Investigation of Anticholinesterase Activity of Chemically Characterised Hieracium s. str. Methanol Extracts and Their Selected Metabolites.

The composition and anticholinesterase activity of the dried MeOH extracts of Hieracium scheppigianum and H. naegelianum underground parts (rhizomes and roots), as well as the anticholinesterase activity of the dried, previously chemically characterised MeOH extracts of the flowering aerial parts of these two and 26 other Hieracium species in the strict sense (s. str.), were investigated. Furthermore, the anticholinesterase activity of 12 selected secondary metabolites of these extracts was evaluated. Using semi-preparative LC-MS, five caffeoylquinic acids and the sesquiterpene lactone crepiside E were isolated from H. scheppigianum underground parts extract. All these compounds were also identified in the underground parts extract of H. naegelianum. Quantitative LC-MS analysis showed that the analysed underground parts extracts were rich in both caffeoylquinic acids (139.77 and 156.62 mg/g of extract, respectively) and crepiside E (126.88 and 116.58 mg/g). In the Ellman method, the tested extracts showed an interesting anti-AChE and/or anti-BChE activity (IC50 =0.56-1.58 mg/mL), which can be explained, at least partially, by the presence of some of their constituents. Among the metabolites tested, the best activity was revealed for the flavonoids apigenin, luteolin and diosmetin, and the sesquiterpene lactone 8-epiixerisamine A (IC50 =68.09-299.37 µM).

Alchemilla viridiflora Rothm.: the potent natural inhibitor of angiotensin I-converting enzyme.

Alchemilla viridiflora Rothm., Rosaceae is a herbaceous plant widespread in central Greece, Bulgaria, North Macedonia and Serbia with Kosovo. Liquid chromatography-mass spectrometry analysis leads to the identification of 20 compounds in methanol extract, mainly ellagitannins and flavonoid glycosides. Given that various plant extracts have traditionally been used to treat hypertension and that some of the analyzed methanol extract constituents have beneficial cardiovascular effects, we hypothesized that some of these effects are achieved by inhibiting angiotensin I-converting enzyme (ACE). The dose-dependent ACE inhibitory activities of A. viridiflora and miquelianin were observed with an IC50 of 2.51 ± 0.00 µg/mL of A. viridiflora extract compared to the IC50 of 5.4139 ± 0.00 µM for miquelianin. The contribution of the single compounds to the tested activity was further analyzed through the in silico experimental approach. Computational docking results showed that tiliroside, ellagic acid pentose and galloyl-hexahydroxydiphenoyl-glucose exhibited even better binding affinity for the ACE active site than miquelianin, for which ACE activity was confirmed by an in vitro assay.

Spasmolytic, Gastroprotective and Antioxidant Activities of Dry Methanol Extract of Ferula heuffelii Underground Parts.

Dry MeOH extract of Ferula heuffelii (Apiaceae) underground parts was tested for spasmolytic, gastroprotective and antioxidant activities. HPLC analysis revealed that chlorogenic acid (CGA; 34.6 mg/g) was its main constituent. Extract in vitro exhibited notable total antioxidant activity (FRAP value=1.0 µmol Fe2+ /mg), and scavenging of DPPH (SC50 =62.5 µg/ml) and • OH radicals (49.5 % at 20 µg/ml in 2-deoxyribose assay). In vitro on isolated rat ileum, extract exhibited significant spasmolytic activity, i. e., it showed 124.6 % of maximal atropine effect on spontaneous contractions (at 100 µg/ml), and reduced spasmogenic effect of KCl (80 mm) to 44.4 % (at 60 µg/ml) and of highest applied concentration of ACh to 26.3 % (at 120 µg/ml). In parallel experiments, spasmolytic effect of CGA was also demonstrated. In acute EtOH-induced gastric ulceration model in rats, extract (100 mg/kg p.o.) showed significant gastroprotective effect (gastric damage score 0.50), similar to ranitidine (20 mg/kg p.o.). Obtained results showed that tested F. heuffelii polar extract represents new herbal preparation with potential use against some gastrointestinal complaints.

