Composition of Leaf n‐Alkanes in Three Satureja montana L. Subspecies from the Balkan Peninsula: Ecological and Taxonomic Aspects

The composition of the epicuticular leaf n‐alkanes of eight populations of three Satureja montana subspecies (S. montana L. subsp. pisidica (Wettst.) ?ili? , S. montana L. subsp. montana, and S. montana L. subsp. variegata (Host ) P. W. Ball ), from central and western areas of the Balkan Peninsula was characterized by GC‐FID and GC/MS analyses. In the leaf waxes, 15 n‐alkane homologs with chain‐lengths ranging from C₂₁ to C₃₅ were identified. The main n‐alkane in almost all samples was n‐nonacosane (C₂₉), but differences in the contents of three other dominant n‐alkanes allowed separating the coastal from the continental populations. The diversity and variability of the epicuticular‐leaf‐n‐alkane patterns and their relation to different geographic and bioclimatic parameters were analyzed by several statistical methods (principal component, discriminant, and cluster analyses as well as the Mantel test). All tests showed a high correlation between the leaf n‐alkane pattern and the geographical distribution of the investigated populations, confirming the differentiation between S. montana subsp. pisidica and the other two subspecies. The S. montana subsp. variegata and S. montana subsp. montana populations are geographically closer and their differentiation according to the leaf‐n‐alkane patterns was not clear, even though there was some indication of discrimination between them. Moreover, most of the bioclimatic parameters related to temperature were highly correlated with the differentiation of the coastal and the continental populations.     

Chemotaxonomic and Micromorphological Traits of Satureja montana L. and S. subspicata Vis. (Lamiaceae)

Satureja montana and S. subspicata are used as spice, pepper substitute, for preparing tea, juice, and as a medicine. Fourteen populations (seven per species) of Satureja montana L. and S. subspicata Vis . growing in Croatia were examined to determine the chemical composition of the essential oil (analyzed by GC‐FID and GC/MS), the content of macroelements (Na, K, Ca, Mg) and trace elements (B, Fe, Cu, Mn, Zn, Al, Pb, Cr, Cd, Ni, Hg, As) analyzed by ICP‐AES, antioxidant compounds (analyzed by UV/VIS spectrophotometer), and the types and distribution of trichomes (analyzed by scanning electron microscopy). The main constituents of the essential oil were carvacrol and thymol in S. montana (all populations belong to one phenol chemotype), while α‐eudesmol, β‐eudesmol, and spathulenol dominated in S. subspicata (three chemotypes could be distinguished). Both species possess considerably higher quantities of Ca and Mg, and moderate concentrations of K and Na, while Hg and As levels were below the limit of quantification. Non‐glandular trichomes, peltate trichomes, and three types of capitate trichomes were observed on leaves, stem, calyx, and corolla.     

Volatile Compounds of Viola odorata Absolutes: Identification of Odorant Active Markers to Distinguish Plants Originating from France and Egypt

Absolutes isolated from Viola odorata leaves, valuable materials for the flavor and fragrance industry, were studied. Violets are mainly cultivated in France and Egypt and extracted locally. The absolutes of the two origins showed different olfactory profiles both in top and heart notes, as evidenced by sensory analysis. The aims of this study were i) to characterize the volatile compounds, ii) to determine the odorant‐active ones, and iii) to identify some markers of the plant origin. Two complementary analytical methods were used for these purposes, i.e., headspace solid‐phase microextraction (HS‐SPME) using different fiber coatings followed by GC/MS analysis and gas chromatography – olfactometry/mass spectrometry (GC‐O/MS) applied to violet leaf extracts. From a total of 70 identified compounds, 61 have never been reported so far for this species, 17 compounds were characterized by both techniques (with seven among them known from the literature), 23 compounds were solely identified by HS‐SPME GC/MS (among them only two being already mentioned as components of violet absolutes in the literature), and, finally, 30 compounds were only identified by GC‐O/MS. According to the HS‐SPME GC/MS analyses, ethyl hexanoate and (2E,6Z)‐nona‐2,6‐dienol were specific volatile compounds of the sample with French origin, while (E,E)‐hepta‐2,4‐dienal, hexanoic acid, limonene, tridecane, and eugenol were specific of the samples with Egyptian origin. Additional compounds that were not detected by HS‐SPME GC/MS analysis were revealed by GC‐O analyses, some of them being markers of origin. Pent‐1‐en‐3‐ol, 3‐methylbut‐2‐enal, 2‐methoxy‐3‐(1‐methylethyl)pyrazine, 4‐ethylbenzaldehyde, β‐phenethyl formate, and 2‐methoxy‐3‐(2‐methylpropyl)pyrazine revealed to be odorant markers of the French sample, whereas cis‐rose oxide, trans‐rose oxide, and 3,5,5‐trimethylcyclohex‐2‐enone were odorant markers of the Egyptian samples.     

