The present study provides, for the first time, a physicochemical and biochemical characterizatio... more The present study provides, for the first time, a physicochemical and biochemical characterization of the redox processes associated with the ripening of Solanum dulcamara L. (bittersweet) berries. Electron Paramagnetic Resonance Spectroscopy (EPRS) and Imaging (EPRI) measurements of reactive oxygen species (ROS) were performed in parallel with the tissue-specific metabolic profiling of major antioxidants and assessment of antioxidant enzymes activity. Fruit transition from the mature green (MG) to ripe red (RR) stage involved changes in the qualitative and quantitative content of antioxidants and the associated cellular oxidation and peroxidation processes. The skin of bittersweet berries, which was the major source of antioxidants, exhibited the highest antioxidant potential against DPPH radicals and nitroxyl spin probe 3CP. The efficient enzymatic antioxidant system played a critical protective role against the deleterious effects of progressive oxidative stress during ripening. ...
The aim of this study was to determine intra- and interspecies variation in the qualitative and q... more The aim of this study was to determine intra- and interspecies variation in the qualitative and quantitative composition of methanol-soluble metabolites in the leaves of three Digitalis species (D. lanata, D. ferruginea, and D. grandiflora) from the central Balkans. Despite the steady use of foxglove constituents for human health as valuable medicinal products, populations of the genus Digitalis (Plantaginaceae) have been poorly investigated to describe their genetic and phenetic variation. Following untargeted profiling using UHPLC-LTQ Orbitrap MS, by which we identified a total of 115 compounds, 16 compounds were quantified using the UHPLC(–)HESI–QqQ-MS/MS approach. In total, 55 steroid compounds, 15 phenylethanoid glycosides, 27 flavonoids, and 14 phenolic acid derivatives were identified across the samples with D. lanata and D. ferruginea showing a great similarity, while 15 compounds were characteristic only for D. grandiflora. The phytochemical composition of methanol extracts...
Evidence-Based Complementary and Alternative Medicine, 2021
Artemisia absinthium L. (Asteraceae) is traditionally used for gastrointestinal ailments and diso... more Artemisia absinthium L. (Asteraceae) is traditionally used for gastrointestinal ailments and disorders linked to numerous risk factors including microbial infections. We aimed to provide contemporary evidence for its ethnopharmacological use and determine its antimicrobial capacity and mode of action, cytotoxicity, and phenolic constituents. Ethnopharmacological survey was conducted using semistructured interviews. Antimicrobial and antibiofilm capacities were determined by microdilution/crystal violet assay, respectively. Modes of action tested include estimation of exopolysaccharide production (congo red binding assay) and interference with membrane integrity (crystal violet uptake and nucleotide leakage assay). Cytotoxicity was determined using crystal violet assay. Polyphenolic profiling was done by advanced liquid chromatography/mass spectrometry (UHPLC-LTQ OrbiTrap MS). Artemisia absinthium in Serbia is traditionally used for gastrointestinal disorders, among others. Further s...
The following study was designed to explore antimicrobial properties of the by-product obtained i... more The following study was designed to explore antimicrobial properties of the by-product obtained in a hydro-distillation process of essential oil from Nepeta nuda L. We strived to develop a novel drink with antimicrobial self-preserving properties based on two components, N. nuda decoct and apple juice. By using 96-well plate microdilution assay it was shown that the N. nuda decoct has antimicrobial potential towards 8 bacterial and 6 fungal species, with the range of minimal inhibitory concentrations 10-300 mg/mL. By using actual food system, such as apple juice, in combination with and without short thermal treatment, we have shown that the decoct of N. nuda can inhibit the growth of food contaminant fungus Penicillium aurantiogriseum. It was determined that 3 volumes of decoct (500 mg/mL) and 22 volumes of apple juice should be mixed in order to obtain self-preserving drink resistant to P. aurantiogriseum contamination. Likewise, when thermal treatment (80 °C for 10 s) is included...
