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.

Elemental Profile of Propolis from Different Areas of Serbia.

Propolis is a resinous natural substance collected by honeybees from different plant sources. The element content of propolis is influenced by the content of elements in the soil, climatic factors, and the degree of pollution. The aim of the study was to characterize element composition and content of toxic elements in 51 propolis collected from different locations in Serbia and to evaluate the differences among them. Determination of the 21 elements were performed using Inductively coupled plasma - optical emission spectrometry. K was the most abundant element, followed by Ca, Mg, Si, Fe, Zn, and Al. The microelements B, Ba, Mn, Na, Pb, and Ti were also present in propolis, while Co, Cr, Cu, Mo, Ni, Sr, and V were found in trace. Toxic elements such as Pb, As, and Cd were found in the trace. Kruskal-Wallis tests differentiate groups of samples by geographical origin. The presence of B, Fe, Sr, Ti, Zn, and As demonstrated statistical significance between six regions of Serbia.

Bioassay-Guided Assessment of Antioxidative, Anti-Inflammatory and Antimicrobial Activities of Extracts from Medicinal Plants via High-Performance Thin-Layer Chromatography.

Natural products and their analogues have contributed significantly to treatment options, especially for anti-inflammatory and infectious diseases. Thus, the primary objective of this work was to compare the bioactivity profiles of selected medicinal plants that are historically used in folk medicine to treat inflammation and infections in the body. Chemical HPTLC fingerprinting was used to assess antioxidant, phenolic and flavonoid content, while bioassay-guided HPTLC was used to detect compounds with the highest antibacterial and anti-inflammatory activities. The results of this study showed that green tea leaf, walnut leaf, St. John's wort herb, wild thyme herb, European goldenrod herb, chamomile flower, and immortelle flower extracts were strong radical scavengers. Green tea and nettle extracts were the most active extracts against E. coli, while calendula flower extract showed significant potency against S. aureus. Furthermore, green tea, greater celandine, and fumitory extracts exhibited pronounced potential in suppressing COX-1 activity. The bioactive compounds from the green tea extract, as the most bioactive, were isolated by preparative thin-layer chromatography and characterized with their FTIR spectra. Although earlier studies have related green tea's anti-inflammatory properties to the presence of catechins, particularly epigallocatechin-3-gallate, the FTIR spectrum of the compound from the most intense bioactive zone showed the strongest anti-inflammatory activity can be attributed to amino acids and heterocyclic compounds. As expected, antibacterial activity in extracts was related to fatty acids and monoglycerides.

Primary Metabolite Chromatographic Profiling as a Tool for Chemotaxonomic Classification of Seeds from Berry Fruits.

Research background: Considering the importance of consumption of berry fruits with proven health-beneficial properties and difficulties in quality control of products of specific botanical and geographic origin, a fingerprint method was developed, based on advanced data analysis (pattern recognition, classification), in order to relate the variability of nutrients in the selected cultivars to primary metabolite profile. Experimental approach: Forty-five samples of genuine berry fruit cultivars (strawberry, raspberry, blackberry, black currant, blueberry, gooseberry, chokeberry, cape gooseberry and goji berry) were characterized according to chromatographic profiles of primary metabolites (sugars, lipids and fatty acids) obtained by three chromatographic techniques (high-performance thin-layer chromatography, gas chromatography coupled to mass spectrometry, and high-performance anion-exchange chromatography with pulsed amperometric detection). Results and conclusions: Comprehensive analysis allowed monitoring and identification of metabolites belonging to polar lipids, mono-, di- and triacylglycerols, free fatty acids, free sterols, sterol esters, mono- to heptasaccharides and sugar alcohols. Chemical fingerprint of berry seeds showed the uniformity of primary metabolites within each fruit species, but revealed differences depending on the botanical origin. All three chromatographic methods provided a discriminative, informative and predictive metabolomics methodology, which proved to be useful for chemotaxonomic classification. Novelty and scientific contribution: A novel methodology for the identification of bioactive compounds from primary metabolites of natural products was described. The proposed untargeted metabolite profiling approach could be used in the future as a routine method for tracing of novel bioactive compounds. The knowledge of metabolite composition obtained in this study can provide a better assessment of genotypic and phenotypic differences between berry fruit species and varieties, and could contribute to the development of new breeding programs.