Antimicrobial and Cytotoxic Activities of Selected Hieracium L. s. str. (Asteraceae) Extracts and Isolated Sesquiterpene Lactones.

Antimicrobial and cytotoxic activities were tested for dried MeOH extracts of Hieracium calophyllum (CAL), H. coloriscapum (COL), H. pseudoschenkii (PSE), H. valdepilosum (VAL) and H. glabratum (GLA) herbs (flowering aerial parts), their 2 sesquiterpene lactones (SLs) 8-epiixerisamine A and crepiside E, and dried CH2 Cl2 extract of H. scheppigianum (SCH) herb. In microdilution test, extracts showed activity on all tested microorganisms (8 bacteria, 10 fungi). The best effect was exhibited by SCH and CAL on Salmonella Typhimurium (MIC=1.7-2.5 mg/mL MBC=3.4-5.0 mg/mL), and SCH and VAL on Candida albicans (MIC=2.5 mg/mL MFC=5.0 mg/mL). SLs showed notable effect on all tested fungi Aspergillus ochraceus, Penicillium funiculosum, C. albicans and C. krusei (MIC=0.15-0.4 mg/mL MFC=0.3-0.8 mg/mL). In MTT test, extracts inhibited growth of all tested cancer cells (HeLa, LS174 and A549), with the best effect on HeLa (IC50 =148.1 µg/mL for SCH, and 152.3-303.2 µg/mL for MeOH extracts); both SLs were active against HeLa cells (IC50 =46.2 µg/mL for crepiside E and 103.8 µg/mL for 8-epiixerisamine A). Extracts and SLs showed good safety profile on normal MRC-5 cells.

Barks of Three Wild Pyrus Taxa: Phenolic Constituents, Antioxidant Activity, and in Vitro and in Silico Investigations of α-Amylase and α-Glucosidase Inhibition.

Dry MeOH extracts of the twig barks of Pyrus communis subsp. pyraster, P. spinosa and their hybrid P.×jordanovii nothosubsp. velenovskyi, collected in wild in Serbia, were analyzed. By LC/MS, the contents of arbutin (99.9-131.0 mg/g), chlorogenic acid (2.2-6.3 mg/g), catechin (1.0-5.3 mg/g) and total dimeric and trimeric procyanidins (42.2-61.3 mg/g), including procyanidin B2 (8.9-17.2 mg/g), were determined. Colorimetrically, high contents of total phenolics (436.2-533.4 mg GAE/g) and tannins (339.4-425.7 mg GAE/g), as well as strong total antioxidant activities (FRAP values 4.5-5.9 mmol Fe2+ /g), and DPPH (SC50 =6.6-7.1 µg/ml) and hydroxyl radical (SC50 =447.1-727.7 µg/ml) scavenging abilities were revealed. In vitro, all extracts exhibited notable inhibition of α-amylase (IC50 =310.8-617.7 µg/ml) and particularly strong inhibition of α-glucosidase (IC50 =2.1-3.7 µg/ml). Molecular docking predicted that among identified compounds procyanidin B2 is the best inhibitor of these carbohydrate-digesting enzymes. Obtained results showed that the barks of investigated Pyrus hybrid and its parent taxa have similar composition and bioactivity.

Chemosystematic Significance of Essential Oil Constituents and Furanocoumarins of Underground Parts and Fruits of Nine Heracleum L. Taxa from Southeastern Europe.

