Phytochemicals and Their Correlation with Molecular Data in Micromeria and Clinopodium (Lamiaceae) Taxa.

A study of the phytochemical and molecular characteristics of ten Micromeria and six Clinopodium taxa (family Lamiaceae) distributed in the Balkan Peninsula was carried out. The phytochemicals detected in essential oils by gas chromatography, mass spectrometry, and molecular data amplified fragment length polymorphism were used to study the taxonomic relationships among the taxa and the correlations between phytochemical and molecular data. STRUCTURE analysis revealed three genetic groups, while Bayesian Analysis of Population Structure grouped the studied taxa into 11 clusters nested in the groups obtained by STRUCTURE. Principal components analysis performed with the 21 most represented compounds in the essential oils yielded results that were partly consistent with those obtained by STRUCTURE and neighbour-joining analyses. However, their geographic distributions did not support the genetic grouping of the studied taxa and populations. The Mantel test showed a significant correlation between the phytochemical and genetic data (r = 0.421, p < 0.001). Genetic distance explained 17.8% of the phytochemical distance between populations. The current taxonomic position of several of the studied taxa is yet to be satisfactorily resolved, and further studies are needed. Such future research should include nuclear and plastid DNA sequences from a larger sample of populations and individuals.

In vitro, in vivo and in silico evaluation of the anti-inflammatory potential of Hyssopus officinalis L. subsp. aristatus (Godr.) Nyman (Lamiaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal properties of hyssop have been used in traditional medicine since ancient times, inter alia, in diseases/conditions with an inherent inflammatory process. AIM OF THE STUDY: Accordingly, the aim of this study was to investigate the anti-inflammatory properties of hyssop herb preparations (essential oil and methanol extracts) in vivo, in vitro and in silico. MATERIALS AND METHODS: For in vitro testing of essential oils and extracts of hyssop herb, the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme assays were used. In vivo anti-inflammatory potential of the extracts (at doses of 50, 100 and 200 mg/kg) was assessed using the carrageenan-induced rat paw edema test. Molecular docking and dynamics were used for in silico testing of the inhibitory activity of chlorogenic (CA) and rosmarinic (RA) acids, as the dominant compounds in the tested methanol extracts against COX-1 and COX-2 enzymes. RESULTS: Significant inhibitory activity was shown in the COX-2 test regarding extracts (essential oils did not exhibit any significant activity). Namely, all analyzed extracts, at a concentration of 20 µg/mL, showed a percentage of inhibition of COX-2 enzyme (54.04-63.04%), which did not indicate a statistically significant difference from the positive control of celecoxib (61.60%) at a concentration of 8.8 µM. In vivo testing showed that all methanol extracts of hyssop herb, at the highest test dose of 200 mg/kg in the third and fourth hours, after carrageenan administration, exhibited a statistically significant (p < 0.05) inhibitory effect on the increase in rat paw edema in relation to control. This activity is comparable or higher in relation to the reference substance, indomethacin, at a concentration of 8 mg/kg. The preliminary in silico results suggest that investigated compounds (RA and CA) showed better inhibitory activity against COX-1 and COX-2 than standard non-steroidal anti-inflammatory drug (NSAID), ibuprofen, as evident from the free binding energy (ΔGbind in kJ mol-1). The binding energies of the docked compounds to COX-1 and -2 were found to be in the range between -47.4 and -49.2 kJ mol-1. Ibuprofen, as the one NSAID, for the same receptors targets, showed remarkably higher binding energy (ΔGbind = -31.3 kJ mol-1 to COX-1, and ΔGbind = -30.9 kJ mol-1 to COX-2). CONCLUSION: The results obtained not only support the traditional use of hyssop herb in inflammatory conditions in folk medicine, but also open the door to and the need for further in vivo testing of extracts in order to examine the molecular mechanism of anti-inflammatory activity in living systems and possibly develop a new anti-inflammatory drug or supplement.

Micromorphological Traits of Balcanic Micromeria and Closely Related Clinopodium Species (Lamiaceae).

