Cuticular Wax Variability of Abies alba, A.×borisii-regis and A.†cephalonica from the Balkans: Chemophenetic and Ecological Aspects.

This is the first study on cuticular wax variability of Abies alba, A.×borisii-regis and A. cephalonica, using 18 native populations from the assumed hybrid zone in the Balkan Peninsula. Presence of 13 n-alkanes with chain-lengths ranging from C21 to C33 , one primary alcohol, two diterpenes, one triterpene and one sterol was determined in hexane extracts of 269 needle samples. The multivariate statistical analyses at the population level entirely failed in supporting circumscription of Balkan Abies taxa and therefore, in identifying hybrid populations. However, performed at the species level, these analyses revealed a certain tendency of differentiation between A. alba and A. cephalonica, while individuals of A.×borisii-regis were largely overlapped by the clouds of both parent species. Finally, the correlation analysis suggested that the observed variation of wax compounds was probably genetically conditioned and that it does not represent an adaptive response to various environmental factors.

Essential Oils of Three Balkan Abies Species: Chemical Profiles, Antimicrobial Activity and Toxicity toward Artemia salina and Drosophila melanogaster.

This study focused on the essential oils (EOs) isolated from needles with twigs of three indigenous Balkan Abies species (A. alba, A. × borisii-regis and A. cephalonica) regarding their chemical composition, antimicrobial activity and toxicity toward crustaceans and insects. Even though distinct phytochemical profiles of dominant volatiles were revealed for each species, ß-pinene and α-pinene represented the first two major volatiles in all three EOs. Antimicrobial activity of EOs has shown inhibitory effect against all 17 studied strains (ATCC and respiratory isolates) in the range of 0.62-20.00 mg/mL (MICs). Further, all three EOs exhibited strong toxicity (LC50 <100 µg/mL) in Artemia salina lethality bioassay, but with significant differences that depended on the EO type. Additionally, tested EOs have shown a certain level of toxicity against Drosophila melanogaster, mostly at the highest tested concentration (3 %) which caused significant prolongation of developmental time, larvicidal effect and pupal mortality. In the three biological assays performed, there was no observed inhibitory effect or weakest activity for A. alba EO. Further, A. cephalonica EO has shown the highest levels of antimicrobial activity and toxicity toward A. salina, while in relation to the insecticidal potential, A. cephalonica and A. × borisii-regis EOs exhibited similar level of toxicity against D. melanogaster.

Essential Oils of Six Achillea Species: Chemical Profiles, Antimicrobial Potential and Toxicity toward Crustaceans.

This is the first comparative study on antimicrobial activity of essential oils (EOs) of six Achillea species (A. crithmifolia, A. distans, A. grandifolia, A. millefolium, A. nobilis, and A. lingulata) against ATCC and clinical microbial strains isolated from human swabs and on their toxicological potential on crustaceans. Oxygenated monoterpenes represented the major compound class in all six EOs, ranging from around 50 % (A. millefolium, A. nobilis, and A. lingulata) up to over 80 % (A. crithmifolia and A. grandifolia). Specific phytochemical profiles of the major volatiles were determined for each species. Still, borneol was the dominant compound in EOs of three yarrow species: A. distans, A. lingulata, and A. millefolium. Other major compounds were 1,8-cineole in A. crithmifolia, camphor in A. grandifolia, and artemisia ketone in A. nobilis EO. Antimicrobial activities of EOs were examined by a microdilution method revealing inhibitory action against all 15 studied strains in the range of 0.07-20.00 mg/mL (MICs). High toxicity (LC50 <100 µg/mL) in Artemia salina acute toxicity bioassay was a shared characteristic, appearing in five out of six tested EOs, i. e., those isolated from A. lingulata, A. millefolium, A. distans, A. nobilis, and A. grandifolia. In both types of biological tests, A. lingulata EO has shown the highest and A. crithmifolia EO the lowest potential. The strongest activities, recorded for EO of A. lingulata, which was the only studied species from the section Anthemoideae, may be explained by its somewhat unusual chemical composition. According to the obtained results, the second dominant compound of this EO is γ-palmitolactone, which may be a significant agent regarding the antimicrobial activity and toxicity to crustaceans, either on its own or in union with other components, and it may be a suitable topic for further research.

