Search

The chromatographic behavior of the selected compounds was studied under conditions of hydrophilic interaction liquid chromatography (HILIC). The effect of mobile phase composition on the retention in different chromatographic systems was systematically examined using high-performance thin-layer chromatography. The sorbents of different polarity and adsorption characteristics were selected and mixtures of water and organic solvents of various compositions, from pure water to pure organic solvent were used as mobile phases. Increasing the amount of water in the mobile phase leads to a conversion of the separation mechanism, and the retention curves have a characteristic “U” shape. The conversion between the adsorption and partition mechanisms is most likely continuous and depends on the chemical nature of separated substances, the stationary phase as well as on organic component of the mobile phase. Silica gel can be considered the most suitable stationary phase for the systematic investigation of the chromatographic behavior of the test compounds, whereas acetonitrile was the most suitable solvent. The obtained results contribute to the understanding of the dominant separation mechanism, the type, and the intensity of the interactions between separated substances with both stationary and mobile phases. Besides, the lipophilicity parameters obtained under HILIC conditions were evaluated and correlated with the calculated values.

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.

The individual steps of HPTLC are technically independent among each other and they provide access to versatile methods and flexibility in the detection of targeted compound groups. After the removal of the mobile phase, the bacterial or enzyme assays can be performed directly in a compatible medium. The bioactivity of the sample can be observed from a more detailed perspective than with the strictly defined fractions separated by elution on a column. The subjects of this study were methanol extracts from peels of 14 apple cultivars (6 autochthonous, 4 standard, and 4 resistant cultivars), with the aim to detect and distinguish the extracted compounds by observing their antimicrobial activity. The extraction was performed with a 0.1% hydrochloric acid solution in methanol. The chromatographic separation was carried out on HTPLC silica gel plates with a mobile phase consisting of ethyl acetate: n-hexane: formic acid: water (48/33/6/3, v/v/v/v). The bioautographic assay was performed with the Escherichia coli (ATCC 35218). Visualisation with p-anisaldehyde – sulfuric acid reagent (ASA) was used to detect phenolic acids, flavonoids and terpenes. Images of plates (both assays) were taken and processed in ImageJ, while R software was used for further processing of the chromatographic data. Antimicrobial activity was observed in zones between RF 0.6 and 0.9. The presence of compounds was determined using the ASA reagent, by comparing the RF values of the active zones and the standards used, and by the colours of the zones after derivatisation. The RF values of oleanolic, ursolic, ferulic, p-coumaric and caffeic acid standards are in the range of the RF values defined by the zones active against E. coli. The prevalence of terpene compounds is striking in the zones with antimicrobial activity. Regarding the antimicrobial activity of the used standard mixture, oleanolic and ursolic acids showed antimicrobial activity.

Strawberries (Fragaria x ananassa Duch.) are renowned for their nutritional benefits, high contents of polyphenols, and strong antioxidant properties. In the numerous global strawberry breeding programs, key factors for selection and breeding include fruit quality, flavor, and aroma, which are crucial for commercial appeal. Additionally, the radical scavenging activity (RSA), which assesses the fruit's ability to neutralize radicals, is an indicator of its antioxidant compound levels and overall quality [1,2]. The results indicated a variation in radical scavenging activity across different cultivation years, with progeny lines demonstrating lower RSA. Additionally, the total phenolic content in strawberry extracts was greater in the first breeding year and was found to be higher in the parental line (Fig 1).

Strawberry (Fragaria × ananassa Duch.) is among the most widely consumed berries globally, prized for their delightful taste. It is rich in micronutrients and phytochemical compounds, including anthocyanins, vitamin C, sugars, acids, and phenolic compounds (Fig 1). The concentrations of these phytochemicals significantly impact both the sensory and the nutritional value of the fruit. Sugars are the primary soluble components in ripe strawberry fruits, with glucose, fructose, and sucrose making up nearly 99% of the total sugar content [1,2].

Bee venom (BV), or apitoxin, is biotoxin naturally produced by the venom glands of honeybees. A major role of BV is defence of the bee colony, while it possesses a wide range of pharmacological and biological activities 1. Several techniques and methods have been used for separation and quantification of BV constituents. Selection is usually done by proportionality of BV components, as melittin has the highest proportion among three most important components, and is followed by phospholipase A2 (PLA2) and apamin 2. So far no method has been standardized for full characterization of BV. The majority of analytical procedures revolve around qualification and separation of melittin, PLA2 and apamin using the HPLC method. The main drawback of this method is that it consumes large amounts of mobile phase and it is a relatively slow process. In this work, we have tried to develop and optimize a more efficient, faster and greener HPTLC method, for the complete separation of three main components of BV 1,2. The separation of BV components was effectively accomplished with each component’s peak areas aligning closely with the RF value ranges of standard solutions for the individual components. The mobile phases were composed of a solution of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN) and a solution of 0.1% TFA in water (H2O) in different ratios. The same solvent ratio was applied for the separation of melittin and PLA2, while a different solvent ratio was necessary for the separation of apamin.