The composition of the essential oils and the furanocoumarin profiles of CH2 Cl2 extracts from underground parts and fruits of nine Heracleum taxa (Apiaceae) from Southeastern Europe were statistically analyzed to evaluate their chemosystematic significance. Eight investigated taxa (H. orphanidis and members of H. sphondylium group: H. sphondylium, H. sibiricum, H. montanum, H. ternatum, H. pyrenaicum subsp. pollinianum, H. pyrenaicum subsp. orsinii and H. verticillatum) belong to the type section of the genus. Additionally analyzed taxon, H. austriacum subsp. siifolium, belongs to H. sect. Wendia. Qualitative and quantitative analysis of the essential oils was performed by GC-FID and GC/MS, and of the furanocoumarins by LC/MS. Furanocoumarins were identified using standards and/or based on UV, MS, 1 H- and ROESY NMR spectra. Multivariate statistics (PCA, nMDS, UPGMA) of analyzed metabolites showed that the investigated taxa were grouped according to their taxonomic classification. PCA revealed the significance of some monoterpenes and (Z)-falcarinol of the underground parts oils, octyl esters of the fruit oils, and most of the total of 12 identified furanocoumarins.

Methanol Extracts of 28 Hieracium Species from the Balkan Peninsula - Comparative LC-MS Analysis, Chemosystematic Evaluation of their Flavonoid and Phenolic Acid Profiles and Antioxidant Potentials.

INTRODUCTION: Hieracium s. str. represents one of the largest and most complex genera of flowering plants. As molecular genetics seems unlikely to disentangle intricate relationships within this reticulate species complex, analysis of flavonoids and phenolic acids, known as good chemosystematic markers, promise to be more reliable. Data about pharmacological activity of Hieracium species are scarce. OBJECTIVE: Evaluation of the chemosystematic significance of flavonoids and phenolic acids of methanol extracts of aerial flowering parts of 28 Hieracium species from the Balkans. Additionally, investigation of antioxidant potentials of the extracts. METHODS: Comparative qualitative and quantitative analysis of flavonoids and phenolic acids was performed by LC-MS. Multivariate statistical data analysis included non-metric multidimensional scaling (nMDS), unweighted pair-group arithmetic averages (UPGMA) and principal component analysis (PCA). Antioxidant activity was evaluated using three colorimetric tests. RESULTS: Dominant phenolics in almost all species were luteolin type flavonoids, followed by phenolic acids. Although the investigated Hieracium species share many compounds, the current classification of the genus was supported by nMDS and UPGMA analyses with a good resolution to the group level. Hieracium naegelianum was clearly separated from the other investigated species. Spatial and ecological distances of the samples were likely to influence unexpected differentiation of some groups within H. sect. Pannosa. The vast majority of dominant compounds significantly contributed to differences between taxa. The antioxidant potential of the extracts was satisfactory and in accordance with their phenolics composition. CONCLUSIONS: Comparative LC-MS analysis demonstrated that flavonoids and phenolic acids are good indicators of chemosystematic relationships within Hieracium, particularly between non-hybrid species and groups from the same location. Copyright © 2017 John Wiley & Sons, Ltd.

Laserpitium zernyi Hayek Flower and Herb Extracts: Phenolic Compounds, and Anti-edematous, Antioxidant, and Antimicrobial Activities.

Phenolic compounds and different biological activities of the dry MeOH extracts of the flowers and the herb (aerial parts without flowers) of Laserpitium zernyi Hayek (Apiaceae) were investigated. The total phenolic contents in the extracts were determined spectrophotometrically using Folin-Ciocalteu reagent. In both extracts, apigenin, luteolin, their 7-O-glucosides, and chlorogenic acid were detected by HPLC. Identified phenolics were quantified in both extracts, except luteolin in L. zernyi herb extract. The extracts (p.o.) were tested for anti-edematous activity in a model of carrageenan (i.pl.) induced rat paw edema. Antioxidant activity of the extracts was assessed by FRAP assay and DPPH and • OH radicals scavenging tests. Antimicrobial activity was investigated using broth microdilution test against five Gram-positive and three Gram-negative bacteria, as well as against two strains of Candida albicans. The polyphenol-richer flower extract exerted higher anti-edematous and antioxidant activities. The herb extract exhibited better antimicrobial effect against Micrococcus luteus, Enterococcus faecalis, Bacillus subtilis, and Pseudomonas aeruginosa, while against other tested microorganisms, the activity of both extracts was identical. Demonstrated biological activities of L. zernyi flower and herb extracts represent a good basis for their further investigation as potential new herbal medicinal raw materials.