Screening of semi-volatile compounds in plants treated with coated cerium oxide nanoparticles by comprehensive two-dimensional gas chromatography.

Literature data about semi-volatile organic compounds in plants and the effect of cerium oxide nanoparticles on them are scarce. Surface modification of nanoparticles may change nanoparticle-environment interaction, and therefore affects compounds in plants. In this research, uncoated and glucose-, levan-, and pullulan-coated cerium oxide nanoparticles were used for wheat and pea treatment during the growth. The aim was the screening of semi-volatile organic compounds from plants' shoots using comprehensive two-dimensional gas chromatography-mass spectrometry, a powerful separation technique allowing to reach unique separation resolution, and investigation of qualitative changes after the treatment with coated cerium oxide nanoparticles. The results were analyzed by the identification of individual peaks and fingerprint analysis by image processing. Wheat samples contained a higher number of semi-volatile organic compounds (108) compared to pea (77) but were less affected by the treatments with coated nanoparticles. The highest number of compounds was detected in wheat after the treatment with levan- and pullulan-coated nanoparticles, and in pea after treatment with levan-coated nanoparticles. This article reports a successful application of a semi-volatile organic compounds profile presented only as categorical variables and unique fingerprint images for the inter-cultivar recognition. This method may be useful in screening nanoparticles' effects on different plants.

Distribution of elements in seeds of some wild and cultivated fruits. Nutrition and authenticity aspects.

BACKGROUND: The compositional, functional, and nutritional properties of fruits are important for defining their quality. Fruit seeds should be better exploited as they are also considered to be a good source of bioactive components. Twenty macro, micro, and trace elements were identified and quantified in the seeds of 70 genuine wild and cultivated fruit species/cultivars by inductively coupled plasma atomic emission spectrometry and inductively coupled plasma mass spectrometry. Sophisticated chemometric techniques were also used to establish criteria for the classification of the analyzed samples. RESULTS: Calcium and P were the most abundant elements, followed by K and Na. The content of microelements and trace elements differed among the different cultivars/genotypes. The content of Ba, Pb, and Sr was significantly higher in wild fruits, whereas Fe, Mg, Mn, Ni and Zn content was higher in cultivated fruits. CONCLUSION: All of the statistical procedures that were used - Kruskal-Wallis, Mann-Whitney U-test, and principal component analysis (PCA) - confirm a unique set of parameters that could be used as phytochemical biomarkers to differentiate fruit-seed samples belonging to different cultivars/genotypes according to their botanical origin. This kind of investigation may contribute to intercultivar/genetic discrimination and may enhance the possibilities of acquiring a valuable authenticity factor. © 2018 Society of Chemical Industry.

Assessment of contamination, environmental risk, and origin of heavy metals in soils surrounding industrial facilities in Vojvodina, Serbia.

Content of potentially toxic elements was examined in soils from Srem (Vojvodina), to evaluate industrial facilities as pollution sources. Based on the distribution of the elements, the results of sequential extraction, enrichment factor (EF), ecological risk factor (Er), ecological risk index (RI), and statistical analysis, the current ecological status of the soils was determined. Elements in soils around the industrial facilities can be grouped into the five significant components derived by the principal component analysis (PCA), which explains 78.435% of the total variance. Al, Fe and Mg, and K and Mn are associated with two lithogenic components, respectively. Anthropogenic origin is identified for Hg and Cd. Mixed sources, geogenic and anthropogenic, are identified within two PCA components; one wich includes As, Pb, B, Zn, and the other: K and Cr, Ni and Cu. Cluster analysis (CA) corroborated the results obtained by PCA. The preliminary results revealed that the soils studied in a vicinity of industrial facilities in Srem have been exposed to different degrees of pollution. Among the characterized studied elements, Pb, Cd, Hg, Cu, Ni, and Cr are the main contaminants. Based on calculated EF, the studied soils show minor to severe enrichment with heavy metals. Ecological risk assessment results indicate that Cd and Hg carry the highest ecological risk level, and Zn and Cr the lowest.

