Patterns of Genetic Variation of Nepeta nuda L. from the Central Balkans: Understanding Drivers of Chemical Diversity.

Nepeta nuda L., a notable medicinal species in the tradition of the Balkan region, is a rich source of bioactive iridoids and phenolics previously described as high-resolution taxonomical classifiers for the genus Nepeta. However, their potential in investigating intra-species differentiation is here described for the first time. The aim was to recognize the sources of natural chemical diversity and their association with the genetic variability both within and among N. nuda populations in the Central Balkans. Chemical diversity was assessed from methanol extracts and essential oils through untargeted and targeted metabolomics using state-of-the-art analytical tools, covering a broad spectrum of compounds that represent the N. nuda metabolome. We found that chemodiversity primarily resides within populations of N. nuda, and similar results were obtained at the DNA level using microsatellite markers. The low genetic and chemical differentiation of the studied N. nuda populations implies that their metabolomic profiles may be less influenced by geographic distance and variable environmental conditions within the Central Balkans, as they are under the pivotal control of their genetic backgrounds. Screening the distribution of the major bioactive compounds belonging to phenolics (phenolic acids and flavonoids) and iridoids (both aglycones and glycosylated forms), within and among N. nuda populations, is able to guarantee mass spectrometry-based tools for the selection of elite representative genotypes with practical importance. The knowledge acquired will allow us to delve deeper into the molecular background of N. nuda chemical diversity, which is the course of our further work.

Variation in the chemical profiles of three foxglove species in the central Balkans.

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, considered here as complex phenotypes, are further examined along multiple levels of biological organization (intra- and interpopulation) and subsequently subjected to chemometric data analysis. The quantitative composition of the selected set of 16 chemomarkers belonging to the classes of cardenolides (3 compounds) and phenolics (13 compounds) pointed to considerable differences between the taxa studied. D. grandiflora and D. ferruginea were found to be richer in phenolics as compared to cardenolides, which otherwise predominate in D. lanata over other compounds. PCA revealed lanatoside C, deslanoside, hispidulin, and p-coumaric acid to be the main compounds contributing to the differences between D. lanata on one side and D. grandiflora and D. ferruginea on the other, while p-coumaric acid, hispidulin, and digoxin contribute to the diversification between D. grandiflora and D. ferruginea. However, quantitative variation in the metabolite content within species was faint with mild population diversification visible in D. grandiflora and particularly in D. ferruginea. This pointed to the highly conserved content and ratio of targeted compounds within the analyzed species, which was not severely influenced by the geographic origin or environmental conditions. The presented metabolomics approach might have, along with morphometrics and molecular genetics studies, a high information value for further elucidation of the relationships among taxa within the genus Digitalis.

Functional iridoid synthases from iridoid producing and non-producing Nepeta species (subfam. Nepetoidae, fam. Lamiaceae).

Iridoids, a class of atypical monoterpenes, exhibit exceptional diversity within the Nepeta genus (subfam. Nepetoidae, fam. Lamiaceae).The majority of these plants produce iridoids of the unique stereochemistry, with nepetalactones (NLs) predominating; however, a few Nepeta species lack these compounds. By comparatively analyzing metabolomics, transcriptomics, gene co-expression, and phylogenetic data of the iridoid-producing N. rtanjensis Diklic & Milojevic and iridoid-lacking N. nervosa Royle & Bentham, we presumed that one of the factors responsible for the absence of these compounds in N. nervosa is iridoid synthase (ISY). Two orthologues of ISY were mined from leaves transcriptome of N. rtanjensis (NrPRISE1 and NrPRISE2), while in N. nervosa only one (NnPRISE) was identified, and it was phylogenetically closer to the representatives of the Family 1 isoforms, designated as P5ßRs. Organ-specific and MeJA-elicited profiling of iridoid content and co-expression analysis of IBG candidates, highlighted NrPRISE2 and NnPRISE as promising candidates for ISY orthologues, and their function was confirmed using in vitro assays with recombinant proteins, after heterologous expression of recombinant proteins in E. coli and their His-tag affinity purification. NrPRISE2 demonstrated ISY activity both in vitro and likely in planta, which was supported by the 3D modeling and molecular docking analysis, thus reclassification of NrPRISE2 to NrISY is accordingly recommended. NnPRISE also displays in vitro ISY-like activity, while its role under in vivo conditions was not here unambiguously confirmed. Most probably under in vivo conditions the NnPRISE lacks substrates to act upon, as a result of the loss of function of some of the upstream enzymes of the iridoid pathway. Our ongoing work is conducted towards re-establishing the biosynthesis of iridoids in N. nervosa.