Gentiana pneumonanthe L. (fam. Gentianaceae) is a herbaceous perennial growing on wet heathlands,... more Gentiana pneumonanthe L. (fam. Gentianaceae) is a herbaceous perennial growing on wet heathlands, grasslands and hay meadows of Europe and Asia. Like other Gentiana species, G. pneumonanthe is characterized by the production of bitter secoiridoid glycosides (SGs) widely used in the pharmaceutical industry. Hairy roots (HRs) of G. pneumonanthe, as an alternative source of these phytochemicals, have been induced by agropine type Agrobacterium rhizogenes strain (A40M70GUS), and were characterized by genetic stability, sizable biomass production, and increased biosynthetic capacity compared to the native plant roots and above ground parts. Although HRs of G. pneumonanthe showed intensive growth without the application of phytohormones, the effect of varying concentrations of cytokinins (BAP – benzylaminopurine; KIN – kinetin) on the morphogenesis and on the content of SGs has been investigated in the present study, with the aim of optimizing in vitro conditions for enhanced production o...
The toxicity of bipyridynium herbicide paraquat (PQ) is related to its rapid generation of supero... more The toxicity of bipyridynium herbicide paraquat (PQ) is related to its rapid generation of superoxide in a chain reaction and the production of reactive oxygen species (ROS) in chloroplasts under light conditions. The nature and magnitude of the cellular responses to oxidative stress effects of five paraquat concentrations (0.05-20 μM) on in vitro grown Nepeta panonnica shoots were investigated. The results revealed that relative abundances of Mn SOD and Cu/Zn SOD isoforms changed dose-dependently, whereas catalase (CAT) and peroxidase (POX) activities increased along with paraquat concentrations. Polyphenol oxidase (PPO) activity was progressively inhibited, and, consequently, phenolic compounds were accumulated. Rosmarinic acid (RA) and an unidentified caffeic acid derivative (UCAD) were found to be the most abundant phenolics in shoots of this species. Paraquat was shown to be a potent elicitor that can stimulate accumulation of potent bioactive compounds in Nepeta pannonica, suc...
A plant’s main mechanism to diminish the effects caused by high free radical levels generated dur... more A plant’s main mechanism to diminish the effects caused by high free radical levels generated during high irradiance is the synthesis of various secondary metabolites. In addition to interspecies differences, their concentrations may be influenced by genetic, ontogenic, morphogenetic or environmental factors. We investigated the influence of genetic (genotypes from different natural habitats) and environmental (contrasting light regimes as well as successive parts of the vegetation period) variability on the accumulation of 10 selected phenolic compounds (phenolic acids, flavonoids, and xanthones) in Iris variegata genotypes. Genotypes originated from either sun-exposed or shaded natural habitats were transplanted to two experimental light treatments (high light intensity with a higher R/FR ratio and low light intensity with a lower R/FR ratio). Significant impacts of both genetic and environmental seasonal variability (spring, summer and fall during the vegetation period) on phenol...
Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characteri... more Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characterized by specialized metabolite profiles predominated by iridoids and phenolics, are differentiated according to the stereochemistry of major iridoid aglycone nepetalactone (NL). For the first time, the present study provides a comparative analysis of antimicrobial and immunomodulating activities of the two Nepeta species and their major iridoids isolated from natural sources—cis,trans-NL, trans,cis-NL, and 1,5,9-epideoxyloganic acid (1,5,9-eDLA), as well as of phenolic acid rosmarinic acid (RA). Methanol extracts and pure iridoids displayed excellent antimicrobial activity against eight strains of bacteria and seven strains of fungi. They were especially potent against food-borne pathogens such as L. monocytogenes, E. coli, S. aureus, Penicillium sp., and Aspergillus sp. Targeted iridoids were efficient agents in preventing biofilm formation of resistant P. aeruginosa strain, and they dis...