Chemical Composition of Volatiles of Eight Geranium L. Species from Vlasina Plateau (South Eastern Serbia).

Geranium species are widely used in traditional medicine of Balkan. The aim of this work was to investigate and compare chemical composition of volatile fractions obtained by hydrodistillation from aerial parts of G. macrorrhizum, G. phaeum, G. sanguineum, G. robertianum, G. palustre, G. pyrenaicum, G. columbinum and G. lucidum as well as from underground parts of G. macrorrhizum and G. phaeum, originated from Vlasina plateau in South Eastern Serbia. The volatiles were analyzed using GC/MS and GC-FID. G. palustre volatiles have been studied for the first time with ß-selinene (18.6 %) as a characteristic compound. The cluster analysis revealed separation of volatiles into two main groups. Volatile fractions of G. macrorrhizum were separated from all other samples due to high sesquiterpene content (92.3 % in aerial and 94.6 % in underground parts). The volatile fractions of other samples were mainly composed of sesquiterpenes (10.8-61.8 %), diterpenes (12.9-43.0 %) and fatty acids and their derivatives (6.6-21.6 %) with the exception of volatile fraction of G. phaeum underground parts which was dominated only by fatty acids and their derivatives (76.6 %). The results presented in this article contribute to the knowledge on the chemistry of this genus and advances the knowledge on flora of southeast Serbia.

Epicuticular Waxes Provide Insights into Phytochemical Differentiation of Natural Populations of Pinus mugo Turra sensu stricto.

Phytochemical diversity and differentiation of nine native populations of Pinus mugo sensu stricto from Julian Alps, Southern Carpathians and Balkan Peninsula were analyzed in regard to their epicuticular wax compounds. GC/MS and GC-FID analyses of the hexane extracts of needle samples, collected from 118 individuals of P. mugo, revealed the presence of six diterpenes, three primary alcohols and 10 n-alkanes ranging from C19 to C29 (except n-alkane C21 ). According to simple linear regression, the contents of epicuticular wax compounds showed generally weak correlations with tested bioclimatic, orographic and geographic parameters, leading to assumption about their genetic conditioning. The multivariate statistical analyses suggested the existence of two chemical entities: the Alpine and the South Carpathian, while the Balkan populations appeared heterogeneous as three of them belonged to the Alpine and one to the South Carpathian group. The obtained results are largely consistent to those previously published in reference to morpho-anatomical and molecular characters of P. mugo, supporting the hypothesis of colonization of Balkan Peninsula from two different glacial refugia: from the Alps (across the Dinarides) and from the Southern Carpathians. Extending this study to the entire range of P. mugo complex should provide a clearer picture of its phytochemical diversity and differentiation.

Chemical characterization, in vitro biological activity of essential oils and extracts of three Eryngium L. species and molecular docking of selected major compounds.

Many Eryngium species have been traditionally used as ornamental, edible or medicinal plants. The gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) analyses have shown that the major compounds in the aerial parts were spathulenol (in E. campestre and E. palmatum oils) and germacrene D (in E. amethystinum oil). The main compounds in the root oil were nonanoic acid, 2,3,4-trimethylbenzaldehyde and octanoic acid for E. campestre, E. amethystinum and E. palmatum, respectively. All the oils expressed the highest potential against Gram-positive bacteria Staphylococcus aureus as well as Gram-negative Klebsiella pneumoniae and Proteus mirabilis. Molecular docking analysis was used for determining a potential antibacterial activity mechanism of compounds present in the essential oils. Molecular docking confirmed that the binding affinity of spathulenol to the active site of tyrosyl-tRNA synthetase was the highest among the tested dominant compounds. Regarding the total phenolic content (determined by the Folin-Ciocalteu assay) and flavonoid content (evaluated using aluminum nitrate nonahydrate), the highest amount was found in the ethyl acetate extract of E. palmatum. The results of DPPH and ABTS assay indicated that the highest antioxidant activity was present in the water extract of E. amethystinum. Extracts of the aerial parts presented as minimum inhibitory concentration (MIC) expressed the activity in the range 0.004-20.00 mg/mL, with the highest activity exhibited by the acetone and ethyl acetate extracts against Proteus mirabilis. The obtained results suggest that Eryngium species may be considered a beneficial native source of the compounds with antioxidant and antimicrobial properties.