Comprehensive Study of Mediterranean (Croatian) Propolis Peculiarity: Headspace,Volatiles, Anti‐Varroa‐Treatment Residue,Phenolics, and Antioxidant Properties

Eight propolis samples from Croatia were analyzed in detail, to study the headspace, volatiles, anti‐Varroa‐treatment residue, phenolics, and antioxidant properties. The samples exhibited high qualitative/quantitative variability of the chemical profiles, total phenolic content (1,589.3–14,398.3 mg GAE (gallic acid equivalent)/l EtOH extract), and antioxidant activity (11.1–133.5 mmol Fe²⁺/l extract and 6.2–65.3 mmol TEAC (Trolox^® equivalent antioxidant capacity)/l extract). The main phenolics quantified by HPLC‐DAD at 280 and 360 nm were vanillin, p‐coumaric acid, ferulic acid, chrysin, galangin, and caffeic acid phenethyl ester. The major compounds identified by headspace solid‐phase microextraction (HS‐SPME), simultaneous distillation extraction (SDE), and subsequent GC‐FID and GC/MS analyses were α‐eudesmol (up to 19.9%), β‐eudesmol (up to 12.6%), γ‐eudesmol (up to 10.5%), benzyl benzoate (up to 28.5%), and 4‐vinyl‐2‐methoxyphenol (up to 18.1%). Vanillin was determined as minor constituent by SDE/GC‐FID/MS and HPLC‐DAD. The identified acaricide residue thymol was ca. three times more abundant by HS‐SPME/GC‐FID/MS than by SDE/GC‐FID/MS and was not detected by HPLC‐DAD.     

Antioxidant Capacity and Chemical Profiles of Satureja montana L. Honey: Hotrienol and Syringyl Derivatives as Biomarkers

The present study is focused on the antioxidant capacity and chemical profiling of eight Croatian Satureja montana L. honey samples. Among the 20 compounds obtained by headspace solid‐phase microextraction (HS‐SPME) and identified by GC‐FID and GC/MS analyses, hotrienol was predominant (75.9–81.7%). The honey matrix volatile/semivolatile profile was investigated by ultrasonic solvent extraction (USE) followed by GC‐FID and GC/MS analyses. The major compounds identified by this latter method were the sinapic‐acid derivatives methyl syringate (36.2–72.8%) and syringaldehyde (2.2–43.1%). Direct, targeted HPLC‐DAD analyses of the native honey samples revealed the presence of methyl syringate (7.10–39.60 mg/kg) and syringic acid (0.10–1.70 mg/kg). In addition, the total phenolic content of the samples was determined by the Folin? Ciocalteu assay (311.0–465.9 mg GAE/kg), and the antioxidant capacity was evaluated by the DPPH radical‐scavenging activity (0.5–1.0 mmol TEAC/kg) and the ferric reducing antioxidant power (2.5–5.1 mmol Fe²⁺/kg).     

Volatiles as Chemosystematic Markers for Distinguishing Closely Related Species within the Pinus mugo Complex

Headspace solid‐phase microextraction (HS‐SPME) coupled to GC/MS analysis was used to identify the constituents of pine‐needle volatiles differentiating three closely‐related pine species within the Pinus mugo complex, i.e., P. uncinata Ramond ex DC., P. uliginosa G.E.Neumann ex Wimm ., and P. mugo Turra . Moreover, chemosystematic markers were proposed for the three analyzed pine species. The major constituents of the pine‐needle volatiles were α‐pinene (28.4%) and bornyl acetate (10.8%) for P. uncinata, δ‐car‐3‐ene (21.5%) and α‐pinene (16.1%) for P. uliginosa, and α‐pinene (20%) and δ‐car‐3‐ene (18.1%) for P. mugo. This study is the first report on the application of the composition of pine‐needle volatiles for the reliable identification of closely‐related pine species within the Pinus mugo complex.     