Strawberry (Fragaria x ananassa Duch.) is highly valued for its distinctive aroma, vibrant red color, juicy texture, and sweetness. The main "sweet" metabolites in strawberry are glucose, fructose, and sucrose. Because the levels and quality of these sugars are closely tied to the fruit's genotype, breeding initiatives are focused on developing new cultivars that meet changing consumer demands for superior fruit quality. Although genetic factors significantly affect sugar levels, cultivation practices and pre-harvest conditions also play an important role [1,2]. In this study, sugar profile of 24 strawberry genotypes was investigated during two years of cultivation. The harvest from the second year exhibits a more diverse sugar profile, with higher levels of individual sugars. Additionally, the progeny line contains greater amounts of disaccharides and trisaccharides, resulting in a richer sugar profile (Fig 1).

For a long time, Reversed-Phase Liquid Chromatography (RPLC) was the most dominant technique for the analysis of pharmaceutical compounds, but with poor efficiency in the separation of small polar molecules. From the efforts to solve the problem of insufficient retention of these molecules, during the last decades, a mode of liquid chromatography named Hydrophilic Interaction Liquid Chromatography (HILIC) has experienced vast expansion. It is based on the use of a highly hydrophilic stationary phase along with an aqueous mobile phase with high organic modifier content. In this review, the characteristics of stationary and mobile phases used in HILIC are described, and corresponding separation mechanisms are discussed. An overview of recently published papers dealing with the application of HILIC in analyzing pharmaceuticals in biological and non-biological samples is provided. Besides, the application of HILIC systems in the determination of the physicochemical properties of compounds is described.

Cilj ove doktorske disertacije je određivanje hemijskog sastava ekstrakata komercijalno prerađenog drveta različitog geografskog i biljnog porekla, koje se koristi za izradu buradi za odležavanje rakija u Srbiji. Analizirani su uzorci hrasta lužnjaka i kitnjaka, cera, belog duda, bagrema, džanarike i divlje trešnje. Analizom mikroskopskih preparata uzoraka drveta izvršena je taksonomska identifikacija ispitivanih vrsta. Antioksidativni potencijal i sadržaj ukupnih polifenola vodeno-etanolnih ekstrakata drveta su određeni spektrofotometrijski. Polifenolni profili ekstrakata ispitivani su primenom tankoslojne hromatografije i tečne hromatografije spregnute sa detektorom sa nizom dioda i masenim detektorom. Spektrofluorimetrijski su dobijeni podaci o prisutnim fluoroforama, dok je CIELab metodom određena boja ekstrakata. Antimikrobna aktivnost je ispitana uz pomoć modifikovane difuzione metode u bunarićima i mikrodilucione metode. Gasna hromatografija spregnuta sa masenom spektrometrijom upotrebljena je za identifikaciju i kvantifikaciju isparljivih jedinjenja. Induktivno spregnuta plazma sa optičkom emisionom spektroskopijom korišćena je za određivanje elementalnog sastava. U drugom delu istraživanja izvršeno je simuliranje odležavanja rakija tokom različitih vremenskih intervala korišćenjem različitih ekstrakcionih sredstava (dve vrste rakije i model-rastvor etanola). Ciklična voltametrija i diferencijalno pulsna voltametrija su primenjene za ispitivanje elektrohemijskog ponašanja i antioksidativnog potencijala etanolnih ekstrakata. Rezultati su pokazali da ispitivane vrste drveta obiluju polifenolima, te da postoji mogućnost upotrebe drvnog otpada iz procesa proizvodnje buradi za odležavanje alkoholnih pića. Pored toga, proširena su saznanja o nedovoljno ispitivanim vrstama (džanarika i cer) i pokazan je potencijal analitičkih tehnika koje do sada nisu primenjivane za analizu uzoraka drveta i njihovih ekstrakata (spektrofluorimetrija, ciklična voltametrija i diferen, The aim of this doctoral dissertation is to determine the chemical composition of extracts made from commercial wood samples of different geographical and plant origin, which is used for making barrels for ageing of fruit brandies in Serbia. The following samples were analysed: pedunculate oak, sessile oak, Turkey oak, white mulberry, black locust, Myrobalan plum and wild cherry. Taxonomic identification of the examined plant species was performed by analysis of microscope slides of wood samples. Spectrophotometry was used for determining the radical scavenging activity and the content of total polyphenols of the aqueous-ethanolic wood extracts. Phenolic profiles of the extracts were obtained by thin-layer chromatography and high-performance liquid chromatography coupled to diode array detector and mass spectrometry. Spectrofluorimetry was used for obtaining data about the fluorophores present, while CIELab method was used for measuring the colour of the extracts. Antimicrobial activity was investigated by modified well-diffusion method and microdilution method. Gas chromatography coupled to mass spectrometry was used for identifying and quantifying volatile compounds. Elemental composition was determined by inductively coupled plasma with optical emission spectroscopy. In the second part of the study, simulation of brandy ageing during different time intervals was performed by using different extraction agents (two fruit brandies and a model-ethanol solution). Cyclic voltammetry and differential pulse voltammetry were used for examining electrochemical behaviour and antioxidant capacity of the ethanolic extracts. The results have shown that the investigated wood samples are rich in polyphenols, therefore there is a potential of reusing wood waste from cooperage. In addition, the knowledge is expanded for the insufficiently tested species (Myrobalan plum and Turkey oak) while the potential for using analytical techniques not used before for the analysis of wood samp