Needle Terpenes as Chemotaxonomic Markers in Pinus: Subsections Pinus and Pinaster.

Chemical compositions of needle essential oils of 27 taxa from the section Pinus, including 20 and 7 taxa of the subsections Pinus and Pinaster, respectively, were compared in order to determine chemotaxonomic significance of terpenes at infrageneric level. According to analysis of variance, six out of 31 studied terpene characters were characterized by a high level of significance, indicating statistically significant difference between the examined subsections. Agglomerative hierarchical cluster analysis has shown separation of eight groups, where representatives of subsect. Pinaster were distributed within the first seven groups on the dendrogram together with P. nigra subsp. laricio and P. merkusii from the subsect. Pinus. On the other hand, the eighth group included the majority of the members of subsect. Pinus. Our findings, based on terpene characters, complement those obtained from morphological, biochemical, and molecular parameters studied over the past two decades. In addition, results presented in this article confirmed that terpenes are good markers at infrageneric level.

Chemical Composition of Volatiles; Antimicrobial, Antioxidant and Cholinesterase Inhibitory Activity of Chaerophyllum aromaticum L. (Apiaceae) Essential Oils and Extracts.

The present study reports the chemical composition of the headspace volatiles (HS) and essential oils obtained from fresh Chaerophyllum aromaticum root and aerial parts in full vegetative phase, as well as biological activities of their essential oils and MeOH extracts. In HS samples, the most dominant components were monoterpene hydrocarbons. On the other hand, the essential oils consisted mainly of sesquiterpenoids, representing 73.4% of the root and 63.4% of the aerial parts essential oil. The results of antibacterial assay showed that the aerial parts essential oil and MeOH extract have no antibacterial activity, while the root essential oil and extract showed some activity. Both of the tested essential oils exhibited anticholinesterase activity (47.65% and 50.88%, respectively); MeOH extract of the root showed only 8.40% inhibition, while aerial part extract acted as an activator of cholinesterase. Regarding the antioxidant activity, extracts were found to be more effective than the essential oils.

Chemotaxonomic Approach to the Central Balkan Sedum Species Based on Distribution of Triterpenoids in Their Epicuticular Waxes.

Triterpenoid distribution in epicuticular waxes of 20 central Balkan Sedum L. species and four out-groups of genera: Hylotelephium H. Ohba, Crassula L., Echeveria DC., and Kalanchoe Adans. were investigated for chemotaxonomic purposes. Identification and quantification of wax triterpenoids were performed by GC/MS and GC-FID analyses. Distribution of identified triterpenoids (oleanane, lupane, and taraxerane series), as a pattern in statistical analysis, indicated very good agreement with phylogeny and systematics, except members of series Rupestria Berger, in which case the distribution of triterpenoids did not support known classification in a satisfactory manner. According to the obtained clustering, Kalanchoe is shown as similar to Sedum samples, while the other out-groups were clearly separated.

Chemical Composition of Ballota macedonica Vandas and Ballota nigra L. ssp. foetida (Vis.) Hayek Essential Oils - The Chemotaxonomic Approach.