Edible wild plant Heracleum pyrenaicum subsp. orsinii as a potential new source of bioactive essential oils.

Many Heracleum L. taxa (Apiaceae) are used as food and spices, and in traditional medicine. In this work, the chemical composition of Heracleum pyrenaicum subsp. orsinii (Guss.) F. Pedrotti and Pignatti root, leaf and fruit essential oils, their antimicrobial activity and cytotoxic effect on malignant and normal cells were investigated for the first time. The composition of the oils was analyzed by GC and GC-MS. Monoterpenes prevailed in the root oil, with ß-pinene (38.6%) being dominant, while in the leaf oil, sesquiterpenes, mostly (E)-nerolidol (20.5%) and (E)-caryophyllene (17.0%), were the most abundant constituents. The fruit oil contained the majority of aliphatic esters, mainly octyl acetate (36.8%) and octyl hexanoate (22.1%). The antimicrobial activity was determined by microdilution method against eight bacteria and eight fungi (standard strains, clinical or food isolates). The best antibacterial activity, better than the activity of ampicillin, was shown by the root oil against Salmonella typhimurium, Escherichia coli and Pseudomonas aeruginosa. The strongest antifungal activity, stronger than the activity of ketoconazole, was exhibited by the leaf and root oils against Trichoderma viride, and by the root oil against Aspergillus ochraceus. The cytotoxic effect of the oils, determined by MTT test, was prominent against malignant HeLa, LS174 and A549 cells (IC50 = 6.49-14.56 µg/mL). On the other hand, the oils did not show toxicity against normal MRC-5 cells at tested concentrations (IC50 > 200.00 µg/mL). It can be concluded that investigated H. pyrenaicum subsp. orsinii oils represent potential new raw materials for food and pharmaceutical industry.

Patterns of rapid diversification in heteroploid Knautia sect. Trichera (Caprifoliaceae, Dipsacoideae), one of the most intricate taxa of the European flora.

BACKGROUND: Polyploidy is one of the most important evolutionary pathways in flowering plants and has significantly contributed to their diversification and radiation. Due to the prevalence of reticulate evolution spanning three ploidy levels, Knautia is considered one of the taxonomically most intricate groups in the European flora. On the basis of ITS and plastid DNA sequences as well as AFLP fingerprints obtained from 381 populations of almost all species of the genus we asked the following questions. (1) Where and when did the initial diversification in Knautia take place, and how did it proceed further? (2) Did Knautia undergo a similarly recent (Pliocene/Pleistocene) rapid radiation as other genera with similar ecology and overlapping distribution? (3) Did polyploids evolve within the previously recognised diploid groups or rather from hybridisation between groups? RESULTS: The diversification of Knautia was centred in the Eastern Mediterranean. According to our genetic data, the genus originated in the Early Miocene and started to diversify in the Middle Miocene, whereas the onset of radiation of sect. Trichera was in central parts of the Balkan Peninsula, roughly 4 Ma. Extensive spread out of the Balkans started in the Pleistocene about 1.5 Ma. Diversification of sect. Trichera was strongly fostered by polyploidisation, which occurred independently many times. Tetraploids are observed in almost all evolutionary lineages whereas hexaploids are rarer and restricted to a few phylogenetic groups. Whether polyploids originated via autopolyploidy or allopolyploidy is unclear due to the weak genetic separation among species. In spite of the complexity of sect. Trichera, we present nine AFLP-characterised informal species groups, which coincide only partly with former traditional groups. CONCLUSIONS: Knautia sect. Trichera is a prime example for rapid diversification, mostly taking place during Pliocene and Pleistocene. Numerous cycles of habitat fragmentation and subsequent reconnections likely promoted hybridisation and polyploidisation. Extensive haplotype sharing and unresolved phylogenetic relationships suggest that these processes occurred rapidly and extensively. Thus, the dynamic polyploid evolution, the lack of crossing barriers within ploidy levels supported by conserved floral morphology, the highly variable leaf morphology and unstable indumentum composition prevent establishing a well-founded taxonomic framework.