A study of the trichomes types and distribution and pollen morphology was carried out in nine Micromeria taxa (M. cristata ssp. cristata, M. cristata ssp. kosaninii, M. croatica, M. graeca ssp. graeca, M. graeca ssp. fruticulosa, M. juliana, M. kerneri, M. longipedunculata and M. microphylla) and five closely related Clinopodium species (C. dalmaticum, C. frivaldszkyanum, C. pulegium, C. serpyllifolium and C. thymifolium) from the Lamiaceae family of the Balkan Peninsula. By scanning electron microscope, non-glandular trichomes, peltate and capitate trichomes were observed on the calyx, leaves and stem of the studied species. Two subtypes of capitate trichomes were observed in Micromeria species: subtype 1 (consisting of a basal epidermal cell and an elliptically shaped head cell) and subtype 2 (consisting of a basal epidermal cell, two to three stalk cells and a round head cell). In Clinopodium species, three types of capitate trichomes were observed: subtype 1, subtype 3 (consisting of a basal epidermal cell, a short peduncle cell, and a single round head cell), and subtype 4 (consisting of a basal epidermal cell, a stalk cell, and an elongated head cell). These results support the recent transfer of Micromeria species from the section Pseudomelissa to the genus Clinopodium.

Antioxidant, Antigenotoxic and Cytotoxic Activity of Essential Oils and Methanol Extracts of Hyssopus officinalis L. Subsp. aristatus (Godr.) Nyman (Lamiaceae).

Hyssopus officinalis L. is a well-known aromatic plant used in traditional medicine and the food and cosmetics industry. The aim of this study is to assess the antioxidant, genotoxic, antigenotoxic and cytotoxic properties of characterized hyssop essential oils and methanol extracts. Chemical composition was analyzed by gas chromatography - mass spectrometry (GC-MS) and liquid chromatography with diode array detection and mass spectrometry (LC-DAD-MS), respectively. Antioxidant activity was examined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) tests; genotoxic and antigenotoxic activity were examined by the comet assay, while cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide dye (MTT) test against tumor cell lines (SW480, MDA-MB 231, HeLa) and non-transformed human lung fibroblast cell lines (MRC-5). The essential oils were rich in monoterpene hydrocarbons (e.g., limonene; 7.99-23.81%), oxygenated monoterpenes (1,8-cineole; 38.19-67.1%) and phenylpropanoids (methyl eugenol; 0.00-28.33%). In methanol extracts, the most abundant phenolics were chlorogenic and rosmarinic acid (23.35-33.46 and 3.53-17.98 mg/g, respectively). Methanol extracts expressed moderate to weak antioxidant activity (DPPH IC50 = 56.04-199.89 µg/mL, FRAP = 0.667-0.959 mmol Fe2+/g). Hyssop preparations significantly reduced DNA damage in human whole blood cells, induced by pretreatment with hydrogen peroxide. Methanol extracts exhibited selective and potent dose- and time-dependent activity against the HeLa cell line. Results of the current study demonstrated notable H. officinalis medicinal potential, which calls for further investigation.

Beach litter along various sand dune habitats in the southern Adriatic (E Mediterranean).

Marine litter accumulates on sandy beaches and is an important environmental problem, as well as a threat to habitat types that are among the most endangered according to EU legislation. We sampled 120 random plots (2 × 2 m) in spring 2017 to determine the distribution pattern of beach litter along the zonation of habitat types from sea to the inland. The most frequent litter items were plastic, polystyrene and glass. A clear increase of litter cover along the sea-inland gradient is evident, and foredunes and pine forests have the highest cover of litter. Almost no litter was present in humid dune slacks. Shoreline and recreational activities are the major source of beach litter, while ocean/waterway activities are more important in the aphytic zone and strandline.

Sorbus aucuparia and Sorbus aria as a Source of Antioxidant Phenolics, Tocopherols, and Pigments.