Sempervivum tectorum L. (common houseleek) (Crassulaceae) has been traditionally used since ancient times for the treatment of various medical conditions, including ear and bladder infections of bacterial origin. The main aim of the current study is to investigate the antibacterial activity of S. tectorum leaf extracts using high-performance thin-layer chromatography– direct bioautography (HPTLC–DB) combined with chemometrics. This work presents a novel approach for the investigation of the antibacterial activity of S. tectorum leaf extracts on four Gram-positive (Bacillus subtilis, Micrococcus lysodeikticus, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus) and two Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) using HPTLC–DB bioassays. Gram-negative bacterial strains showed higher sensitivity in comparison with Gram-positive bacterial ones, due to the fact that numerous bands that represent active compounds are observed on biochromatograms. Principal component analysis (PCA) confirmed the discrimination between Gram-positive and Gram-negative biochromatograms by highlighting the main markers responsible for the separation. The proposed technique represents a fast and efficient way to separate and identify compounds with antibacterial activity in complex natural matrices.

Besides being one of the most commercially grown berry fruits, strawberries (Fragaria x annanassa Duch.) are known for their nutritional value, richness in polyphenols, and antioxidant capacity. New directions of strawberry breeding and selection are set towards premium fruit quality, high sugars content, and desirable sugars/total acids ratio (sweet index, SI), which represent some of the main prerequisites for selecting new breeding materials [1]. With this intention, a set of 24 strawberry genotypes was cultivated, including 12 parental varieties and 12 of their descendants - perspective candidates obtained by crossing the parental varieties mentioned above. A total of eleven sugars were quantified using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). Results implied that glucose, sorbitol, sucrose, and melibiose content were the most important sugars in separating the fruits of parental varieties and their progeny [2]. Results showed that old, parental varieties had a significantly higher content of glucose and sucrose, while new perspective genotypes had higher sorbitol and melibiose content (Fig.2). PCA analysis confirmed that parental (samples 1-12) and descendant genotypes (samples 13-24), could be discriminated according to sugars profile i.e. that these four sugars have an effect on their differentiation (Fig.1). This was in accordance with Mann-Whitney U test results (Fig.2). Fig.1. Principal Component Analysis - The difference between parental and descendant genotypes; score plot (A) – parental samples (PS) 1-12, descendant (DS) samples 13-24, and loading plot (B) – sugar components. Acknowledgments: This work has been supported by the Ministry of Education, Science, Technological Development and Innovation of Republic of Serbia, Contract number: 451-03-47/2023-01/200168 and 451-03-47/2023-01/200288., as well as company “Zeleni hit d.o.o.” from Belgrade, Serbia and “BerryLab” breeding consortium from Italy.

Considering the health-benefits of berry fruits consumption and increased market demands for food authenticityas one of the most important quality assurances, phenolic profiling by high-performance thin layer chroma-tography and ultra-high-performance liquid chromatography hyphenated with mass spectrometry was combinedwith multivariate analysis for phytochemical characterization and intercultivar discrimination of cultivatedberry seeds. The phenolic profiles of 45 berry seeds from nine genuine Serbian cultivated fruit species (straw-berry, 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 forthe classification, a total of 103 phenolic compounds were identified, including 53 phenolic acids and theirderivatives, 26 flavonoids and 24 glycosides. Biomarkers derived from the phenolic profile of berry seeds provedto be a powerful tool in the authentication of botanical origin, and may be useful in detection of frauds in berry-based seed-containing product.