A Geometric Morphometrics Approach to the Study of Natural Variations and Hybrid Detection in Populations of Alnus incana (L.) Moench and Alnus rohlenae Vít, Douda and Mandák.

Landmark-based geometric morphometrics (GM) was used to examine, for the first time, spontaneous hybridization between Alnus incana (L.) Moench and Alnus rohlenae Vít, Douda and Mandák, and to assess inter- and intrapopulation variability in leaf shape, leaf size and venation in natural populations in Serbia (Western Balkans). Two geographically distant (30 km) and two close (1.2 km) populations were selected to examine hybridization. The variability in leaf shapes was assessed by canonical variate analysis and linear discriminant analysis performed on the symmetric component of variation. Covariation between the symmetric component of shape variation and the number of pairs of secondary leaf veins was investigated with partial least squares analysis. Static allometry was examined for the first time in the genus Alnus Mill. A higher proportion of A. incana leaves was classified as A. rohlenae in geographically close populations, which is in accordance with the hypothesis about spontaneous hybridization. No single leaf of A. rohlenae was classified as A. incana, indicating that putative hybrids can only be found in grey alder populations. This study demonstrates that GM is a powerful tool for species delimitation and hybrid detection in the genus Alnus and it can be used for preliminary screening in hybrid zones.

Population-Related Variability in Qualitative and Quantitative Secondary Metabolite Profile of Gentianella austriaca (A. & J. Kern.) Holub.

Phytochemical profiling of six natural populations of Gentianella austriaca was performed by HPLC identification and quantification of a number of secondary metabolites, and evaluation of time series of peak areas by chemometric analysis. Phytochemical analysis of G. austriaca revealed the presence of iridoids, flavone-C-glucosides and xanthones. Twelve secondary metabolites were identified in the aerial parts, roots and seeds, including swertiamarin (SWM), gentiopicrin (GP), sweroside (SWZ), isoorientin (ISOOR), swertisin (SWE), demethylbellidifolin-8-O-glucoside (DMB-8-O-glc), bellidifolin-8-O-glucoside (BDF-8-O-glc), mangiferin (MGF), corymbiferin (CBF), corymbiferin-1-O-glucoside (CBF-1-O-glc), bellidifolin (BDF) and campestroside. Multivariate statistical analyses showed relatively low variability among populations according to secondary metabolite content. However, some pharmacologically important compounds were found in higher amounts in a few populations, which could be useful for conservation and future biotechnological procedures.

Ecophysiological and Growth-Related Traits of Two Geophytes Three Years after the Fire Event in Grassland Steppe.

Deliblato Sands is the single largest expanse of sand in Europe. It is the most fire-prone area of Serbia due to the absence of surface water, sandy soils, specific microclimate conditions, and vegetation composition. Post-fire regeneration is a long-term process that includes many aspects of vegetation regrowth and habitat recovery. In the third year following one of the disastrous fires, the growth dynamics of two geophyte species in unburned and burned sites were studied. During the growing season, burned and unburned populations of Crocus reticulatus Steven ex Adam and Iris pumila L. were assessed for growth parameters (biomass production, specific leaf area, leaf area index) and leaf-level ecophysiological traits (photosystem II efficiency, chlorophyll amount, relative water content). Species acclimated differently to changed abiotic and biotic site conditions after the fire event. C. reticulatus burned and unburned populations differed significantly in terms of flowering phenology and ecophysiological traits, whereas I. pumila burned and unburned populations differed significantly in terms of growth parameters. The findings support the assertion that geophytes are generally well adapted to environmental disturbances. Species, however, responded differently to fire-induced changes in the physicochemical and biotic environment, depending on their ecological requirements and adaptive capacity.

Gentiana asclepiadea L. from Two High Mountainous Habitats: Inter- and Intrapopulation Variability Based on Species' Phytochemistry.