The essential oils isolated from fresh aerial parts of Ballota macedonica (two populations) and Ballota nigra ssp. foetida were analyzed by GC and GC/MS. Eighty five components were identified in total; 60 components in B. macedonica oil (population from the Former Yugoslav Republic of Macedonia), 34 components in B. macedonica oil (population from the Republic of Serbia), and 33 components in the oil of B. nigra ssp. foetida accounting for 93.9%, 98.4%, and 95.8% of the total oils, respectively. The most abundant components in B. macedonica oils were carotol (13.7 - 52.1%), germacrene D (8.6 - 24.6%), and (E)-caryophyllene (6.5 - 16.5%), while B. nigra ssp. foetida oil was dominated by (E)-phytol (56.9%), germacrene D (10.0%), and (E)-caryophyllene (4.7%). Multivariate statistical analyses (agglomerative hierarchical cluster analysis and principal component analysis) were used to compare and discuss relationships among Ballota species examined so far based on their volatile profiles. The chemical compositions of B. macedonica essential oils are reported for the first time.

Geographically Related Variation in Epicuticular Wax Traits of Pinus nigra Populations from Southern Carpathians and Central Balkans - Taxonomic Considerations.

The chemical composition of epicuticular waxes of nine populations from three Pinus nigra J. F. Arnold subspecies (namely subsp. nigra, subsp. banatica (Borbás) Novák, and subsp. pallasiana (Lamb.) Holmboe) from Southern Carpathians and central Balkan Peninsula were analyzed using GC/MS and GC/FID chromatography, and multivariate statistical techniques with respect to biogeography and taxonomy. In the needle waxes, four primary alcohols and 14 n-alkanes ranging from C21 to C33 were identified, and the most abundant compounds were the four odd-numbered n-alkanes C27 , C25 , C23 , and C29. Multivariate statistical analyses (CDA and CA) have shown existence of three P. nigra groups and suggested clinal differentiation as a mechanism of genetic variation across a geographic area: the first group consisted of the southernmost populations of subsp. pallasiana from Macedonia, the second consisted of the northernmost subsp. banatica populations from Romania, while all populations in Serbia described as three different subspecies (nigra, banatica, and pallasiana) formed the third group together with subsp. nigra population from Bosnia and Herzegovina. According to simple linear regression, geographic latitude and four bioclimatic parameters were moderately correlated with the contents of epicuticular wax compounds that are important in population discrimination, while stepwise multiple regression showed that latitude participated in most of the regression models for predicting the composition of the epicuticular waxes. These results agree with CDA and CA analysis, and confirmed the possibility of recognition of fine geographic differentiation of the analyzed P. nigra populations.

Distribution and variability of n-alkanes in epicuticular waxes of sedum species from the central Balkan Peninsula: chemotaxonomic importance.

For the first time, the n-alkane distribution and variability of the epicuticular waxes within 22 Sedum taxa was reported with focus on the chemotaxonomy of native Sedum representatives from the central Balkan Peninsula, compared to their relations with four other species of the Crassulaceae family. By GC/MS and GC-FID identification and quantification, it was established that n-alkanes C27 , C29 , C31 , C33 , and C35 were the dominant constituents of the examined epicuticular wax samples. Applying multivariate statistical analyses including agglomerative hierarchical clustering (AHC) and principal component analysis (PCA), the relation according to the n-alkane composition between the examined samples was established. It was shown that the n-alkane variability of the central Balkan Sedum species was considerable and that n-alkanes might not be very reliable taxonomic markers for these species.

Inferring the origin of rare fruit distillates from compositional data using multivariate statistical analyses and the identification of new flavour constituents.

BACKGROUND: In Serbia, delicatessen fruit alcoholic drinks are produced from autochthonous fruit-bearing species such as cornelian cherry, blackberry, elderberry, wild strawberry, European wild apple, European blueberry and blackthorn fruits. There are no chemical data on many of these and herein we analysed volatile minor constituents of these rare fruit distillates. Our second goal was to determine possible chemical markers of these distillates through a statistical/multivariate treatment of the herein obtained and previously reported data. RESULTS: Detailed chemical analyses revealed a complex volatile profile of all studied fruit distillates with 371 identified compounds. A number of constituents were recognised as marker compounds for a particular distillate. Moreover, 33 of them represent newly detected flavour constituents in alcoholic beverages or, in general, in foodstuffs. With the aid of multivariate analyses, these volatile profiles were successfully exploited to infer the origin of raw materials used in the production of these spirits. It was also shown that all fruit distillates possessed weak antimicrobial properties. CONCLUSION: It seems that the aroma of these highly esteemed wild-fruit spirits depends on the subtle balance of various minor volatile compounds, whereby some of them are specific to a certain type of fruit distillate and enable their mutual distinction.

