Antioxidative and anti-inflammatory activities of Erica spiculifolia extracts and fractions.

There is little data on the phytochemical/pharmacological properties of Erica spiculifolia Salisb. (syn. Bruckentalia spiculifolia (Salisb.) Rchb.). This study examines the antioxidative and anti-inflammatory activities of different extracts and fractions of E. spiculifolia in vitro on isolated rat peritoneal macrophages, in the carrageenan-induced rat paw oedema test, BSA test, and two complementary antioxidant assays. Ethanolic extracts of leaves, flowers, and aboveground parts, and petroleum ether, ether, ethyl acetate, and water fractionations of the ethanol extract of E. spiculifolia applied at doses of 50-200 mg/kg p.o. exhibited dose-dependent anti-inflammatory activity comparable with indomethacin. All tested samples, except for the petroleum ether fraction, exerted excellent in vitro antioxidant activity, and all of them exhibited significant and similar inhibition of BSA denaturation comparable with diclofenac. Ethanolic extract of the aboveground parts obtained by percolation, ethyl acetate and water fractions had the highest efficiency, attenuating inflammation by more than 50% in the lowest applied concentration alongside exceptional radical scavenging activity.

Antidiabetic effects of polyherbal mixture made of Centaurium erythraea, Cichorium intybus and Potentilla erecta.

ETHNOPHARMACOLOGICAL RELEVANCE: The polyherbal mixture made of Centaurium erythraea aerial parts and Cichorium intybus roots and Potentilla erecta rhizomes has been used for centuries to treat both the primary and secondary complications of diabetes. AIM OF THE STUDY: As a continuation of our search for the most effective herbal mixture used as an ethnopharmacological remedy for diabetes, this study aimed to compare the in vitro biological activities of this polyherbal mixture and its individual ingredients, and, most importantly, to validate the ethnopharmacological value of the herbal mixture through evaluation of its phytochemical composition, its potential in vivo toxicity and its effect on diabetes complications. MATERIALS AND METHODS: Phytochemical analysis was performed using HPLC-UV. Antioxidant activity was estimated via the DPPH test. Potential cytotoxicity/anticytotoxicity was assessed using an in vitro RBCs antihemolytic assay and an in vivo sub-chronic oral toxicity method. Antidiabetic activity was evaluated using an in vitro α-amylase inhibition assay and in vivo using a chemically induced diabetic rat model. RESULTS: The HPLC-UV analysis revealed the presence of p-hydroxybenzoic acid, p-hydroxybenzoic acid derivative, catechin, five catechin derivatives, epicatechin, isoquercetin, hyperoside, rutin, four quercetin derivatives, caffeic acid, and four caffeic acid derivatives in the polyherbal mixture decoction. Treatment with the decoction has shown no toxic effects. The antioxidant and cytoprotective activities of the polyherbal mixture were higher than the reference's ones. Its antidiabetic activity was high in both in vitro and in vivo studies. Fourteen days of treatment with the decoction (15 g/kg) completely normalized blood glucose levels of diabetic animals, while treatments with insulin and glimepiride only slightly lowered glycemic values. In addition, lipid status of treated animals as well as levels of serum AST, ALT, ALP, creatinine, urea and MDA were completely normalized. In addition, the polyherbal mixture completely restored the histopathological changes of the liver, kidneys and all four Cornu ammonis regions of the hippocampus. CONCLUSIONS: The polyherbal mixture was effective in the prevention of both primary and secondary diabetic complications such as hyperlipidemia, increased lipid peroxidation, non-alcoholic fatty liver disease, nephropathy and neurodegeneration.

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.

Volatile Organic Compound Composition and Glandular Trichome Characteristics of In Vitro Propagated Clinopodium pulegium (Rochel) Bräuchler: Effect of Carbon Source.

Clinopodium pulegium (Rochel) Bräuchler (Lamiaceae) is an endangered species endemic to the Southern Carpathians. It is characterized by the production of high amounts of essential oils, which emit volatile organic compounds (VOCs) that have an essential role in biotic and abiotic stress responses and in plant-plant and plant-insect interactions. The present study was initiated to phytochemically examine the influence of different carbon sources in the nutrition medium on VOC emissions of micropropagated C. pulegium plants, using gas chromatography-mass spectrometry analysis of headspace VOCs. The volatile profiles were subjected to multivariate analysis with respect to the presence, concentration and type of carbon source in the nutrient medium. In addition, the effect of different carbohydrates on the density and size of the leaf glandular trichomes, the main structures involved in the emission of VOCs, was determined. A total of 19 VOCs, primarily belonging to mono- and sesquiterpenes previously described in plants, were tentatively identified. Six VOCs were produced at levels higher than 2% of the total VOC emission, dominated by pulegone, ß-pinene and menthone. Inclusion of the carbohydrates in the culture media affected the production of the main leaf trichome-associated volatile allelochemicals although the qualitative composition of the volatiles changed only slightly. Multivariate analysis showed that the concentration, rather than the carbohydrate type, influenced the VOC profile.

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.

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.

Phytochemistry, Toxicology and Therapeutic Value of Petasites hybridus Subsp. Ochroleucus (Common Butterbur) from the Balkans.