Phosphinothricin (PPT) is one of the most widely used herbicides. PTT targets glutamine synthetas... more Phosphinothricin (PPT) is one of the most widely used herbicides. PTT targets glutamine synthetase (GS) activity in plants, and its phytotoxicity is ascribed to ammonium accumulation and reactive oxygen species bursts, which drives rapid lipid peroxidation of cell membranes. In agricultural fields, PPT is extensively sprayed on plant foliage; however, a portion of the herbicide reaches the soil. According to the present study, PPT absorbed via roots can be phytotoxic to Arabidopsis, inducing more adverse effects in roots than in shoots. Alterations in plant physiology caused by 10 days exposure to herbicide via roots are reflected through growth suppression, reduced chlorophyll content, perturbations in the sugar and organic acid metabolism, modifications in the activities and abundances of GS, catalase, peroxidase, and superoxide dismutase. Antagonistic interaction of Nepeta rtanjensis essential oil (NrEO) and PPT, emphasizes the existence of complex control mechanisms at the trans...
Centaurium erythraea Rafn produces and accumulates various biologically active specialized metabo... more Centaurium erythraea Rafn produces and accumulates various biologically active specialized metabolites, including secoiridoid glucosides (SGs), which help plants to cope with unfavorable environmental conditions. Specialized metabolism is commonly modulated in a way to increase the level of protective metabolites, such as SGs. Here, we report the molecular background of the wounding-induced changes in SGs metabolism for the first time. The mechanical wounding of leaves leads to a coordinated up-regulation of SGs biosynthetic genes and corresponding JA-related transcription factors (TFs) after 24 h, which results in the increase of metabolic flux through the biosynthetic pathway and, finally, leads to the elevated accumulation of SGs 96 h upon injury. The most pronounced increase in relative expression was detected for secologanin synthase (CeSLS), highlighting this enzyme as an important point for the regulation of biosynthetic flux through the SG pathway. A similar expression patte...
Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As t... more Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As the result of allopolyploid hybridization, offspring may gain different ploidy levels in comparison to parental species, which can provide them instant reproductive isolation. Two tetraploid sister species, Centaurium erythraea and C. littorale, readily cross-fertilize, resulting in hybrids of various ploidy. In northern Serbia, two stable populations of a hexaploid taxon C. pannonicum have been documented. It has been proposed previously that this taxon emerged after an interspecific hybridization event between two tetraploid sister-species: C. erythraea and C. littorale subsp. compressum. The existing populations of the hybridogenic taxon, as well as neighboring populations of the two parental taxa were here characterized by both morphometrics and molecular markers (EST-SSR and trnL-F). Three leaf and two flower characteristics were found to be informative in delimitation of the parenta...
Available scientific literature reports very few microsatellite markers derived from tetraploid g... more Available scientific literature reports very few microsatellite markers derived from tetraploid genomes using de novo transcriptome sequencing, mostly because their gain usually represents a major computational challenge due to complicated combinatorics during assembly of sequence reads. Here we present a novel approach for mining polymorphic microsatellite loci from transcriptome data in a tetraploid species with no reference genome available. Pairs of 114 bp long de novo sequenced transcriptome reads of Centaurium erythraea were merged into short contigs of 170-200 bp each. High accuracy assembly of the pairs of reads was accomplished by a minimum of 14 bp overlap. Sequential bioinformatics operations involved fully free and open-source software and were performed using an average personal computer. Out of the 13 150 candidate contigs harboring SSR motifs obtained in a final output, we randomly chose 16 putative markers for which we designed primers. We tested the effectiveness of...
The main principles of C. pulchellum (Sw.) Druce, secoiridoid glycosides, have been studied as po... more The main principles of C. pulchellum (Sw.) Druce, secoiridoid glycosides, have been studied as potent bioactive compounds. Here we scored their content in extracts of ten populations of this species. Antibacterial and antifungal assays of the extracts and pure secoiridoid glycosides were performed against eight bacterial strains and five fungal species. Methanol extracts from both aerial parts and roots exhibited excellent antibacterial (0.05-0.2 mg mL−1) and very good antifungal (0.1-2 mg mL−1) activity. Pure secoiridoid glycosides isolated from these extracts demonstrated very strong antibacterial (0.01-0.04 mg mL−1) and especially antifungal (0.001-0.1 mg mL−1) activity.