Chemical Composition, Antimicrobial and Cytotoxic Activity of Heracleum verticillatum Pancic and H. ternatum Velen. (Apiaceae) Essential Oils.

In this work, the chemical composition, antimicrobial and cytotoxic activity of Heracleum verticillatum Pancic and H. ternatum Velen. root, leaf, and fruit essential oils were investigated. The composition was analyzed by GC and GC/MS. Heracleum verticillatum and H. ternatum root oils were dominated by monoterpenes, mostly ß-pinene (23.5% and 47.3%, respectively). Heracleum verticillatum leaf oil was characterized by monoterpenes, mainly limonene (20.3%), and sesquiterpenes, mostly (E)-caryophyllene (19.1%), while H. ternatum leaf oil by the high percentage of phenylpropanoids, with (Z)-isoelemicin (35.1%) being dominant constituent. Both fruit oils contained the majority of aliphatic esters, mostly octyl acetate (42.3% in H. verticillatum oil and 49.0% in H. ternatum oil). The antimicrobial activity of the oils was determined by microdilution method against eight bacterial and eight fungal strains. The strongest effect was exhibited by H. verticillatum root oil, particularly against Staphylococcus aureus, Salmonella typhimurium (MICs = 0.14 mg/ml, MBCs = 0.28 mg/ml), and Trichoderma viride (MIC = 0.05 mg/ml, MFC = 0.11 mg/ml). Cytotoxic effect was determined by MTT test against malignant HeLa, LS174, and A549 cells (IC50 = 5.9 - 146.0 µg/ml), and against normal MRC-5 cells (IC50 > 120.1 µg/ml). The best effect was exhibited by H. verticillatum root oil on A549 cells (IC50 = 5.9 µg/ml), and H. ternatum root oil against LS174 cells (IC50 = 6.7 µg/ml).

Antimicrobial and Cytotoxic Activity of Extracts of Ferula heuffelii Griseb. ex Heuff. and Its Metabolites.

The antimicrobial and cytotoxic activities of isolates (CHCl3 and MeOH extracts and selected metabolites) obtained from the underground parts of the Balkan endemic plant Ferula heuffelii Griseb. ex Heuff. were assessed. The CHCl3 and MeOH extracts exhibited moderate antimicrobial activity, being more pronounced against Gram-positive than Gram-negative bacteria, especially against Staphylococcus aureus (MIC=12.5 µg/ml for both extracts) and Micrococcus luteus (MIC=50 and 12.5 µg/ml, resp.). Among the tested metabolites, (6E)-1-(2,4-dihydroxyphenyl)-3,7,11-trimethyl-3-vinyldodeca-6,10-dien-1-one (2) and (2S*,3R*)-2-[(3E)-4,8-dimethylnona-3,7-dien-1-yl]-2,3-dihydro-7-hydroxy-2,3-dimethylfuro[3,2-c]coumarin (4) demonstrated the best antimicrobial activity. Compounds 2 and 4 both strongly inhibited the growth of M. luteus (MIC=11.2 and 5.2 µM, resp.) and Staphylococcus epidermidis (MIC=22.5 and 10.5 µM, resp.) and compound 2 additionally also the growth of Bacillus subtilis (MIC=11.2 µM). The cytotoxic activity of the isolates was tested against three human cancer cell lines, viz., cervical adenocarcinoma (HeLa), chronic myelogenous leukemia (K562), and breast cancer (MCF-7) cells. The CHCl3 extract exhibited strong cytotoxic activity against all cell lines (IC50 <11.0 µg/ml). All compounds strongly inhibited the growth of the K562 and HeLa cell lines. Compound 4 exhibited also a strong activity against the MCF-7 cell line, comparable to that of cisplatin (IC50 =22.32±1.32 vs. 18.67±0.75µM).

