compounds, the aim of this study was to determine the phenolic profile and antioxidant activity of twenty-four bee-pollen samples collected from different regions of Serbia. High-performance thin-layer chromatographic (HPTLC) fingerprinting was used for profiling of bee-pollen samples according to the botanical type. HPTLC hyphenated with image analysis and a pattern recognition technique confirmed the grouping of samples caused by the specific phenolic composition of pollens of different botanical origin. Flavonoid glycosides in bee-pollen samples were identified by applying ultra-high-performance liquid chromatography (UHPLC) coupled with linear ion trap-Orbitrap mass spectrometry (LTQ Orbitrap MS). Eight out of twenty-seven flavonol glycosides were identified in bee-pollen samples for the first time. All analyzed bee-pollen samples showed a high number of phenolic compounds which may have therapeutic potential.

Anti-quorum sensing activity, toxicity in zebrafish (Danio rerio) embryos and phytochemical characterization of Trapa natans leaf extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Trapa natans L. (water chestnut or water caltrop) is a widespread aquatic plant, which has been cultivated for food and traditional medicine since ancient times. Pharmacological studies showed that water chestnut exhibits the wide range of biological activities, such as antimicrobial, antioxidative, analgesic, anti-inflammatory, as well as antiulcer. AIM OF THE STUDY: Evaluation of anti-virulence potential and toxicity of T. natans methanol (TnM), acetone (TnA) and ethyl acetate (TnEA) leaf extracts. MATERIALS AND METHODS: The anti-quorum sensing activity of Tn extracts was addressed by measuring their effects on biofilm formation, swarming motility and pyocyanin and elastase production in Pseudomonas aeruginosa. Specific P. aeruginosa biosensors were used to identify which of the signaling pathways were affected. The lethal and developmental toxicity of extracts were addressed in vivo using the zebrafish (Danio rerio) model system. The phenolic composition of T. natans leafs extracts was analyzed by a linear ion trap-OrbiTrap hybrid mass spectrometer (LTQ OrbiTrapMS) and UHPLC system configured with a diode array detector (DAD) hyphenated with the triple quadrupole mass spectrometer. RESULTS: Subinhibitory concentrations of Tn leaf extracts (0.2 MIC) inhibited pyocyanin and elastase production up to 50% and 60%, respectively, and reduced swarming zones, comparing to non-treated P. aeruginosa. TnA inhibited biofilm formation by 15%, TnM showed a stimulatory effect on biofilm formation up to 20%, while TnEA showed no effect. The bioactive concentrations of TnM and TnA were not toxic in the zebrafish model system. Twenty-two phenolic compounds were tentatively identified in TnM, where thirteen of them were identified in T. natans for the first time. Tn extracts, as well as their major components, ellagic and ferulic acids, demonstrated the ability to interfere with P. aeruginosa Las and PQS signaling pathways. CONCLUSIONS: This study demonstrates anti-virulence potential of Tn leaf extracts against medically important pathogen P. aeruginosa and confirms the ethnopharmacological application of this plant against microbial infections.

Quality assessment of marketed chamomile tea products by a validated HPTLC method combined with multivariate analysis.

Chamomile tea composed of dried flower heads of Matricaria recutita L. (Asteraceae) is one of the most popular single ingredient herbal teas. Tea industries, spice shops or public bazaars are mostly supplied chamomile as a raw material via cultivation or through nature-picking. However, one of the drawbacks of nature-picking is adulteration. This could be either due to false authentication of the plant materials by ingenuous pickers or intentional/unintentional substitution with other flowers resembling to chamomile in appearance during harvesting. Therefore, quality control of raw chamomile materials before marketing should be carefully considered not only by quantification of apigenin 7-O-glucoside (active marker) but also by fingerprinting of chemical composition. This work presents both quantification of apigenin 7-O-glucoside and chemical fingerprinting of commercial chamomile tea products obtained from different food stores and spice shops by a validated HPTLC method. In addition, HPTLC profiles of investigated chamomile tea samples were compared with HPLC method stated in the European Pharmacopoeia and it was found that HPTLC method was superior to HPLC method in the field of adulteration confirmation. Therefore, fingerprint profiles performed on the silica gel 60 NH2 F254s HPTLC plates combined with pattern recognition techniques of these marketed products were comparatively evaluated with wild and cultivar chamomile samples and also chamomile-like species from Asteraceae. Consequently, not chamomile tea bags but crude flowers sold on market were found to be adulterated with other plant materials.

The polyphenolics and carbohydrates as indicators of botanical and geographical origin of Serbian autochthonous clones of red spice paprika.