Due to its nutritive and medicinal properties, berries of some Sorbus species are used for the preparation of jams and jelly as well as in traditional medicine. On the other hand, their chemical composition is not much studied especially of those grown in Balkan Peninsula. We have analyzed individual phenolics, tocopherols, carotenoids and chlorophylls using HPLC in berries from Sorbus aucuparia and Sorbus aria collected in different localities in Serbia and Montenegro together with the amounts of total phenolics and proanthocyanidins as well as their radical scavenging activity against DPPH radical. Berries of S. aucuparia were richer source of polyphenolics in comparision with S. aria and, regardless the species and locality, caffeoylquinic acids such as neochlorogenic and chlorogenic acid were the most abundant compounds. Among analyzed tocopherols the most abundant in all samples was α-tocopherol (0.48 - 19.85 µg/g dw) as it was ß-carotene among carotenoids (mean concentration of 0.98 µg/g dw in S. aucuparia and 0.40 µg/g dw in S. aria, respectively). Correlation between total phenolics and DPPH radical scavenging activity was noticed. Our study represents comprehensive report on chemical composition of S. aucuparia and S. aria which could contribute to a better understanding of their quality.

Genetic Diversity and Demographic History of Wild and Cultivated/Naturalised Plant Populations: Evidence from Dalmatian Sage (Salvia officinalis L., Lamiaceae).

Dalmatian sage (Salvia officinalis L., Lamiaceae) is a well-known aromatic and medicinal Mediterranean plant that is native in coastal regions of the western Balkan and southern Apennine Peninsulas and is commonly cultivated worldwide. It is widely used in the food, pharmaceutical and cosmetic industries. Knowledge of its genetic diversity and spatiotemporal patterns is important for plant breeding programmes and conservation. We used eight microsatellite markers to investigate evolutionary history of indigenous populations as well as genetic diversity and structure within and among indigenous and cultivated/naturalised populations distributed across the Balkan Peninsula. The results showed a clear separation between the indigenous and cultivated/naturalised groups, with the cultivated material originating from one restricted geographical area. Most of the genetic diversity in both groups was attributable to differences among individuals within populations, although spatial genetic analysis of indigenous populations indicated the existence of isolation by distance. Geographical structuring of indigenous populations was found using clustering analysis, with three sub-clusters of indigenous populations. The highest level of gene diversity and the greatest number of private alleles were found in the central part of the eastern Adriatic coast, while decreases in gene diversity and number of private alleles were evident towards the northwestern Adriatic coast and southern and eastern regions of the Balkan Peninsula. The results of Ecological Niche Modelling during Last Glacial Maximum and Approximate Bayesian Computation suggested two plausible evolutionary trajectories: 1) the species survived in the glacial refugium in southern Adriatic coastal region with subsequent colonization events towards northern, eastern and southern Balkan Peninsula; 2) species survived in several refugia exhibiting concurrent divergence into three genetic groups. The insight into genetic diversity and structure also provide the baseline data for conservation of S. officinalis genetic resources valuable for future breeding programmes.

Antibacterial effect of Herniaria hirsuta, Prunus avium, Rubia tinctorum and Sempervivum tectorum plant extracts on multiple antibiotic resistant Escherichia coli / Efeito antibacteriano de Herniaria hirsuta, Prunus avium, Rubia tinctorum e Sempervivum tectorum extratos vegetais em vários resistentes a antibióticos Escherichia coli

The emergence of Escherichia coli isolates with multiple antibiotic resistant phenotypes is considered as a severe health concern. In the present work the antibacterial effect of following plants (Herniaria hirsuta, Prunus avium, Rubia tinctorum and Sempervivum tectorum) was examined. The bacterial model used for estimation of bacterial susceptibility is hospital multiple antibiotic resistant E. coli strain. E. coli ATCC 25922 was used for standard comparison of bacterial susceptibility. Leaves of H. hirsuta, R. tinctorum and S. tectorum as well as petioles of P. avium were collected. Ethanol and aqueous extract of each plant was prepared. Antibacterial activity was examined using the agar well diffusion method. Concentration of total phenols, flavonoids, tannins, antocyanins and saponins was determined in plant extracts. E. coli strain is resistant to four unrelated families of antibiotics. Antibacterial effect is proven for all examined plants. Ethanol extracts of H. hirsuta and P. avium have a more potent antibacterial effect than their aqueous extracts. Aqueous extracts of R. tinctorum and S. tectorum have higher antibacterial potential than theirs ethanol extracts. Examined plant extracts represent good candidates for more extensive research in view of their application in the treatment of multiple antibiotic resistant E.coli strains.