Composition and antimicrobial activity of the essential oils of Laserpitium latifolium L. and L. ochridanum Micevski (Apiaceae).

The chemical composition and antimicrobial activity of essential oils of Laserpitium latifolium and L. ochridanum were investigated. The essential oils were isolated by steam distillation and characterized by GC-FID and GC/MS analyses. All essential oils were distinguished by high contents of monoterpenes, and α-pinene was the most abundant compound in the essential oils of L. latifolium underground parts and fruits (contents of 44.4 and 44.0%, resp.). The fruit essential oil was also rich in sabinene (26.8%). Regarding the L. ochridanum essential oils, the main constituents were limonene in the fruit oil (57.7%) and sabinene in the herb oil (25.9%). The antimicrobial activity of these essential oils as well as that of L. ochridanum underground parts, whose composition was reported previously, was tested by the broth-microdilution method against four Gram-positive and three Gram-negative bacteria and two Candida albicans strains. Except the L. latifolium underground-parts essential oil, the other investigated oils showed a high antimicrobial potential against Staphylococcus aureus, S. epidermidis, Micrococcus luteus, or Candida albicans (minimal inhibitory concentrations of 13.0-73.0 µg/ml), comparable to or even higher than that of thymol, which was used as reference compound.

Escaping to the summits: phylogeography and predicted range dynamics of Cerastium dinaricum, an endangered high mountain plant endemic to the western Balkan Peninsula.

The Balkans are a major European biodiversity hotspot, however, almost nothing is known about processes of intraspecific diversification of the region's high-altitude biota and their reaction to the predicted global warming. To fill this gap, genome size measurements, AFLP fingerprints, plastid and nuclear sequences were employed to explore the phylogeography of Cerastium dinaricum. Range size changes under future climatic conditions were predicted by niche-based modeling. Likely the most cold-adapted plant endemic to the Dinaric Mountains in the western Balkan Peninsula, the species has conservation priority in the European Union as its highly fragmented distribution range includes only few small populations. A deep phylogeographic split paralleled by divergent genome size separates the populations into two vicariant groups. Substructure is pronounced within the southeastern group, corresponding to the area's higher geographic complexity. Cerastium dinaricum likely responded to past climatic oscillations with altitudinal range shifts, which, coupled with high topographic complexity of the region and warmer climate in the Holocene, sculptured its present fragmented distribution. Field observations revealed that the species is rarer than previously assumed and, as shown by modeling, severely endangered by global warming as viable habitat was predicted to be reduced by more than 70% by the year 2080.

Chloroform extract of underground parts of Ferula heuffelii: secondary metabolites and spasmolytic activity.

Plants from the genus Ferula L. (Apiaceae) were used for various purposes in traditional medicine of different nations throughout the history. Ferula heuffelii Griseb. ex Heuffel is a perennial species endemic for Balkan peninsula. Ten compounds which belong to classes of prenyl-furocoumarin-, prenyl-dihydrofurochromone-, prenyl-benzoyl- and prenyl-benzoylfuranone-type sesquiterpenoids, as well as sesquiterpene coumarins and phenylpropanoids, were, for the first time, isolated from the CHCl3 extract of the underground parts of this plant and identified. Furthermore, extract and three isolated compounds, i.e., latifolone (1), dshamirone (4), and (2S*,3R*)-2-[(3E)-4,8-dimethylnona-3,7-dien-1-yl]-2,3-dihydro-7-hydroxy-2,3-dimethylfuro[3,2-c]coumarin (6) were, for the first time, evaluated for their in vitro antispasmodic activities in three experimental models: spontaneous contraction, and ACh- and KCl-induced contraction of an isolated rat ileum. The extract (0.1-1.3 mg/ml) and compound 6 (1-10 µg/ml) exhibited dose-dependent effect in all three models. Compound 1 (1-6 µg/ml) affected spontaneous contractions and those induced by KCl, while compound 4 (8 µg/ml) displayed only moderate activity with ACh-induced contractions. It can be concluded that tested compounds contribute to exhibited antispasmodic activity of crude extract. Additionally, extract (0.1-1.3 mg/ml) was tested for in vitro relaxant activity on an isolated rat trachea, and relaxed the KCl-induced contractions in a dose-dependent manner.