Bioaccumulation and effects of metals on oxidative stress and neurotoxicity parameters in the frogs from the Pelophylax esculentus complex.

Metals are involved in the formation of reactive oxygen species and can induce oxidative stress. The aim of this study was to assess the effects of several metals on oxidative stress in the skin and muscle of the Pelophylax esculentus "complex" frogs (parental species Pelophylax ridibundus, Pelophylax lessonae, and their hybrid Pelophylax esculentus) that inhabit the wetland Obedska Bara in Serbia, and the potential use of these species as bioindicator organisms in biomonitoring studies. The biomarkers of oxidative stress (SOD, CAT, GSH-Px, GR, GST activities and GSH, SH concentrations) and cholinesterase activity were investigated. The concentrations of nine metals (Fe, Cu, Zn, As, Cd, Cr, Hg, Ni, and Pb) were measured in the water and tissues. Correlations were established between metals and biomarkers in the tissues. The results of metal accumulation distinguished the skin of P. lessonae and muscle of P. ridibundus from other P. esculentus complex species. The oxidative stress biomarkers observed in P. ridibundus and P. esculentus had greater similarity than in P. lessonae. The P. lessonae displayed the highest number of correlations between biomarkers and metals. The results of tissue responses revealed that skin was more susceptible to metal-induced oxidative stress, with only exception of As. In the light of these findings, we can suggest the use of P. esculentus complex species as a biomonitoring species in studies of metal accumulation and metal-induced oxidative stress, but with special emphasis on P. lessonae.

Ultrahigh-performance liquid chromatography and mass spectrometry (UHPLC-LTQ/Orbitrap/MS/MS) study of phenolic profile of Serbian poplar type propolis.

INTRODUCTION: Propolis is a resinous natural substance collected by honeybees from different plant sources. Due to the presence of various phytochemicals, this bee-product exhibits numerous biological activities, including anti-bacterial, anti-viral, anti-inflammatory, anti-oxidant, immunostimulating and anti-tumour effects. As the chemical composition and biological activity of propolis depend on its botanical and geographical origin, searching for new bioactive substances in various types of propolis from unexplored regions is of great importance. OBJECTIVE: The aim of this study is the evaluation of the phenolic profile of poplar propolis samples in order to characterise Serbian propolis, to identify possible new constituents and to specify the phenolic components relevant for differentiation of poplar propolis samples into two subgroups through simultaneous analysis of poplar bud extracts. METHODS: Ethanolic extracts of propolis and poplar buds were comprehensively analysed using ultrahigh-performance liquid chromatography coupled with hybrid mass spectrometry, which combines the linear trap quadrupole and Orbitrap MS/MS mass analyser together with chemometric methods. RESULTS: Extensive fingerprint analysis of Serbian propolis was achieved for the first time. Seventy-five phenolic compounds were detected. Eight of them were identified in propolis for the first time. Pattern-recognition methods applied to the content of ten quantified phenolics verified the existence of two subgroups of propolis, with galangin, chrysin and pinocembrin as the most influential distinguishing factors. CONCLUSION: The phenolic composition of the analysed propolis samples confirm their affiliation to the European poplar type propolis and the existence of two subgroups according to botanical origin.

Sugar Profile of Kernels as a Marker of Origin and Ripening Time of Peach (Prunus persicae L.).