Antimicrobial and Immunomodulating Activities of Two Endemic Nepeta Species and Their Major Iridoids Isolated from Natural Sources.

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 displayed additive antimicrobial interaction. Iridoids are, to a great extent, responsible for the prominent antimicrobial activities of the two Nepeta species, although are probably minor contributors to the moderate immunomodulatory effects. The analyzed iridoids and RA, individually or in mixtures, have the potential to be used in the pharmaceutical industry as potent antimicrobials, and in the food industry to increase the shelf life and safety of food products.

Phenotypic and Genetic Variation of an Interspecific Centaurium Hybrid (Gentianaceae) and Its Parental Species.

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 parental taxa and in their discernment from hybrid individuals, the latter having intermediate values. Eight microsatellite markers were found to have good ability to distinguish studied taxa, placing C. pannonicum in closer relationship with C. erythraea. Conversely, trnL-F plastid marker nominated C. littorale subsp. compressum to be the donor of the C. pannonicum plastid DNA. Reproductive isolation of the hexaploid hybrid individuals from the parental species should be examined as the next logical step in describing the new species.

Chemodiversity of two closely related tetraploid Centaurium species and their hexaploid hybrid: Metabolomic search for high-resolution taxonomic classifiers.

Species within the genus Centaurium readily hybridize and polyploid complexes are often seen in natural populations. We describe phytochemical profiles of newly discovered allohexaploid hybrid, here named Centaurium pannonicum, and its parental tetraploid species C. erythraea and rare C. littorale ssp. compressum. Our aim was to examine chemodiversity of these taxa in the area of Vojvodina (North Serbia) and to perform metabolomics search for chemical classifiers which would provide high resolution discrimination of parental and hybrid individuals. In sum, UHPLC-MS/MS Orbitrap metabolomics fingerprinting revealed seventy compounds in methanol extracts. Despite the lack of qualitative chemical novelty in hybrid plants, UHPLC-qqqMS targeted metabolomics approach, aimed at three secoiridoid compounds and seventeen phenolics, pointed to considerable differences in quantitative composition of these dominant compounds among the plant taxa studied. In addition to the difference in the ploidy levels, the hybrid taxon was well distinguished from both parental species based on metabolite profiles, and, for most individuals, positioned intermediately to the parental taxa in both PCA and hierarchical clustering. After optimizing and comparing several statistical learning methods, it was possible to narrow the number of taxonomic classifiers to five (three xanthones, one secoiridoid glycoside, and one phenolic acid), while increasing the differentiation resolution. The presented metabolomics approach will certainly, along with morphometrics and molecular genetics studies, have high impact on further elucidation of complex relationships among taxa within the genus Centaurium.

Simultaneous UHPLC/DAD/(+/-)HESI-MS/MS analysis of phenolic acids and nepetalactones in methanol extracts of Nepeta species: a possible application in chemotaxonomic studies.