Chemical Analysis and Investigation of Antimicrobial and Antibiofilm Activities of Prangos trifida (Apiaceae).

Plants of the genus Prangos are intensively investigated as potential new sources of bioactive isolated products. In this work, the chemical composition of volatile constituents (essential oils and headspace volatiles) and dichloromethane extracts, as well as antimicrobial and antibiofilm activities of essential oils and MFDEs (methanol fractions of dichloromethane extracts) of Prangos trifida from Serbia, were investigated. Volatiles of roots, leaves, stems and fruits, and fatty acids and phytosterols in dichloromethane extracts of roots and fruits were analyzed by GC-FID-MS, whereas coumarins in MFDEs by LC-MS and some isolated coumarins by 1H-NMR. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations/minimum fungicidal concentrations (MBCs/MFCs) of essential oils and MFDEs were determined against 13 microorganisms. Antibiofilm activity was assessed against four microorganisms. Additionally, congo red and ergosterol binding assays were conducted to elucidate selected mechanisms of antibiofilm action in the case of Candida albicans. Total of 52 volatile constituents, 16 fatty acids, eight phytosterols and 10 coumarins were identified. Essential oils demonstrated significant activity, surpassing that of commercial food preservatives, against six tested molds from the Aspergillus, Penicillium and Trichoderma genera, as well as against bacteria Staphylococcus aureus and Bacillus cereus. Most of the oils strongly inhibited the formation of biofilms by S. aureus, Listeria monocytogenes and Escherichia coli. MFDEs exhibited noteworthy effects against B. cereus and the tested Aspergillus species, particularly A. niger, and significantly inhibited C. albicans biofilm formation. This inhibition was linked to a marked reduction in exopolysaccharide production, while antifungal mechanisms associated with ergosterol remained unaffected.

Genetic Variability in Balkan Paleoendemic Resurrection Plants Ramonda serbica and R. nathaliae Across Their Range and in the Zone of Sympatry.

The genus Ramonda includes three Paleoendemic and Tertiary relict species that survived in refugial habitats of the Balkan Peninsula (R. nathaliae and R. serbica) and the Iberian Peninsula (R. myconi). They are all "resurrection plants," a rare phenomenon among flowering plants in Europe. Ramonda myconi and R. nathaliae are diploids (2n = 2x = 48), while R. serbica is a hexaploid (2n = 6x = 144). The two Balkan species occur in sympatry in only two localities in eastern Serbia, where tetraploid potential hybrids (2n = 4x = 96) were found. This observation raised questions about the existence of gene flow between the two species and, more generally, about the evolutionary processes shaping their genetic diversity. To address this question, genetic markers (AFLP) and an estimate of genome size variation were used in a much larger sample and at a larger geographic scale than previously. The combination of AFLP markers and genome size results suggested ongoing processes of interspecific and interploidy hybridization in the two sites of sympatry. The data also showed that interspecific gene flow was strictly confined to sympatry. Elsewhere, both Ramonda species were characterized by low genetic diversity within populations and high population differentiation. This is consistent with the fact that the two species are highly fragmented into small and isolated populations, likely a consequence of their postglacial history. Within sympatry, enormous variability in cytotypes was observed, exceeding most reported cases of mixed ploidy in complex plant species (from 2x to >8x). The AFLP profiles of non-canonical ploidy levels indicated a diversity of origin pathways and that backcrosses probably occur between tetraploid interspecific hybrids and parental species. The question arises whether this diversity of cytotypes corresponds to a transient situation. If not, the question arises as to the genetic and ecological mechanisms that allow this diversity to be maintained over time.