Large amounts of fruit seeds, especially peach, are discarded annually in juice or conserve producing industries which is a potential waste of valuable resource and serious disposal problem. Regarding the fact that peach seeds can be obtained as a byproduct from processing companies their exploitation should be greater and, consequently more information of cultivars' kernels and their composition is required. A total of 25 samples of kernels from various peach germplasm (including commercial cultivars, perspective hybrids and vineyard peach accessions) differing in origin and ripening time were characterized by evaluation of their sugar composition. Twenty characteristic carbohydrates and sugar alcohols were determined and quantified using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD). Sucrose, glucose and fructose are the most important sugars in peach kernels similar to other representatives of the Rosaceae family. Also, high amounts of sugars in seeds of promising hybrids implies that through conventional breeding programs peach kernels with high sugar content can be obtained. In addition, by the means of several pattern recognition methods the variables that discriminate peach kernels arising from diverse germplasm and different stage of maturity were identified and successful models for further prediction were developed. Sugars such as ribose, trehalose, arabinose, galactitol, fructose, maltose, sorbitol, sucrose, iso-maltotriose were marked as most important for such discrimination.

Leaching of major and minor elements during the transport and storage of coal ash obtained in power plant.

In power plant, coal ash obtained by combustion is mixed with river water and transported to the dump. Sequential extraction was used in order to assess pollution caused by leaching of elements during ash transport through the pipeline and in the storage (cassettes). A total of 80 samples of filter ash as well as the ash from active (currently filled) and passive (previously filled) cassettes were studied. Samples were extracted with distilled water, ammonium acetate, ammonium oxalate/oxalic acid, acidic solution of hydrogen-peroxide, and a hydrochloric acid. Concentrations of the several elements (Al, As, Cd, Co, Cu, Cr, Fe, Ba, Ca, Mg, Ni, Pb, and Zn) in all extracts were determined by inductively coupled plasma atomic emission spectrometry. Pattern recognition method was carried out in order to provide better understanding of the nature of distribution of elements according to their origins. Results indicate possible leaching of As, Ca, Cd, Cu, Zn, and Pb. Among these elements As, Cd, and Pb are toxicologically the most important but they were not present in the first two phases with the exception of As. The leaching could be destructive and cause negative effects on plants, water pollution, and damage to some life forms.

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.

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.

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.

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.

Efficiency of Natural Deep Eutectic Solvents to Extract Phenolic Compounds from Agrimonia eupatoria: Experimental Study and In Silico Modelling.

To replace common organic solvents that present inherent toxicity and have high volatility and to improve the extraction efficiency, a range of natural deep eutectic solvents (NADESs) were evaluated for the extraction of phenolic compounds from Agrimonia eupatoria. Screening of NADES efficiency was carried out based on the total phenolic and flavonoid content and radical-scavenging activity, determined by spectrophotometry, as well as phenolic compounds quantified, obtained using ultra-high-performance liquid chromatography with a diode array detector and a triple-quadrupole mass spectrometer. Increased extraction efficiency when compared with organic solvent was achieved using NADES mixtures choline chloride (ChCl):urea 1:2 and choline chloride:glycerol 1:1. Flavonol glycosides were the most abundant compounds in all extracts. The COSMO-RS model provided insights into the most important intermolecular interactions that drive the extraction process. Moreover, it could explain the extraction efficiency of flavonol glycosides using ChCl:glycerol NADES. The current article offers experimental evidence and mechanistic insights for the selection of optimal NADES to extract bioactive components from Agrimonia eupatoria.

Comprehensive electrophoretic profiling of proteins as a powerful tool for authenticity assessment of seeds of cultivated berry fruits.

Product authentication is one of the most important food quality assurances. Considering the importance of consumption of berry fruits with proven health-beneficial properties, high sensory values and rich composition in bioactive substances, the aim of this study was to evaluate a straightforward and simple procedure for the protein fingerprinting of berry seeds. For this purpose, protein profiles of 45 samples of genuine berry fruit cultivars (strawberry, raspberry, blackberry, black currant, blueberry, gooseberry, chokeberry, cape gooseberry, and gojiberry) were analyzed by SDS-PAGE electrophoresis in combination with advanced chemometric tools. The most important parameters for discrimination among berry seeds were polypeptides at 12.8; 15.1; 25.0; 26.4; 30.0; 41.8; 44.4; 46.0; 48.5; 52.3 and 56.4 kDa. Biomarkers obtained from the protein profile of berry seeds proved to be a powerful tool in the authentication of their botanical origin, as well as for potential detection of berry-based products adulteration.