Spice peppers (Capsicum annuum L.) var. Lemeska and Lakosnicka paprika were investigated to evaluate their polyphenolic and carbohydrate profiles and antioxidant activity. A total of forty-nine polyphenolics were identified using ultrahigh-performance liquid chromatography (UHPLC) coupled to LTQ OrbiTrap mass analyzer. Twenty-five of them were quantified using available standards, while the other compounds were confirmed by exact mass search of their deprotonated molecule [M-H](-) and its MS(4) fragmentation. Thirteen carbohydrates were quantified using high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). Radical scavenging activity (RSA) ranged from 17.32 to 48.34mmol TE (Trolox equivalent)/kg DW (dry weight) and total phenolics content (TPC) was ranged between 7.03 and 14.92g GAE (gallic acid equivalents)/kg DW. To our best knowledge, five polyphenolic compounds were for the first time tentatively identified in paprika: 5-O-p-coumaroylquinic acid, luteolin 7-O-(2″-O-pentosyl-4″-O-hexosyl)hexoside, quercetin 3-O-(2″-O-hexosyl)rhamnoside, isorhamnetin 3-O-[6″-O-(5-hydroxyferuloyl)hexoside]-7-O-rhamnoside, and luteolin 7-O-[2″-O-(5'″-O-sinapoyl)pentosyl-6″-O-malonyl]hexoside.

Chemical profile of major taste- and health-related compounds of Oblacinska sour cherry.

BACKGROUND: Oblacinska sour cherry, an autochthonous cultivar, is the most planted cultivar in Serbian commercial orchards. Owing to its long cultivation under different agro-ecological conditions, it is a mixture of different clones. To obtain comprehensive information on Oblacinska sour cherry fruit, the total carbohydrate composition, total phenolic content, total anthocyanin content, antioxidant activity and polyphenolic profiles of 39 clones were investigated. RESULTS: Fructose was found to be the dominant sugar, followed by glucose, sorbitol and sucrose. Rutin and chlorogenic acid were the most abundant polyphenols. Some clones contained pinobanksin, hesperetin and galangin, which, to best knowledge, is the first time these compounds have been reported in sour cherry fruit. CONCLUSION: The data obtained showed huge variation within the germplasm studied, confirming the fact that Oblacinska sour cherry is not a cultivar but a population.

Poplar-type Propolis: Chemical Composition, Botanical Origin and Biological Activity.

Propolis is one of the most used natural products known for centuries for its beneficial effects. Due to significant differences in chemical composition of samples originating from different geographic and climatic zones it is crucial to characterize reliably each type of propolis. This article comprises the latest findings concerning the poplar type propolis, i.e. it gives a cross section of chemical composition, botanical origin and biological activity of poplar type propolis in order to encourage further investigations that would indicate its beneficial effects.

Amino acids profile of Serbian unifloral honeys.

BACKGROUND: The free amino acids profile of 192 samples of seven different floral types of Serbian honey (acacia, linden, sunflower, rape, basil, giant goldenrod, and buckwheat) from six different regions was analysed in order to distinguish honeys by their botanical origin. RESULTS: The most abundant amino acids were proline, alanine, phenylalanine, threonine and arginine. Based on the established amino acids profiles, some important differences have been identified among studied honey samples relying on the basic descriptive statistics data, and confirmed by multivariate chemometric methods. Principal component analysis revealed that basil honey samples form a well-defined cluster imposed with phenylalanine content. The model obtained by linear discriminant analysis might be used to distinguish basil honey from the rest of the samples, and has moderate predictive power to separate genuine acacia, linden, sunflower and rape honeys. New data for the amino acids profile of giant goldenrod and buckwheat honey samples are presented. CONCLUSIONS: The floral origin of honey could be successfully evaluated by its amino acids profile coupled with chemometric analysis.

Chemical composition of two different extracts of berries harvested in Serbia.

Total phenolic content (TPC), total anthocyanin content (TAC), free and total ellagic acid content, sugars, minerals, and radical-scavenging activity were determined in nine berries harvested in Serbia. More than 30 phenolic compounds were identified; among them, 11 polyphenols and cis,trans-abscisic acid were quantified using UHPLC coupled with an LTQ-Orbitrap XL mass analyzer. For the first time chrysin, naringenin, pinocembrin, and galangin were quantified in some of the investigated berry species. The extraction efficiency of the two extraction systems, methanol and acetone, was investigated. It was found that acetone is a better extracting solvent for TPC, whereas more TAC was extracted by methanol. TPC in acetone extracts ranged from 177.51 to 459.71 mg gallic acid equiv/100 g frozen weight. TAC ranged from 5.39 to 96.94 mg cyanidin-3-glucoside/100 g frozen weight in methanol extracts. The amounts of both free and total ellagic acid were found to be higher in the acetone extract in comparison to the methanol extract.

Lipophilicity of some guaianolides isolated from two endemic subspecies of Amphoricarpos neumayeri (Asteraceae) from Montenegro.

In this study 10 guaianolide-type sesquiterpene gamma-lactones named amphoricarpolides, isolated from the aerial parts of two endemic subspecies of Amphoricarpos neumayeri (ssp. neumayeri and ssp. murbeckii Bosnjak), were investigated by means of reversed-phase thin-layer chromatography. Methanol-water and tetrahydrofuran-water binary mixtures were used as mobile phase in order to determine lipophilicity parameters R (0) (M) and C(0). Some of the investigated compounds were screened for their cytotoxic activity against HeLa and B16 cells. Chromatographically obtained lipophilicity parameters were correlated with calculated logP values and IC(50) values. Principal component analysis identified the dominant pattern in the chromatographically obtained data.