Natural populations of Gentiana asclepiadea L., located at two mountainous sites, were HPLC-analyzed regarding the contents of six representative secondary metabolites. The contents of swertiamarin (SWM), gentiopicrin (GP), sweroside (SWZ), mangiferin (MGF), isoorientin (ISOOR), and isovitexin (ISOV) were determined in six populations (three per study site), and separately for aboveground and belowground plant parts. PCA showed a clear separation of four groups according to the contents of the analyzed secondary metabolites. Out of six analyzed compounds, five were present in all samples and only one (SWZ) was found in Golija populations (belowground parts) but not in Vlasina populations, and its presence can be indicative of the geolocation of populations. Clear separation of groups was mostly affected by the different contents of chemical compounds in plant parts (aboveground versus belowground) and by the differences related to population origin (higher content of SWM and GP in belowground parts of individuals from Vlasina populations and higher content of MGF and ISOOR of individuals from Golija populations). The results of this study contribute to the spatiochemical profiling of G. asclepiadea populations and a better understanding of inter- and intrapopulation variability of pharmacologically important compounds.

Composition, Antioxidant Potential, and Antimicrobial Activity of Helichrysum plicatum DC. Various Extracts.

Helichrysum plicatum DC. is widely used in folk medicine in treating a variety of health disorders. The aim of this study was to examine the influence of different extraction solvents on the chemical composition, antioxidant potential, and antimicrobial activities of H. plicatum. Aerial parts were separately extracted with ethanol, dichloromethane, and sunflower oil. The oil extract (OE) was re-extracted with acetonitrile. A total of 142 compounds were tentatively identified in ethanolic (EE), dichloromethane (DCME), and acetonitrile (ACNE) extracts using HPLC-DAD/ESI-ToF-MS. The dominant compound class in all extracts were α-pyrones, alongside flavonoids in EE, terpenoids in DCME and ACNE, and phloroglucinols in DCME. The antioxidant potential of the extracts was assessed by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay. EE and DCME possessed the most potent radical scavenging capacity. Antimicrobial activity was investigated on eight bacterial, two yeast, and one fungal species. All extracts exhibited high antifungal and notable antibacterial activities compared to control substances, with DCME being the most potent. DCME exhibited stronger antimicrobial activity against P. aeruginosa than the standard chloramphenicol.

Chemical and Morphological Inter- and Intrapopulation Variability in Natural Populations of Gentiana pneumonanthe L.

Inter- and intrapopulation variability in six natural populations of the rare species Gentiana pneumonanthe was examined based on morphological and chemical data. Population size and linear morphometric parameters differed significantly among populations, but without a clear connection to habitat conditions, i. e. water supply and light availability. Leaf shape varied from ovate to lanceolate in all populations, and one population was distinctive in having the largest number of leaves of transitional shape. HPLC analyses of six secondary metabolites were performed separately for belowground parts, and aboveground vegetative and reproductive parts of individual plants (6 populations ×7 individuals ×3 plant parts, n=126) in order to examine differences at the population and individual levels. Three secoiridoids (swertiamarin (SWM), sweroside (SWZ), and gentiopicrin (GP)), one xanthone (mangiferin (MGF)), and two flavones (isoorientin (IO) and isovitexin (IV)) were detected and quantified in the analyzed samples: sweroside dominated in the aboveground reproductive part, mangiferin in the aboveground vegetative part, and gentiopicrin in the belowground part. At the population level, differences in contents of the analyzed chemicals among populations were significant only for a few metabolites. At the individual level, a pronounced organ-dependent distribution of secondary metabolites was revealed. The results of this study contribute to a better understanding of natural variability within populations of the rare and threatened G. pneumonanthe, and provide data on the contents and within-plant distribution of secondary metabolites, which are important as pharmacologically active compounds and may be useful for further biotechnological procedures regarding this species.

Bioactive Pentacyclic Triterpene Ester Derivatives from Alnus viridis ssp. viridis Bark.

Seven derivatives of pentacyclic triterpene acids (1-7) were isolated from the bark of Alnus viridis ssp. viridis using a combination of column chromatography and semipreparative HPLC. Compounds 1-3, 6, and 7 were determined to be new after spectroscopic data interpretation and were assigned as 27-hydroxyalphitolic acid derivatives (1-3), a 27-hydroxybetulinic acid derivative (6), and a 3-epi-maslinic acid derivative (7), respectively. Pentacyclic triterpenoids with a C-27 hydroxymethyl group have been found in species of the genus Alnus for the first time. These compounds were subjected to cytotoxicity testing against a number of cancer cell lines. Also, selected pentacyclic triterpenoids were selected as potential inhibitors of topoisomerases I and IIα for an in silico investigation.

Chemical Composition and Interpopulation Variability of Essential Oils of Taxus baccata L. from Serbia.