Structure-retention relationship study of arylpiperazines by linear multivariate modeling.

A quantitative structure-retention relationship study has been performed to correlate the retention of 33 newly synthesized arylpiperazines with their molecular characteristics, using thin-layer chromatography. Principal component analysis followed by multiple linear regression (MLR), principal component regression (PCR) and partial least squares (PLS) was performed to identify the most important factors, to quantify their influences, and to select descriptors that best describe the behavior of the compounds investigated. The best statistical performance was achieved by applying PLS regression, leading to the lowest value of the standard error (root mean square errors of calibration of 0.159 and cross-validated value RMSE cross-validation=0.231 units), followed by the PCR (root mean square errors of calibration=0.195 and RMSE cross-validation=0.305) and MLR (R(adj)(2)=0.9499, F=102.017, mean square error=0.052 and predicted residual error sum of squares=2.23). Two factors of the highest influence: surface tension and hydrophilic-lipophilic balance appear as the part of obtained models. In addition, polar surface area and hydrophilic surface area are included by both PLS and PCR models. Moreover, logP has been added to the PLS model. Besides, PCR model includes following descriptors: hydrogen bond acceptor, hydrogen bond donor and LUMO energy, whereas topological descriptors: connectivity indices 0 and 2, and valence index 3 are included in the MLR model.

Two aspects of honeydew honey authenticity: Application of advance analytical methods and chemometrics.

Taking into account a growing market and small number of articles related to honeydew honey, a metabolomic approach associated with multivariate analysis and modelling was proposed to discriminate five varieties of honey. Advanced analytical techniques were used for determination of 20 elements, 14 carbohydrates and stable carbon isotope ratio. No chemical marker has been found within sugar compounds, but several elements (Ba, Ca, Mg, Sr, Mn, Al, Co, Ni, Se) were marked as characteristic of honey type and allow classification of three botanical origins (Abies alba, Quercus frainetto, Quercus ilex). Sugars turanose, trehalose, arabinose and raffinose, elements Ba, Sr, P, Cd and Se, and δ13C values of honey, have different concentrations in honeys of the same botanical origin but harvested in different season. In addition to a confirmation of authenticity in terms of production, the values of δ13C of protein could be a good indicator of botanical origin.

Physicochemical analysis and phenolic profile of polyfloral and honeydew honey from Montenegro.

The research subject of this paper was a detail physicochemical analysis of 28 honey samples from the northern part of Montenegro. The honey from Montenegro has not been previously studied in such detail. Differentiation between samples, such as honeydew honey and polyfloral honey, was based on electrical conductivity, which was higher than 0.8 mS cm-1 for honeydew honey, as was expected. Other investigated physicochemical parameters (water content, free acids, diastase activity, hydroxymethylfurfural (HMF) content and sugar content) have shown great similarity for all honey samples. The main interest of this study was the identification and quantification of phenolic compounds using ultra-high performance liquid chromatography (UHPLC) with mass spectrometry detection. The results show that honey samples are very rich in phenolic compounds, especially quercetin. Among the 31 quantified phenolic compounds, the most dominant were phenolic acids. The highlight was based on p-hydroxybenzoic acid, p-coumaric acid, caffeic acid and ferulic acid. Considering polyphenolic compounds and sugar content, a high nutritional value can be observed in all samples, with an emphasis on polyfloral honeys, as was confirmed with principal component analysis (PCA). In addition, all honey samples were tested for total phenolic content (TPC) and radical scavenging activity (RSA). The results indicate the higher antioxidant ability of honeys from Montenegro in comparison to some honey samples from other countries in the region.