O surgimento de Escherichia coli isoladas com vários fenótipos resistentes aos antibióticos é considerado como um grave problema de saúde. No presente trabalho o efeito antibacteriano das seguintes plantas (Herniaria hirsuta, Prunus avium, Rubia tinctorum e Sempervivum tectorum) foi analisado. O agente bacteriano modelo utilizado para estimativa de susceptibilidade bacteriana é o hospital vários resistentes a antibióticos E. coli. E. coli ATCC 25922 padrão foi utilizado para comparação de antibiogramas. Folhas de H. hirsuta, R. tinctorum e S. tectorum bem como pecíolos de P. avium foram coletados. Etanol e extrato aquoso de cada planta foi preparado. Atividade antibacteriana foi analisada através do método de difusão em ágar-bem. Total Concentração de fenóis, flavonóides, taninos e saponinas antocyanins determinou-se em extratos de plantas. E. coli estirpe é resistente às quatro famílias de antibióticos independentes. Efeito antibacteriano é comprovado para todas as plantas examinadas. Os extratos etanólicos de H. hirsuta e P. avium têm um efeito mais potente antibacteriano de seus extratos aquosos. Extratos aquosos de R. tinctorum e S. tectorum têm maior potencial antibacteriano que os extratos etanólicos. Extratos vegetais examinados representam bons candidatos para pesquisa mais ampla em vista de sua aplicação no tratamento de vários antibióticos resistentes a cepas de E. coli.

Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae).

Analysis of the morphological traits of leaves, genetic variability (analyzed by AFLP) and chemical composition of essential oils (analyzed by GC-MS) was conducted on eleven populations of the endemic Illyric-Balcanic species Teucrium arduini L. in Croatia, Bosnia and Herzegovina, and Montenegro. Average blade length and width ranged from 20.00 to 31.47mm and from 11.58 to 15.66mm, respectively. Multivariate analysis (PCA, UPGMA) of morphological traits distinguished two continental Bosnian populations from the remaining populations. AFLP analysis separated the investigated populations into two groups based primarily on geographical distance. Essential oil analysis showed a total of 52 compounds, with two chemotypes distinguished based on the essential oil profile. The first was a sesquiterpene chemotype, with ß-caryophyllene, germacrene D or caryophyllene oxide as the major compounds, while the second was an oxygenated monoterpene chemotype, with pulegone and piperitone oxide as the main components. The Mantel test showed a stronger correlation between the morphological traits and AFLP than between the essential oil profile and AFLP. The test also showed a stronger association between the essential oil profile and geographical position than between the morphological traits and geographical position.

Lichenized and lichenicolous fungi from the Albanian Alps (Kosovo, Montenegro).

396 taxa (381 species) of lichenized and 45 species of lichenicolous fungi from the upper montane, subalpine and alpine belts of the Albanian Alps (= Prokletije Mountain Range, Bjeshkët e Nemuna) are presented. 92 lichenized and 26 lichenicolous fungi are new to Montenegro, 165 lichenized and 24 lichenicolous fungi are new to Kosovo, and 25 lichenized fungi (23 species) are new for the Balkan Peninsula.

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 Krivosije (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.

Chemotype diversity of indigenous Dalmatian sage (Salvia officinalis L.) populations in Montenegro.