The present study provides, for the first time, a physicochemical and biochemical characterizatio... more The present study provides, for the first time, a physicochemical and biochemical characterization of the redox processes associated with the ripening of Solanum dulcamara L. (bittersweet) berries. Electron Paramagnetic Resonance Spectroscopy (EPRS) and Imaging (EPRI) measurements of reactive oxygen species (ROS) were performed in parallel with the tissue-specific metabolic profiling of major antioxidants and assessment of antioxidant enzymes activity. Fruit transition from the mature green (MG) to ripe red (RR) stage involved changes in the qualitative and quantitative content of antioxidants and the associated cellular oxidation and peroxidation processes. The skin of bittersweet berries, which was the major source of antioxidants, exhibited the highest antioxidant potential against DPPH radicals and nitroxyl spin probe 3CP. The efficient enzymatic antioxidant system played a critical protective role against the deleterious effects of progressive oxidative stress during ripening. ...
The aim of this study was to determine intra- and interspecies variation in the qualitative and q... more The aim of this study was to determine intra- and interspecies variation in the qualitative and quantitative composition of methanol-soluble metabolites in the leaves of three Digitalis species (D. lanata, D. ferruginea, and D. grandiflora) from the central Balkans. Despite the steady use of foxglove constituents for human health as valuable medicinal products, populations of the genus Digitalis (Plantaginaceae) have been poorly investigated to describe their genetic and phenetic variation. Following untargeted profiling using UHPLC-LTQ Orbitrap MS, by which we identified a total of 115 compounds, 16 compounds were quantified using the UHPLC(–)HESI–QqQ-MS/MS approach. In total, 55 steroid compounds, 15 phenylethanoid glycosides, 27 flavonoids, and 14 phenolic acid derivatives were identified across the samples with D. lanata and D. ferruginea showing a great similarity, while 15 compounds were characteristic only for D. grandiflora. The phytochemical composition of methanol extracts...
Evidence-Based Complementary and Alternative Medicine, 2021
Artemisia absinthium L. (Asteraceae) is traditionally used for gastrointestinal ailments and diso... more Artemisia absinthium L. (Asteraceae) is traditionally used for gastrointestinal ailments and disorders linked to numerous risk factors including microbial infections. We aimed to provide contemporary evidence for its ethnopharmacological use and determine its antimicrobial capacity and mode of action, cytotoxicity, and phenolic constituents. Ethnopharmacological survey was conducted using semistructured interviews. Antimicrobial and antibiofilm capacities were determined by microdilution/crystal violet assay, respectively. Modes of action tested include estimation of exopolysaccharide production (congo red binding assay) and interference with membrane integrity (crystal violet uptake and nucleotide leakage assay). Cytotoxicity was determined using crystal violet assay. Polyphenolic profiling was done by advanced liquid chromatography/mass spectrometry (UHPLC-LTQ OrbiTrap MS). Artemisia absinthium in Serbia is traditionally used for gastrointestinal disorders, among others. Further s...
The following study was designed to explore antimicrobial properties of the by-product obtained i... more The following study was designed to explore antimicrobial properties of the by-product obtained in a hydro-distillation process of essential oil from Nepeta nuda L. We strived to develop a novel drink with antimicrobial self-preserving properties based on two components, N. nuda decoct and apple juice. By using 96-well plate microdilution assay it was shown that the N. nuda decoct has antimicrobial potential towards 8 bacterial and 6 fungal species, with the range of minimal inhibitory concentrations 10-300 mg/mL. By using actual food system, such as apple juice, in combination with and without short thermal treatment, we have shown that the decoct of N. nuda can inhibit the growth of food contaminant fungus Penicillium aurantiogriseum. It was determined that 3 volumes of decoct (500 mg/mL) and 22 volumes of apple juice should be mixed in order to obtain self-preserving drink resistant to P. aurantiogriseum contamination. Likewise, when thermal treatment (80 °C for 10 s) is included...
