Mirko Ruščić

Antiphytoviral activity of sesquiterpene-rich essential oils from four croatian teucrium species.

The purpose of this study was to compare the essential oil profiles of four Croatian Teucrium species (Lamiaceae), as determined by GC and GC/MS, with their antiphytoviral efficiency. A phytochemical analysis showed that T. polium, T. flavum, T. montanum and T. chamaedrys are characterized by similar essential oil compositions. The investigated oils are characterized by a high proportion of the sesquiterpene hydrocarbons β-caryophyllene (7.1–52.0%) and germacrene D (8.7–17.0%). Other important components were β-pinene from T. montanum and α-pinene from T. flavum. The investigated essential oils were proved to reduce lesion number in the local host Chenopodium quinoa Willd. infected with Cucumber Mosaic Virus (CMV), with reductions of 41.4%, 22.9%, 44.3% and 25.7%, respectively.

Chemical composition and antimicrobial activity of the essential oil of endemic Dalmatian black pine (Pinus nigra ssp. dalmatica).

The chemical composition and the antimicrobial activity of the essential oil isolated from the needles of endemic Dalmatian black pine (Pinus nigra ssp. dalmatica) from Croatia were investigated. The chemical composition of the essential oil was determined by GC and GC/MS analyses, and the main compounds identified were α‐pinene, β‐pinene, germacrene D, and β‐caryophyllene. Disc‐diffusion and broth‐microdilution assays were used for the in vitro antimicrobial screening. The Dalmatian black pine essential oil exhibited a great potential of antibacterial activity against Gram‐positive bacteria (MIC=0.03–0.50% (v/v)) and a less pronounced activity against Gram‐negative bacteria (MIC=0.12–3.2% (v/v)). The volatile compounds also inhibited the growth of all fungi tested, including yeast.

Phytochemical and Cytogenetic Characterization of Centaurea solstitialis L. (Asteraceae) from Croatia

The cytogenetic characterization of Centaurea solstitialis L. (Asteraceae) showed a chromosome number of 2n = 16. Karyotype is composed by four pairs of metacentric, two pairs of submetacentric and two pairs of subtelocentric chromosomes. Physical mapping of two rDNA probes revealed two loci of 35S and one locus of 5S rRNA genes. Chromomycin fluorochrome banding revealed that all rDNA loci were GC rich. The genome size (2C‐value) of 1.95 pg classes this species in the group of very small genomes. Chemical composition of C. solstitialis volatile oil (VO) from Croatia, studied with gas chromatography–mass spectrometry showed dominant components as it follows: hexadecanoic acid, α‐linolenic acid, germacrene D and heptacosane. Antioxidant capacity, measured by ferric reducing power assay and 2,2‐diphenyl‐1‐picrylhydrazyl methods, as well as inhibition of acetyl‐ and butyrylcholinesterase of VO was lower comparing to a standard solutions. Volatile oil tested with disc diffusion method showed good inhibitory potential against Pseudomonas aeruginosa, Escherichia coli and all tested fungi: Candida albicans, Penicillium funiculosum and Aspergillus fumigatus. The microdilution method showed best activity against Chronobacter sakazakii and A. fumigatus.

Micromorphological traits and essential oil contents of Micromeria kerneri Murb. and M. juliana (L.) Benth. (Lamiaceae)

The chemical composition of the essential oil (analysed by GC and GC-MS), the types and distribution of trichomes and pollen morphology (analysed by scanning electron microscopy) were investigated in two closely related species, Micromeria kerneri Murb. and Micromeria juliana (L.) Benth. (Lamiaceae) from Southeast Europe as a contribution to their taxonomy. The essential oil of M. kerneri was characterized by a high concentration of oxygenated sesquiterpenes, with caryophyllene-oxide as the major compound. Caryophyllene-oxide was also the major component of the essential oil of M. juliana from all localities, except from Mt Krivošije (Montenegro), where piperitone oxide was the major constituent. Non-glandular trichomes, peltate trichomes, and two types of capitate trichomes (type 1 composed of one basal epidermal cell, and one head cell with subcuticular space; type 2 composed of one basal epidermal cell, two stalk cells, and one head cell with subcuticular space) were observed on leaves, the calyx and on the stem. Pollen of both species had six apertures (hexacolpate) set in the equatorial pollen belt (zonocolpate) and showed medium reticulate ornamentation. Multivariate analysis (PCA and UPGMA) of essential oil components clearly separated the investigated M. kerneri and M. juliana populations, and confirmed the opinion that they are different taxa. On the other hand, micromorphological traits between these species were the same. Nevertheless, definitive conclusions about the taxonomic relationships among these species will require genetic analysis.

Composition and antiviral activity of the essential oils of Eryngium alpinum and E. amethystinum.

The chemical compositions of the essential oils obtained by hydrodistillation of the aerial parts of Croatian Eryngium alpinum L. and E. amethystinum L. were characterized by GC‐FID and GC/MS analyses. The main components identified were the sesquiterpene β‐caryophyllene (19.7%) in the oil of E. amethystinum and the oxygenated sesquiterpene caryophyllene oxide (21.6%) in the oil of E. alpinum. Overall, 32 and 35 constituents were detected in the essential oils of the aerial parts of E. alpinum and E. amethystinum, respectively, representing 92.4 and 93.1% of the total oil compositions. The essential oils of both Eryngium species were proved to reduce the number of lesions in the local host Chenopodium quinoa infected with cucumber mosaic virus and an associated satellite. This is the first investigation of antiphytoviral activity of essential oils of Eryngium species.