Investigation of antihyperalgesic and antiedematous activities of three Hieracium species.

Present study investigated triterpene profile, antihyperalgesic and antiedematous activities of Hieracium scheppigianum flowering aerial parts dichloromethane extract (SCH), and antihyperalgesic and antiedematous activities of previously chemically characterised polyphenol-rich H. glabratum and H. calophyllum flowering aerial parts methanol extracts (GLA and CAL, respectively). α- and ß-Amyrin and their acetates, and lupeol acetate were identified and quantified in SCH by GC-FID and GC-MS. In carrageenan-induced localised inflammation model in rats, SCH and GLA (50-200 mg/kg, p.o.) produced significant and dose-dependent antihyperalgesic effect of 26.9%-56.2% (ED50=163.0 ± 26.5 mg/kg) and 25.3%-51.6% (ED50=211.6 ± 70.6 mg/kg), respectively, and CAL (200 mg/kg, p.o.) exhibited effect of 38.1%. Extracts did not significantly reduce paw edema. SCH and GLA, which demonstrated higher (over 50%) antihyperalgesic efficacy, were tested in a rotarod test (200 mg/kg, p.o.) and no alteration of motor coordination was observed. Also, acute administration of SCH and GLA in mice (2000 mg/kg, p.o.) caused neither mortality nor toxicity.

The antiinflammatory, gastroprotective and antioxidant activities of Hieracium gymnocephalum extract.

The present study investigated the antiinflammatory, gastroprotective and antioxidant activities of a CH(2)Cl(2) extract of western Balkan endemic Hieracium gymnocephalum Griseb. ex Pant. (Compositae). The carrageenan-induced rat paw oedema test was used as an experimental model for screening the antiinflammatory activity. The extract was administrated p.o. in doses of 25, 50, 100 and 200 mg/kg to rats and its effects compared with indomethacin, used p.o. as a reference drug. The results showed that the investigated extract reduced the oedema in a concentration-dependent manner. The obtained antiinflammatory effect was 5.9%, 11.7%, 31.2% and 44.1% at doses of 25, 50, 100 and 200 mg/kg, respectively, being statistically significant at a dose of 100 mg/kg. Indomethacin had a strong antiinflammatory effect of 73.4% at a dose of 8 mg/kg, but caused large gastric lesions. When the plant extract in the highest tested dose (200 mg/kg) was concomitantly given with indomethacin, the antiinflammatory effect was slightly enhanced, but the gastric lesions were significantly reduced. The antioxidant activity of the H. gymnocephalum extract, investigated using DPPH radical assay, OH-radical assay and TBA-test, was not substantial.

Sesquiterpene lactones from the methanol extracts of twenty-eight Hieracium species from the Balkan Peninsula and their chemosystematic significance.

Four sesquiterpene lactones (SLs), including three undescribed proline-SL conjugates, the guaianolides calophyllamine A and 8-epiixerisamine A, and the eudesmanolide calophyllamine B, were isolated from the methanol extract of Hieracium calophyllum R. Uechtr. (Compositae) flowering heads. Another known guaianolide, crepiside E, was detected in Hieracium L. species for the first time. Their structures were elucidated using extensive 1D and 2D NMR spectroscopy in combination with HRMS. The isolated SLs were used as external standards for qualitative and quantitative LC-MS analysis of the dry methanol extracts of the flowering aerial parts of 28 Hieracium species from the Balkan Peninsula. Guaianolides were the dominant SLs in 27 species studied. The chemosystematic significance of detected SLs was evaluated using multivariate statistics (PCA, nMDS and UPGMA). Differentiation between the main groups was well supported. All four compounds significantly and equally contributed to the differences between the species. In addition, the eudesmanolide calophyllamine B could be a significant chemosystematic marker for H. sect. Villosa (Griseb.) Gremli s.l. and Glauciformia (Freyn) Zahn-Italica (Fr.) Av. Touv.