Gentiana pneumonanthe L. (fam. Gentianaceae) is a herbaceous perennial growing on wet heathlands,... more Gentiana pneumonanthe L. (fam. Gentianaceae) is a herbaceous perennial growing on wet heathlands, grasslands and hay meadows of Europe and Asia. Like other Gentiana species, G. pneumonanthe is characterized by the production of bitter secoiridoid glycosides (SGs) widely used in the pharmaceutical industry. Hairy roots (HRs) of G. pneumonanthe, as an alternative source of these phytochemicals, have been induced by agropine type Agrobacterium rhizogenes strain (A40M70GUS), and were characterized by genetic stability, sizable biomass production, and increased biosynthetic capacity compared to the native plant roots and above ground parts. Although HRs of G. pneumonanthe showed intensive growth without the application of phytohormones, the effect of varying concentrations of cytokinins (BAP – benzylaminopurine; KIN – kinetin) on the morphogenesis and on the content of SGs has been investigated in the present study, with the aim of optimizing in vitro conditions for enhanced production o...
The toxicity of bipyridynium herbicide paraquat (PQ) is related to its rapid generation of supero... more The toxicity of bipyridynium herbicide paraquat (PQ) is related to its rapid generation of superoxide in a chain reaction and the production of reactive oxygen species (ROS) in chloroplasts under light conditions. The nature and magnitude of the cellular responses to oxidative stress effects of five paraquat concentrations (0.05-20 μM) on in vitro grown Nepeta panonnica shoots were investigated. The results revealed that relative abundances of Mn SOD and Cu/Zn SOD isoforms changed dose-dependently, whereas catalase (CAT) and peroxidase (POX) activities increased along with paraquat concentrations. Polyphenol oxidase (PPO) activity was progressively inhibited, and, consequently, phenolic compounds were accumulated. Rosmarinic acid (RA) and an unidentified caffeic acid derivative (UCAD) were found to be the most abundant phenolics in shoots of this species. Paraquat was shown to be a potent elicitor that can stimulate accumulation of potent bioactive compounds in Nepeta pannonica, suc...
A plant’s main mechanism to diminish the effects caused by high free radical levels generated dur... more A plant’s main mechanism to diminish the effects caused by high free radical levels generated during high irradiance is the synthesis of various secondary metabolites. In addition to interspecies differences, their concentrations may be influenced by genetic, ontogenic, morphogenetic or environmental factors. We investigated the influence of genetic (genotypes from different natural habitats) and environmental (contrasting light regimes as well as successive parts of the vegetation period) variability on the accumulation of 10 selected phenolic compounds (phenolic acids, flavonoids, and xanthones) in Iris variegata genotypes. Genotypes originated from either sun-exposed or shaded natural habitats were transplanted to two experimental light treatments (high light intensity with a higher R/FR ratio and low light intensity with a lower R/FR ratio). Significant impacts of both genetic and environmental seasonal variability (spring, summer and fall during the vegetation period) on phenol...
Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characteri... more Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characterized by specialized metabolite profiles predominated by iridoids and phenolics, are differentiated according to the stereochemistry of major iridoid aglycone nepetalactone (NL). For the first time, the present study provides a comparative analysis of antimicrobial and immunomodulating activities of the two Nepeta species and their major iridoids isolated from natural sources—cis,trans-NL, trans,cis-NL, and 1,5,9-epideoxyloganic acid (1,5,9-eDLA), as well as of phenolic acid rosmarinic acid (RA). Methanol extracts and pure iridoids displayed excellent antimicrobial activity against eight strains of bacteria and seven strains of fungi. They were especially potent against food-borne pathogens such as L. monocytogenes, E. coli, S. aureus, Penicillium sp., and Aspergillus sp. Targeted iridoids were efficient agents in preventing biofilm formation of resistant P. aeruginosa strain, and they dis...