LC-MS profiling of glucosinolates in the seeds of Brassica elongata Ehrh., and of the two stenoendemic B. botteri Vis and B. cazzae Ginzb. & Teyber.

Abstract The glucosinolates (GLs) present in seed extracts of Brassica elongata Ehrh., B. botteri Vis and B. cazzae Ginzb. & Teyber from Croatia were identified by LC–MS. 4-Hydroxyindol-3-ylmethyl GL (3) was the major GL in the seeds of B. elongata, along with the four minor GLs 2-(R)-hydroxy-3-butenyl- (1), 3-butenyl- (2), 4-pentenyl- (4) and indol-3-ylmethyl (5). The seeds of B. botteri (Vis island) and B. cazzae (Sušac island) contained 2 as the major GL as well as 1, 3, 5 and 4-methoxyindol-3-ylmethyl GL (6). However, the GLs in B. botteri (Palagruža island) differed from other varieties having 2-propenyl GL (7) as the major GL in the seeds, and the four minor GLs 2, 3, 5 and 6. This first report of the GL content in the seeds of B. elongata, B. botteri and B. cazzae indicates that the unique GL profiles could be specific to the geographical origin of the plant.

Diversity, knowledge and spatial distribution of the vascular flora of Croatia

The Balkan Peninsula is recognized as an important centre of plant diversity. Despite the fact that Croatia contains more than 50% of all Balkan species of vascular flora, the knowledge of the spatial distribution, α-diversity and relation to the conservation efforts have never been summarized and presented. A spatial analysis was performed on several data-sets containing a number of records and a number of species per grid cell. Results show that the Croatian flora consists of 4507 species and 1159 subspecies. The residuals around the linear regression used as a measure of the species richness indicate that Croatia had the highest residual value among the 40 European countries sampled and highest stress on the biodiversity value of the Apennine, the Iberian and the Balkan peninsulas. On the basis of half a million findings and their spatial distributions, we observed that within one grid cell (35 km2) 542 species could be expected. A number of records based on the herbarium specimens, literature and field observation and related spatial distribution were discussed. The spatial distribution of α-diversity indicates that the national hot spots were more consistent with the important plant areas network than with the network of officially protected areas.

Glucosinolates, volatile constituents, and acetylcholinesterase inhibitory activity of Alyssoides utriculata

Glucosinolates (GLs) and volatiles from all plant parts of Alyssoides utriculata (L.) Medik were determined for the first time. Total GLs determined by ferricyanide assay were 45.7, 12.2, 7.3, 12.5 and 15.7 μmol/g dry weight in the seed, root, stem, leaf and flower, respectively. Qualitative determination of individual GLs was done indirectly by GC/MS analysis of volatiles isolated by hydrodistillation, as well as by CH2Cl2 extraction after enzyme hydrolysis. The extracts were shown to contain mostly volatiles originating from degradation of gluconapin, glucoerucin, and glucoraphanin. The volatile isolates, tested for their acetylcholinesterase inhibition according to Ellman method, were dose-dependent reaching 53.9% and 53.3% at the final concentration of 320 μg/mL, for seed CH2Cl2 extract and hydrodistillate of the whole plant, respectively.

Ammi visnaga (L.) Lam. (Apiaceae), a new taxon in Croatian flora

Ammi visnaga (L.) Lam. (Apiaceae), a new taxon in Croatian flora During floristic research into the island of Brač (Dalmatia, Croatia) in 2010, Ammi visnaga (L.) Lam. (Apiaceae), a new neophyte for Croatia was found in several localities and natural habitats. At the altitude of 380-460 m above sea level, mostly in habitats disturbed by humans, the located populations were composed of numerous and vital specimens in blooms and with fruits. The gradual and successful integration of this species into the natural vegetation was noticed, particularly in grasslands of the association Brachypodio retuso-Trifolietum stellati Horvatić 1958 andmacchia of the association Fraxino orno-Quercetum ilicis Horvatić (1956) 1958.

The Chrysopogono grylli-Nerietum oleandri association in Croatia as compared with other Rubo ulmifolii-Nerion oleandri communities (Tamaricetalia, Nerio-Tamaricetea) in the Mediterranean

Abstract A new association, Chrysopogono grylli-Nerietum oleandri, situated in the south of Croatia, is proposed. According to its floristic structure, physiognomy and ecology, it belongs to the Rubo ulmifolii-Nerion oleandri alliance (Tamaricetalia, Nerio-Tamaricetea). It is the only association with Nerium oleander in Croatia. This community has developed along a temporary watercourse as a vegetation exclave (extrazonal stand) within the climazonal evergreen forest vegetation of the Oleo-Ceratonion alliance. The association has a thermophilous character and a large number of companions from the Quercetalia ilicis and Scorzonero-Chrysopogonetalia communities. This report compares the floristic composition of this association with others of the Rubo ulmifolii-Nerion oleandri alliance from other Mediterranean areas. The Croatian association is most similar to the Spartio-Nerietum oleandri association from the Ionian side of Calabria (south Italy). The syntaxonomic status of the Montenegrin association Andropogono distachyi-Nerietum oleandri was determined, and the status of the stands with Nerium oleander is discussed.