Petasites hybridus (Common butterbur) is extensively used in traditional medicine, and is currently gaining interest and popularity as a food supplement and for its medicinal properties. It contains a large number of active compounds of potential therapeutic activity, but also toxic pyrrolizidine alkaloids. Science-based information is needed to support the developing modern use of this plant, and to direct continued safe practice in traditional medicine. The present study focused on the essential oils from leaves and rhizomes of the understudied P. hybridus ssp. ochroleucus from the Balkans, and evaluated its phytochemistry and potential therapeutic activities (antimicrobial, antioxidant, anti-cholinesterase and anti-inflammatory), as well its toxicology potential (acute toxicity in insects and mice). We studied the essential oils, which are not commonly used in traditional practices, but have a potential for safe use since the toxic pyrrolizidine alkaloids, which are non-volatiles, are usually not present in the distilled essential oils. Pyrrolizidine alkaloids were indeed not detected in the essential oils; ingestion of the essential oils did not induce toxicity signs in mice, and topical application did not elicit skin irritation in humans. The essential oils had no antimicrobial properties against 20 pathogenic bacterial strains, but demonstrated good local anti-inflammatory activity in a Carrageenan-induced paw edema test. An insect toxicity test demonstrated that the leaf essential oil is an efficient insect repellent, and the demonstrated anti-cholinesterase activity suggests a potential for the treatment of neurological conditions. Isopetasin, a sesquiterpene found in plants of the genus Petasites, known to have anti-inflammatory effects, was present only in the rhizomes essential oil (3.9%), and sesquiterpene lactones concentrations were high, likely contributing to the antioxidant activity.

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.

Robust kHz-linewidth distributed Bragg reflector laser with optoelectronic feedback.

We demonstrate a combination of optical and electronic feedback that significantly narrows the linewidth of distributed Bragg reflector lasers (DBRs). We use optical feedback from a long external fiber path to reduce the high-frequency noise of the laser. An electro-optic modulator placed inside the optical feedback path allows us to apply electronic feedback to the laser frequency with very large bandwidth, enabling robust and stable locking to a reference cavity that suppresses low-frequency components of laser noise. The combination of optical and electronic feedback allows us to significantly lower the frequency noise power spectral density of the laser across all frequencies and narrow its linewidth from a free-running value of 1.1 MHz to a stabilized value of 1.9 kHz, limited by the detection system resolution. This approach enables the construction of robust lasers with sub-kHz linewidth based on DBRs across a broad range of wavelengths.

Phytochemical composition and biological activities of native and in vitro-propagated Micromeria croatica (Pers.) Schott (Lamiaceae).

MAIN CONCLUSION: In vitro culture conditions and kinetin induced quantitative modifications in the production of the major volatile constituents in Micromeria croatica plantlets. Antimicrobial activity of methanolic extracts obtained from micropropagated and wild-growing plants was evaluated. Micromeria spp. are aromatic plants, many of which were shown to exhibit various biological effects. The present study aimed to determine the content and the composition of the essential oil of in vitro-cultured Micromeria croatica (Pers.) Schott and to evaluate the in vitro antimicrobial activity of its methanolic extract, in order to compare its phytochemical profile and biological activity with wild-growing plants. Shoots regenerated on MS medium without plant growth regulators (PGRs) or supplemented with kinetin were used for phytochemical analysis. Essential oils from both native plant material and in vitro-cultivated M. croatica plants, with a total of 44 identified constituents, were dominated by oxygenated monoterpenes. Borneol was the main component detected in wild-growing plants (25.28%) and micropropagated plants grown on PGR-free medium (20.30%). Kinetin treatment led to increased oil yield and favored the production of oxygenated monoterpenes, dominated by geranial (33.53%) and cis-p-mentha-1(7),8-dien-ol (23.69%). The percentage of total sesquiterpenoids in micropropagated plant material was considerably lower than in wild-growing plants. In vitro culture conditions and PGRs affected the production of essential oils, inducing quantitative modifications in the production of the major volatile constituents in M. croatica plantlets. The antimicrobial activity of M. croatica methanolic extracts was investigated using the broth microdilution method. Extracts obtained from in vitro cultures generally exhibited greater antibacterial potential, compared to wild-growing plants. Among six bacterial strains tested, Bacillus cereus and Staphylococcus aureus were the most sensitive microorganisms. The present study provided evidence that in vitro culture conditions might favorably affect the antimicrobial activity of M. croatica methanolic extracts.

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.

Hypogymnia tubulosa extracts: chemical profile and biological activities.

This study reports for the first time in the chemical composition of acetone, ether, ethyl acetate and dichloromethane extracts of Hypogymnia tubulosa determined by HPLC-UV, GC-FID and GC-MS as well as effect of H. tubulosa acetone extract on micronucleus distribution on human lymphocytes and on cholinesterase activity. Additionally, antioxidant (estimated via DPPH, ABTS, TRP, CUPRAC and TPC assays) and antibacterial activity against two Gram-positive and three Gram-negative bacteria were also determined. The HPLC-UV analysis revealed the presence of depsidones, 3-hydroxyphysodic, 4-O-methyl physodic acid, physodic and physodalic acid together with two depsides, atranorin and chloroatranorin. GC-FID and GC-MS analyses enabled the identification of atranol, chloroatranol, atraric acid, olivetol, olivetonide and 3-hydroxyolivetonide as the main components. The results of present study show that H. tubulosa acetone extract is a promising candidate for in vivo experiments considering antioxidant activity.

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.

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 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.

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.

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.