Phosphinothricin (PPT) is one of the most widely used herbicides. PTT targets glutamine synthetas... more Phosphinothricin (PPT) is one of the most widely used herbicides. PTT targets glutamine synthetase (GS) activity in plants, and its phytotoxicity is ascribed to ammonium accumulation and reactive oxygen species bursts, which drives rapid lipid peroxidation of cell membranes. In agricultural fields, PPT is extensively sprayed on plant foliage; however, a portion of the herbicide reaches the soil. According to the present study, PPT absorbed via roots can be phytotoxic to Arabidopsis, inducing more adverse effects in roots than in shoots. Alterations in plant physiology caused by 10 days exposure to herbicide via roots are reflected through growth suppression, reduced chlorophyll content, perturbations in the sugar and organic acid metabolism, modifications in the activities and abundances of GS, catalase, peroxidase, and superoxide dismutase. Antagonistic interaction of Nepeta rtanjensis essential oil (NrEO) and PPT, emphasizes the existence of complex control mechanisms at the trans...
Centaurium erythraea Rafn produces and accumulates various biologically active specialized metabo... more Centaurium erythraea Rafn produces and accumulates various biologically active specialized metabolites, including secoiridoid glucosides (SGs), which help plants to cope with unfavorable environmental conditions. Specialized metabolism is commonly modulated in a way to increase the level of protective metabolites, such as SGs. Here, we report the molecular background of the wounding-induced changes in SGs metabolism for the first time. The mechanical wounding of leaves leads to a coordinated up-regulation of SGs biosynthetic genes and corresponding JA-related transcription factors (TFs) after 24 h, which results in the increase of metabolic flux through the biosynthetic pathway and, finally, leads to the elevated accumulation of SGs 96 h upon injury. The most pronounced increase in relative expression was detected for secologanin synthase (CeSLS), highlighting this enzyme as an important point for the regulation of biosynthetic flux through the SG pathway. A similar expression patte...
Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As t... more Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As the result of allopolyploid hybridization, offspring may gain different ploidy levels in comparison to parental species, which can provide them instant reproductive isolation. Two tetraploid sister species, Centaurium erythraea and C. littorale, readily cross-fertilize, resulting in hybrids of various ploidy. In northern Serbia, two stable populations of a hexaploid taxon C. pannonicum have been documented. It has been proposed previously that this taxon emerged after an interspecific hybridization event between two tetraploid sister-species: C. erythraea and C. littorale subsp. compressum. The existing populations of the hybridogenic taxon, as well as neighboring populations of the two parental taxa were here characterized by both morphometrics and molecular markers (EST-SSR and trnL-F). Three leaf and two flower characteristics were found to be informative in delimitation of the parenta...
Available scientific literature reports very few microsatellite markers derived from tetraploid g... more Available scientific literature reports very few microsatellite markers derived from tetraploid genomes using de novo transcriptome sequencing, mostly because their gain usually represents a major computational challenge due to complicated combinatorics during assembly of sequence reads. Here we present a novel approach for mining polymorphic microsatellite loci from transcriptome data in a tetraploid species with no reference genome available. Pairs of 114 bp long de novo sequenced transcriptome reads of Centaurium erythraea were merged into short contigs of 170-200 bp each. High accuracy assembly of the pairs of reads was accomplished by a minimum of 14 bp overlap. Sequential bioinformatics operations involved fully free and open-source software and were performed using an average personal computer. Out of the 13 150 candidate contigs harboring SSR motifs obtained in a final output, we randomly chose 16 putative markers for which we designed primers. We tested the effectiveness of...
The main principles of C. pulchellum (Sw.) Druce, secoiridoid glycosides, have been studied as po... more The main principles of C. pulchellum (Sw.) Druce, secoiridoid glycosides, have been studied as potent bioactive compounds. Here we scored their content in extracts of ten populations of this species. Antibacterial and antifungal assays of the extracts and pure secoiridoid glycosides were performed against eight bacterial strains and five fungal species. Methanol extracts from both aerial parts and roots exhibited excellent antibacterial (0.05-0.2 mg mL−1) and very good antifungal (0.1-2 mg mL−1) activity. Pure secoiridoid glycosides isolated from these extracts demonstrated very strong antibacterial (0.01-0.04 mg mL−1) and especially antifungal (0.001-0.1 mg mL−1) activity.
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Papers by Danijela Mišić