INTRODUCTION: Nepeta species contain a variety of secondary metabolites, including iridoid monoterpenes - nepetalactones and phenolic acids - that are considered the main bioactive constituents. This work represents the first attempt to comparatively explore variations in these two major groups of secondary metabolites within the genus. OBJECTIVE: To develop an efficient analytical methodology for simultaneous analysis of nepetalactones and phenolic acids in methanol extracts of selected Nepeta species, and to evaluate its potential application in chemotaxonomic studies. MATERIAL AND METHODS: A UHPLC combined with linear-trap quadrupole (LTQ) orbitrap MS method was used to characterise chemical diversity and complexity of phenolics among 12 selected Nepeta species. A targeted metabolomic approach using UHPLC coupled to a diode array detector (DAD) and combined with (+/-) heated electrospray ionisation (HESI) MS/MS was developed and validated for quantitative analysis of six hydroxycinnamic acid derivatives and four nepetalactones. RESULTS: Phenolic profiling provided a valuable database of bioactive compounds in the plant group studied, including phenolic acids (hydroxybenzoic and hydroxycinnamic acids) and flavonoids (flavones, flavonols and flavanones). Principal component analysis and cluster analysis suggested the applicability of 10 targeted compounds as chemomarkers for chemotaxonomic studies. Pearson's correlation analysis revealed significant positive correlations between metabolites involved in different biosynthetic pathways (phenylpropanoid or monoterpenoid). CONCLUSION: The described targeted metabolomic approach proved to be highly beneficial in designing a phytochemical overview of the genus Nepeta, and might have applications in further clarification of phylogenetic relations. Furthermore, it has the potential to be implemented in a routine quality control of plant material and herbal preparations.

Centauries as underestimated food additives: antioxidant and antimicrobial potential.

Methanol extracts of aerial parts and roots of five centaury species (Centaurium erythraea, C. tenuiflorum, C. littorale ssp. uliginosum, C. pulchellum, and Schenkia spicata) were analysed for their main secondary metabolites: secoiridoid glycosides, a group of monoterpenoid compounds, and phenolics (xanthones and flavonoids), and further investigated for antioxidant capacity and antimicrobial activity. The results of ABTS, DPPH, and FRAP assays showed that above ground parts generally displayed up to 13 times higher antioxidant activity compared to roots, which should be related to higher phenolics content, especially flavonoids, in green plant organs. Secoiridoid glycosides showed no antioxidant activity. All the tested extracts demonstrated appreciative antibacterial (0.05-0.5 mg ml(-1)) and strong antifungal activity (0.1-0.6 mg ml(-1)). Our results imply that above ground parts of all centaury species studied, could be recommended for human usage as a rich source of natural antioxidants and also in food industry as strong antimicrobial agents for food preservation.

Sugars and acid invertase mediate the physiological response of Schenkia spicata root cultures to salt stress.

A heterotrophic model system was established in our studies in order to differentiate the effect of high salt concentrations in external medium on growth and sugar metabolism in roots from the effect of reduced sugar availability resulting from decreased photosynthesis under salinity. Soluble sugar content and the activity of acid invertase in root cultures of salt-tolerant (ST) and salt-sensitive (SS) Schenkia spicata (L.) Mansion genotypes were investigated during exposure to different NaCl concentrations (0-200 mM). Their response to severe salinity was characterized by a metabolic adjustment that led to the accumulation of sucrose (Suc) in root tissues. There was clear evidence that cell wall invertase (CW-Inv) is the major contributor to the Suc/hexose ratio in roots during exposure to elevated salinity. The results of CW-Inv activity and immunodetection assays in our study suggest that the regulation of CW-Inv expression is most likely achieved in a salt stress dependent manner. Also, NaCl modulated soluble acid invertase (SA-Inv) expression differentially in SS and ST genotypes of S. spicata. Regardless of the salt treatment, genotype, or the amount of enzyme, SA-Inv activity was generally low, indicating regulation at the posttranslational level. The results suggest no direct role of SA-Inv in the regulation of the root tissue carbohydrate pool and therefore in the control of the availability of glucose and fructose for the primary metabolism and/or osmotic adjustment in the present heterotrophic model system.