The composition of the essential oil of the twigs and needles of Taxus baccata L. from three natural populations in Serbia has been determined by GC/MS analysis. Of the 91 detected compounds, 87 were identified. The most abundant compound classes were aliphatic alcohols, terpenes, aliphatic hydrocarbons, and aliphatic aldehydes, which together comprised ca. 86.92% of the total oil composition. The dominant constituents were oct-1-en-3-ol (23.48%), (3Z)-hex-3-en-1-ol (11.46%; aliphatic alcohols), and myrtenol (11.38%; oxygenated monoterpene). The PCA of 22 selected compounds revealed differentiations of populations based on geographic distribution. The CA showed that Populations I and II from the Dinaric Alps were similar, and that Population III from the Balkan mountain system was distinct. This was the first investigation of interpopulation variability of T. baccata populations based on essential oil composition. The results of this study were compared with those of previous studies concerning volatile compounds produced by Taxus species. The results indicate that the essential oil content of T. baccata populations from this study is unique, mostly resembling the population from southeast Serbia.

Essential-Oil Variability in Natural Populations of Pinus mugo Turra from the Julian Alps.

The composition and variability of the terpenes and their derivatives isolated from the needles of a representative pool of 114 adult trees originating from four natural populations of dwarf mountain pine (Pinus mugo Turra) from the Julian Alps were investigated by GC-FID and GC/MS analyses. In total, 54 of the 57 detected essential-oil components were identified. Among the different compound classes present in the essential oils, the chief constituents belonged to the monoterpenes, comprising an average content of 79.67% of the total oil composition (74.80% of monoterpene hydrocarbons and 4.87% of oxygenated monoterpenes). Sesquiterpenes were present in smaller amounts (average content of 19.02%), out of which 16.39% were sesquiterpene hydrocarbons and 2.62% oxygenated sesquiterpenes. The most abundant components in the needle essential oils were the monoterpenes δ-car-3-ene, ß-phellandrene, α-pinene, ß-myrcene, and ß-pinene and the sesquiterpene ß-caryophyllene. From the total data set of 57 detected compounds, 40 were selected for principal-component analysis (PCA), discriminant analysis (DA), and cluster analysis (CA). The overlap tendency of the four populations suggested by PCA, was as well observed by DA. CA also demonstrated similarity among the populations, which was the highest between Populations I and II.

Ethnobotany and herbal medicine in modern complementary and alternative medicine: An overview of publications in the field of I&C medicine 2001-2013.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnobotanical knowledge and traditional medicinal practices from different parts of the world are of global importance and documentation of ethnobotanical, and ethnopharmacological data is a key prerequisite for further research in the area of herbal medicine and its implementation in clinical practice. AIMS: An attempt was made to evaluate the scientific output of research related to ethnobotany and herbal medicine in journals indexed in the subject area "Integrative and Complementary Medicine" in the period 2001-2013, in order to ascertain research trends in both subdisciplines. MATERIALS AND METHODS: All articles related to ethnobotany and herbal medicine, extracted from journals included in the field of I&C Medicine and published in the period 2001-2013, have been analyzed for general bibliometric data, and specific data: ethnobotanical data (geographic, floristic, pharmacological, sociological and other relevant data) and phytotherapeutic data (type of applied herbal medicine, plant species studied, pharmacological activity of studied plant species and disease and disorder type studied on a particular model). RESULTS: In the studied period, the number of articles dealing with ethnobotany and herbal medicine increased 6.3-fold. Articles related to ethnobotanical studies documented medicinal flora from 81 countries, either giving an overview of overall medicinal flora, or presenting the ethnomedicinal aspect of the use of plants for the treatment of ailments typical to the studied area. Additionally, the authors provided significant information on the methods of use and herbal preparations. In herbal medicine studies, plants, traditional plant remedies, herbal medicinal products and active herbal compounds were tested for many of pharmacological activities (146), with the curative activity emerging as most frequently tested. Out of 39 model systems, most of the studies were carried out under controlled in vitro conditions (4589 articles), followed by rat in vivo (2320), human in vivo (1285), mouse in vivo (955), and on agents of pathogenic diseases (887); more than 800 medical disorders were treated. CONCLUSIONS: The study revealed the regions most studied for new records of floristic and ethnomedicinal diversity, the most frequently studied plant species, and the most promising therapeutic indications for the integration of herbal remedies in the curative process, as ascertained from the selected bibliographic databases.