Chemical and biological evaluation of Hypericum maculatum Crantz essential oil.

The chemical composition and the antimicrobial activity of the essential oil isolated from the aerial parts of Hypericum maculatum Crantz were determined. In total, 109 compounds were identified, with germacrene D (21.5%), nonane (6.5%), (E)-ß-farnesene (5.3%), δ-cadinene (4.5%), and ledol (4.4%) as the main constituents. The chemical compositions of this oil and of four previously studied H. maculatum oils were compared using multivariate statistical analyses, viz., agglomerative hierarchical cluster and principal component analyses. Based on the results, the interrelationship among the hitherto studied H. maculatum oil samples, including the oil characterized here, was discussed. The study of the antimicrobial potential of the oil against five bacterial and two fungal strains showed that the oil had mainly moderate antimicrobial effects.

Chemical composition and antimicrobial activity of the essential oil of Stachys officinalis (L.) Trevis. (Lamiaceae).

Characterization by GC-FID and GC/MS analyses of the Stachys officinalis (L.) Trevis. essential oil obtained by hydrodistillation of the aerial parts allowed the identification of 190 components that represented 97.9% of the total oil content. The main constituents identified were germacrene D (19.9%), ß-caryophyllene (14.1%), and α-humulene (7.5%). Terpenoids were by far predominant (89.4%), with sesquiterpene hydrocarbons (69.1%) and oxygenated sesquiterpenes (14.8%) being the most abundant compounds detected in the oil. Based on the present and previously published results, multivariate statistical comparison of the chemical composition of the essential oils was performed within the species. Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) of the data on the volatile profiles of S. officinalis taxa revealed no pronounced differences among the samples originated from the Balkan Peninsula. Additionally, the oil was screened for in vitro antibacterial and antifungal activity using the broth microdilution assay. The oil's best antimicrobial activities were obtained against the mold Aspergillus niger (minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of 2.5 and 5.0 mg/ml, resp.) and the yeast Candida albicans (MIC and MFC of 5.0 mg/ml).

Chemical composition and antioxidant and antimicrobial activities of essential oil of Allium sphaerocephalon L. subsp. sphaerocephalon (Liliaceae) inflorescences.

BACKGROUND: Allium sphaerocephalon is a wild-growing plant commonly used as an onion substitute. In this study the essential oil obtained from A. sphaerocephalon subsp. sphaerocephalon inflorescences was analysed by gas chromatography and gas chromatography/mass spectrometry and also evaluated for in vitro antioxidant and antimicrobial activities. The obtained results are reported for the first time and discussed with respect to the sulfur compounds detected in the essential oil. RESULTS: Ninty-one compounds were identified, representing 91.6% (w/w) of the total oil. Major constituents of the analysed sample were shyobunol (15.3%), ß-caryophyllene (8.1%), α-cadinol (7.8%), 3,5-diethyl-1,2,4-trithiolane (isomer II, 5.9%) and δ-cadinene (5.2%). The diluted oil had an antioxidant capacity of 160 000 ± 111 micromol α-tocopherol acetate equivalents g(-1). Its antimicrobial activity was evaluated against five bacterial and two fungal strains using the broth microdilution assay. Among the micro-organisms tested, the most susceptible strains were Pseudomonas aeruginosa (minimal inhibitory/bactericidal concentration = 0.08/2.5 mg mL(-1)) and Aspergillus niger (minimal inhibitory/fungicidal concentration = 0.31/0.63 mg mL(-1)). CONCLUSION: The results showed that the examined species had strong antioxidant and antimicrobial properties and are in accordance with the popular use of plants belonging to the genus Allium in traditional medicine, emphasising the necessity of further detailed study of the active principles in Allium species.

