Isolation of Secondary Metabolites from Achillea grandifolia Friv. (Asteraceae) and Main Compounds' Effects on a Glioblastoma Cellular Model.

This study aims at the isolation and structural determination of the secondary metabolites of the herbaceous perennial plant Achillea grandifolia Friv. (Asteraceae). The examination of the non-volatile content of the leaves and flowers of A. grandifolia afforded the isolation of sixteen secondary metabolites. On the basis of NMR spectra, the identified compounds included ten sesquiterpene lactones; three guaianolides-rupicolin A (1), rupicolin B (2), and (4S,6aS,9R,9aS,9bS)-4,6a,9-trihydroxy-9-methyl-3,6-dimethylene-3a,4,5,6,6a,9,9a,9b-octahydro-3H-azuleno [4,5-b]furan-2-one (3); two eudesmanolides-artecalin (4) and ridentin B (5); two sesquiterpene methyl esters-(1S,2S,4αR,5R,8R,8αS)-decahydro-1,5,8-trihydroxy-4α,8-dimethyl-methylene-2-naphthaleneacetic acid methylester (6) and 1ß, 3ß, 6α-trihydroxycostic acid methyl ester (7); three secoguaianolides-acrifolide (8), arteludovicinolide A (9), and lingustolide A (10); and an iridoid-loliolide (11). Moreover, five known flavonoids, i.e., apigenin, luteolin, eupatolitin, apigenin 7-O-glucoside, and luteolin 7-O-glucoside (12-16) were also purified from the aerial parts of the plant material. We also investigated the effect of rupicolin A (1) and B (2) (main compounds) on U87MG and T98G glioblastoma cell lines. An MTT assay was performed to define cytotoxic effects and to calculate the IC50, while flow cytometry was employed to analyze the cell cycle. The IC50 values of reduced viability during the 48 h treatment for compound (1) and (2) were 38 µM and 64 µM for the U87MG cells and 15 µM and 26 µM for the T98G cells, respectively. Both rupicolin A and B induced a G2/M cell cycle arrest.

Ecological Preferences and Diversity of Essential Oil Composition in Endangered Wild-Growing Populations of Sideritis sipylea Boiss. (Lamiaceae) of the East Aegean Islands (Greece): Evidencing Antioxidant Potential, Antimicrobial and Cytotoxic Activities.

Plants from the genus Sideritis (Lamiaceae) have been widely used in folk medicine for a long time and consequently are a focus of the scientific community. Despite this interest, explicit data about the essential oils (EOs) of the Endangered Sideritis sipylea have not been readily available to date. In this study, we investigated the ecological preferences of Greek S. sipylea and the chemical composition of the essential oils of wild-growing S. sipylea populations from two Greek islands (S1: Samos, S2: Lesvos); we explored concomitant associations with environmental factors; and we assessed their (i) antioxidant potential (two tests), (ii) antimicrobial activity against six microbial and two fungal strains, and (iii) cytotoxic effect in two human and one murine cell lines. We compiled an ecological profile in R based on all known Greek localities of S. sipylea, outlining for the first time its preferences regarding temperature (3.48 ± 1.53 °C to 30.70 ± 1.11 °C) and the precipitation regimes (5.92 ± 2.33 mm to 136 ± 11.43 mm) shaping its natural occurrence in the wild. The chemical analysis (42 compounds in total) confirmed the domination of monoterpene hydrocarbons in both samples (with quantitative and qualitative differences) and identified 12 new constituents reported in S. sipylea for the first time (e.g., Bicyclogermacrene and Cumacrene). Dominant compounds in S1 (39 constituents) were ß-Myrcene (20.4%) followed by ß-caryophyllene (11.8%), bicyclogermacrene (7.1%), ß-pinene (6.3%), carvacrol (6.2%) and α-terpinene (6.1%), whereas in S2 (26 constituents) the main ones were α-pinene (37.3%), ß-pinene (15.1%) and sabinene (12.1%), followed by ß-caryophyllene (5.6%) and bicyclogermacrene (5.5%). The strong antioxidant capacity and cytotoxic activity of S. sipylea EOs are reported herein for the first time, while new insight is provided regarding their effect on bacterial and fungal strains (four ones originally tested herein). The biological activity analysis demonstrated variation among samples, with S2 being more potent than S1. Altogether, the results of the present study demonstrate the high biological potential of S. sipylea EOs with an interesting antioxidant capacity and antimicrobial and cytotoxic effects and reveal associations of natural chemodiversity with climatic factors.