To identify how many chemotypes of Salvia officinalis exist in Montenegro, the chemical composition of the essential oils of 12 wild-growing populations was determined by GC-FID and GC/MS analyses. Among the 40 identified constituents, the most abundant were cis-thujone (16.98-40.35%), camphor (12.75-35.37%), 1,8-cineol (6.40-12.06%), trans-thujone (1.5-10.35%), camphene (2.26-9.97%), borneol (0.97-8.81%), viridiflorol (3.46-7.8%), limonene (1.8-6.47%), α-pinene (1.59-5.46%), and α-humulene (1.77-5.02%). The composition of the essential oils under study did not meet the ISO 9909 requirements, while the oils of populations P02-P04, P09, and P10 complied with the German Drug Codex. A few of the main essential-oil constituents appeared to be highly intercorrelated. Strong positive correlations were observed between α-pinene and camphene, camphene and camphor, as well as between cis-thujone and trans-thujone. Strong negative correlations were evidenced between cis-thujone and α-pinene, cis-thujone and champhene, cis-thujone and camphor, as well as between trans-thujone and camphene. Multivariate analyses allowed the grouping of the populations into three distinct chemotypes, i.e., Chemotype A, rich in total thujones, Chemotype B, with intermediate contents of thujones, α-pinene, camphene, and camphor and high borneol contents, and Chemotype C, rich in camphor, camphene, and α-pinene. The chemotypes did not significantly differ in the total essential-oil content and the cis/trans-thujone ratio.

Lichenized fungi of a chestnut grove in Livari (Rumija, Montenegro).

Sixty taxa (59 species and 1 variety) of lichenized fungi are reported from a chestnut grove in Livari. The majority of them (55 species and 1 variety) occurred on Castanea sativa. The recently described Xylographa soralifera is new to the Balkan Peninsula. The lichenicolous fungus Monodictys epilepraria growing on Lepraria rigidula is new to Montenegro. The lichen mycota is compared with similar localities in Italy and Switzerland. The species composition in Livari is most similar to the Montieri site in Tuscany.

Composition and antimicrobial activity of Seseli montanum subsp. tommasinii essential oil.

The essential oil isolated from Seseli montanum L. subsp. tommasinii Reich. f. was analyzed by GC/MS and the most abundant components were beta-pinene (30.2%), germacrene D (10.1%), sabinene (8.0%), alpha-pinene (7.2%) and limonene (6.6%). The antimicrobial effect of the essential oil was evaluated against four bacterial and four fungal species among which were food contaminants, spoilage fungi, and plant, fungal and animal pathogens. The oil possessed moderate to strong antimicrobial activity.

Epiphytic lichens and lichenicolous fungi from the northern part of Montenegro.

A list of 120 taxa of lichens (117 species) and three species of lichenicolous fungi from the northern part of Montenegro is presented. The lichens Biatora chrysantha, Caloplaca monacensis, Candelariella efflorescens, Loxospora elatina, Micarea adnata, Ochrolechia arborea, O. microstictoides, Phaeophyscia nigricans, Physconia enteroxantha, Ph. grisea, Rinodina capensis, R. polysporoides, R. pyrina, Scoliciosporum umbrinum var. corticolum, Xanthoria candelaria, X. ulophyllodes and the lichenicolous fungi Lichenodiplis lecanorae, Telogalla olivieri, and Xanthoriicola physciae are new to Montenegro.

Cytotoxic guaianolide from Anthemis segetalis (Asteraceae).

Two epimeric guaianolides were isolated from the aerial parts of Anthemis segetalis. These compounds have not been detected in any Anthemis species so far. One of them was tested for cytotoxicity against human cervical cancer (HeLa) and murine melanoma (B16) cell lines showing appreciable activity.

New or otherwise interesting lichenized and lichenicolous fungi from Montenegro.

A list of 256 taxa of lichens (252 species) and 2 species of lichenicolous fungi from Montenegro is presented, including 58 taxa (57 species) new to Montenegro. The list is based on specimens kept in the lichen collections of the herbaria GZU, H, Podgorica, and in the private herbarium of Klaus Kalb, and on recent field work in various parts of the country. The genera Biatoridium, Carbonea, Cercidospora, Heppia, Hyperphyscia, Hypocenomyce, Leprocaulon, Lethariella, Megalospora, Orphniospora, Psorinia and Vahliella are reported from Montenegro for the first time.