Needle volatiles as chemophenetic markers in differentiation of natural populations of Abies alba, A. x borisii-regis, and A. cephalonica.

Seventeen native fir populations from the presumed zones of natural hybridization in the Balkan Peninsula were analyzed using headspace needle volatiles and GC-MS/FID analyses. The multivariate statistical analyses revealed clear differentiation between populations of Abies alba and A. cephalonica, suggesting that Silver and Greek Fir are characterized by distinct volatile profiles. Three populations from northern and central Greece identified as A. x borisii-regis shared the volatile profiles with both of the supposed parent species, whereby profile frequencies changed clinally along the latitudinal gradient connecting the ranges of parent species. Therefore, observed geographical distribution of volatile entities support the hypothesis that studied A. x borisii-regis populations are of secondary origin, formed by hybridization between A. alba and A. cephalonica. All three populations should be included in conservation projects, as the genetic constitution of these polymorphic fir populations may contain useful genetic variation necessary for survival regarding the future environmental challenges in this area.

Chemical Composition and Antibacterial Activity of the Bulgarian Endemic Species Achillea thracica from its Natural Habitat, and in vitro Propagated and ex vitro Established Plants.

The present study reports the chemical composition of headspace volatiles (HS) and acetone extracts of the endemic Bulgarian species Achillea thracica Velen. from its natural habitat (N), in vitro propagated (IN) and ex vitro established (EX) plants. Additionally, acetone extracts were tested by a disk diffusion method for antibacterial activity. Irregular monoterpenes were the most abundant HS volatile constituents, while 0,0-dimethyl quercetin was the most abundant flavonoid in the acetone extracts. The secondary metabolites of A. thracica grown in its natural habitat (N), propagated in vitro (IN) and ex vitro established (EX) showed that the qualitative composition is mutually similar, but there are differences in the quantitative composition. Considering antibacterial activity, IN and EX samples showed moderate activity against Pseudomonas aeruginosa and Escherichia coli.

Essential Oil Chemical Composition and Headspace Volatiles Profile of Achillea coarctata from Serbia.

This study reports the essential oil composition and headspace volatiles profile of Achillea coarctata Poir. from Serbia. The inflorescences, stems and leaves, and the aerial parts of A. coarctata were analyzed separately. Germacrene D, α-terpineol and 1,8-cineole were the main constituents of the aerial parts essential oil; 1,8-cineole, cis-cadin-4-en-7-ol and α-terpineol were the most dominant compounds in the inflorescence essential oil, while the most abundant components in the stem and leaf oil were germacrene D, cis-cadin-4-en-7-ol and ledol. The percentages of monoterpenoids and sesquiterpenoids in the aerial parts were the same, while there were differences in distribution of these compound classes in inflorescence and stem and leaf essential oils. The major components of the headspace volatiles were the same for aerial parts, inflorescence and stem and leaves: 1,8-cineole, ß-pinene and α-pinene.

Distribution and Taxonomic Significance of Secondary Metabolites Occurring in the Methanol Extracts of the Stonecrops (Sedum L., Crassulaceae) from the Central Balkan Peninsula.

The present study is engaged in the chemical composition of methanol extracts of Sedum taxa from the central part of the Balkan Peninsula, and representatives from other genera of Crassulaceae (Crassula, Echeveria and Kalanchoe) considered as out-groups. The chemical composition of extracts was determined by HPLC analysis, according to retention time of standards and characteristic absorption spectra of components. Identified components were considered as original variables with possible chemotaxonomic significance. Relationships of examined plant samples were investigated by agglomerative hierarchical cluster analysis (AHC). The obtained results showed how the distribution of methanol extract components (mostly phenolics) affected grouping of the examined samples. The obtained clustering showed satisfactory grouping of the examined samples, among which some representatives of the Sedum series, Rupestria and Magellensia, are the most remote. The out-group samples were not clearly singled out with regard to Sedum samples as expected; this especially applies to samples of Crassula ovata and Echeveria lilacina, while Kalanchoe daigremontiana was more separated from most of the Sedum samples.