Exploring the Ecological Preferences and Essential Oil Variability in Wild-Growing Populations of the Endangered Local Greek Endemic Thymus holosericeus (Lamiaceae).

Thymus holosericeus Celak. (Lamiaceae) is a taxonomically isolated and endangered local endemic thyme species which is geographically isolated in four Ionian Islands (West Greece). The present study investigated the essential oil (EO) composition, the ecological preferences, and their correlations in three T. holosericeus wild-growing populations from Zakynthos (ΤH-Z), Cephalonia (ΤH-C) and Lefkada (ΤH-L). The variations in essential oil yield and the composition of T. holosericeus populations were evaluated using hydrodistillation, GC/MS, TLC and NMR analysis. The climatic conditions of each sample were organized and analyzed in RStudio with the raster package and in SPSS with Pearson's Canonical Correlation Analysis (CCA), respectively. The aerial parts of the plants varied in EO yields from 1.92 to 2.28% w/v. The analysis of EO constituents revealed noteworthy qualitative and quantitative inter-population variation. The composition of EOs revealed the presence of linalool (82.77%) and borneol (5.95%) as major compounds in ΤH-Z, while carvacrol (35.34%), geraniol (23.98%), linalool (14.37%), borneol (5.66%), thymol (4.27%) and p-cymene (4.08%) were the main compounds in ΤH-C and linalool (40.37%), geraniol (39.42%) and borneol (5.20%) were dominant components in ΤH-L. The results of the gas chromatography procedure have also been confirmed by 1H and 13C-NMR and TLC analysis. The ecological profile showed an average annual precipitation of 942 ± 18.33 mm and the temperature limits in which T. holosericeus seems to adapt to are 6.80± 1.08 °C 27.70 ± 0.70 °C. Regarding the examined samples, TH-C was adapted to the driest summer and coldest winter conditions, TH-Z was adapted to the lowest annual precipitation with the most complex climatic conditions, and TH-L was adapted to the highest summer temperatures with the lowest precipitation in the wettest period of the year. For each sampled population, the CCA identified the association of the samples' EOs composition with the prevailing local environmental conditions.

Metabolomic Fingerprinting and Molecular Characterization of the Rock Samphire Germplasm Collection from the Balkan Botanic Garden of Kroussia, Northern Greece.

The traditionally edible aerial parts of rock samphire (Crithmum maritimum L.) could be a valuable functional food or feed ingredient due to their high antioxidant capacity, ascorbic acid content, and rich content in secondary metabolites such as phenolics and flavonoids. The first objective of this study was to evaluate eighteen genotypes derived from different regions of Greece regarding the phytochemical contents of their soluble extracts in total phenolics, total flavonoids, and individual polyphenols as determined by LC-MS analysis, as well as ascorbic acid content and their antioxidant capacity as determined by different assays, including ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity), and FRAP (ferric reducing antioxidant power) assays. The second objective of the study was the molecular characterization of native Greek C. maritimum genotypes. Great variation among genotypes was observed in terms of the antioxidant capacity, ascorbic acid content, and phenolic compounds (total phenolic content and total flavonoid content), as well as in caffeolquinic acids and flavonoids. The principal component analysis highlighted genotypes with a higher potential in antioxidants and polyphenolics. The most promising genotypes were G9 from Kefalonia, followed by G4 from Ikaria, where both clearly exhibited a similar response with high values of evaluated traits. The molecular characterization of genotypes revealed low variability and low to moderate genetic diversity between populations. Our data indicated that the rock samphire germplasm collection from the Balkan Botanic Garden of Kroussia could serve as an important source of documented genetic material and, thus, it is suggested for further investigation to provide insight regarding cultivation and agro-processing aspects, artificial selection, or plant breeding aimed at developing C. maritimum genotypes of high-bioactive value.

Spatial and Ecological Drivers of Genetic Structure in Greek Populations of Alkanna tinctoria (Boraginaceae), a Polyploid Medicinal Herb.

Background and Aims: Quantifying genetic variation is fundamental to understand a species' demographic trajectory and its ability to adapt to future changes. In comparison with diploids, however, genetic variation and factors fostering genetic divergence remain poorly studied in polyploids due to analytical challenges. Here, by employing a ploidy-aware framework, we investigated the genetic structure and its determinants in polyploid Alkanna tinctoria (Boraginaceae), an ancient medicinal herb that is the source of bioactive compounds known as alkannin and shikonin (A/S). From a practical perspective, such investigation can inform biodiversity management strategies. Methods: We collected 14 populations of A. tinctoria within its main distribution range in Greece and genotyped them using restriction site-associated DNA sequencing. In addition, we included two populations of A. sieberi. By using a ploidy-aware genotype calling based on likelihoods, we generated a dataset of 16,107 high-quality SNPs. Classical and model-based analysis was done to characterize the genetic structure within and between the sampled populations, complemented by genome size measurements and chromosomal counts. Finally, to reveal the drivers of genetic structure, we searched for associations between allele frequencies and spatial and climatic variables. Key Results: We found support for a marked regional structure in A. tinctoria along a latitudinal gradient in line with phytogeographic divisions. Several analyses identified interspecific admixture affecting both mainland and island populations. Modeling of spatial and climatic variables further demonstrated a larger contribution of neutral processes and a lesser albeit significant role of selection in shaping the observed genetic structure in A. tinctoria. Conclusion: Current findings provide evidence of strong genetic structure in A. tinctoria mainly driven by neutral processes. The revealed natural genomic variation in Greek Alkanna can be used to further predict variation in A/S production, whereas our bioinformatics approach should prove useful for the study of other non-model polyploid species.

Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites.

Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of certain plants from the Boraginaceae family such as Lithospermum spp. and Alkanna spp. They possess antimicrobial, anti-tumoral and wound healing properties. The production of secondary metabolites by Alkanna tinctoria might be influenced by its endomicrobiome. To study the interaction between this medicinal plant and its bacterial endophytes, we isolated bacteria from the roots of wild growing Alkanna tinctoria collected near to Athens and Thessaloniki in Greece. Representative strains selected by MALDI-TOF mass spectrometry were identified by partial 16S rRNA gene sequence analysis. In total, 197 distinct phylotypes of endophytic bacteria were detected. The most abundant genera recovered were Pseudomonas, Xanthomonas, Variovorax, Bacillus, Inquilinus, Pantoea, and Stenotrophomonas. Several bacteria were then tested in vitro for their plant growth promoting activity and the production of cell-wall degrading enzymes. Strains of Pseudomonas, Pantoea, Bacillus and Inquilinus showed positive plant growth properties whereas those of Bacteroidetes and Rhizobiaceae showed pectinase and cellulase activity in vitro. In addition, bacterial responses to alkannin and shikonin were investigated through resistance assays. Gram negative bacteria were found to be resistant to the antimicrobial properties of A/S, whereas the Gram positives were sensitive. A selection of bacteria was then tested for the ability to induce A/S production in hairy roots culture of A. tinctoria. Four strains belonging to Chitinophaga sp., Allorhizobium sp., Duganella sp., and Micromonospora sp., resulted in significantly more A/S in the hairy roots than the uninoculated control. As these bacteria can produce cell-wall degrading enzymes, we hypothesize that the A/S induction may be related with the plant-bacteria interaction during colonization.