Essential Oil Composition of Three Globularia Species

The chemical composition of the essential oils obtained by hydrodistillation from the aerial parts of Globularia cordifolia L., G. meridionalis (Podp.) O.Schwarz , and G. punctata Lapeyr . was characterized by GC‐FID and GC/MS analyses. Among the 33 identified compounds, the most abundant present in all investigated samples were oct‐1‐en‐3‐ol (2.9–47.0%), 6‐(1,5‐dimethylhex‐4‐enyl)‐3‐methylcyclohex‐2‐enone (8.2–40.9%), and fukinanolid (7.4–31.6%). Multivariate statistical analyses (PCA and HCA) of the hitherto studied Globularia volatile compounds confirmed to some extent the assumed phylogenetic relationships of the Globularia species studied, including the close relationship between the morphologically similar species G. cordifolia and G. meridionalis, but also evidenced several discrepancies in the current classification of Globularia species.     

Variation in Volatile Leaf Oils of Twelve Eucalyptus Species Harvested from Hajeb Layoun Arboreta (Tunisia)

Hydrodistillation of the dried leaves of twelve species of the genus Eucalyptus L' Hér ., i.e., E. brockwayi C. A. Gardn ., E. gracilis F. Muell ., E. gillii Maiden , E. largiflorens F. Muell ., E. loxophleba Benth ., E. occidentalis Endl ., E. oldfieldii F. Muell ., E. salmonophloia F. Muell ., E. sargentii Maiden , E. stricklandii Maiden , E. torquata Luehm ., and E. woodwardii Maiden , harvested from Hajeb Layoun arboreta (region of Kairouan, central Tunisia) in January 2005, afforded essential oils in yields varying from 0.5±0.1 to 5.7±0.5%, dependent on the species. E. sargentii and E. brockwayi provided the highest and the lowest percentage of essential oil amongst all the species examined, respectively. Analysis by GC (RI) and GC/MS allowed the identification of 133 components, representing 92.9–98.8% of the total oil. The contents of the different samples varied according to the species. The main components were 1,8‐cineole, terpinen‐4‐ol, α‐pinene ( 2 ), p‐cymene, aromadendrene ( 1 ), globulol ( 5 ), trans‐pinocarveol ( 6 ), spathulenol ( 7 ), β‐eudesmol, torquatone ( 3 ), and 4‐methylpentan‐2‐yl acetate ( 8 ). The principal component analysis and the hierarchical clustering indicated that the volatile leaf oil composition of the twelve Eucalyptus species could be clearly differentiated.     

Variability of n‐Alkanes and Nonacosan‐10‐ol in Natural Populations of Picea omorika

This is the first report of population variability of the contents of n‐alkanes and nonacosan‐10‐ol in the needle epicuticular waxes of Serbian spruce (Picea omorika). The hexane extracts of needle samples originated from three natural populations in Serbia (Vranjak, Zmajeva?ki potok, and Mile?evka Canyon) were investigated by GC and GC/MS analyses. The amount of nonacosan‐10‐ol varied individually from 50.05 to 74.42% (65.74% in average), but the differences between the three investigated populations were not statistically confirmed. The results exhibited variability of the composition of n‐alkanes in the epicuticular waxes with their size ranging from C₁₈ to C₃₅. The most abundant n‐alkanes were C₂₉, C₃₁, and C₂₇ (35.22, 13.77, and 12.28% in average, resp.). The carbon preference index of all the n‐alkanes (CPI_total) of the P. omorika populations (average of populations I–III) ranged from 3.3 to 11.5 (mean of 5.9), while the average chain length (ACL) ranged from 26.6 to 29.2. The principal component and cluster analyses of the contents of nine n‐alkanes showed the greatest difference for the population growing in the Mile?evka Canyon. The obtained results were compared with previous literature data given for other Picea species, and this comparison was briefly discussed.     

Chemical composition and antimicrobial activity of volatiles from Degenia velebitica, a European stenoendemic plant of the Brassicaceae family

Free and glucosidic bound leaf volatiles of Degenia velebitica were isolated and fractionated simultaneously into H₂O‐soluble, H₂O‐insoluble, and highly volatile compounds by hydrodistillation–adsorption (HDA) and analyzed by GC/MS. Among the 24 constituents identified, the main compounds obtained by the HDA method were S‐ and/or N‐atom containing compounds, i.e., 6‐(methylsulfanyl)hexanenitrile ( 10 ; 26.78%), dimethyl trisulfide ( 6 ; 26.35%), 3,4,5‐trimethylpyrazole ( 17 ; 13.33%), hex‐5‐enenitrile ( 2 ; 10.11%), dimethyl tetrasulfide ( 8 ; 4.93%), and pent‐4‐enyl isothiocyanate ( 7 ; 4.45%). In addition, O‐glycosidically bound volatiles and free volatiles were isolated by solvent extraction. Sixteen volatile O‐aglycones and twelve free volatile components were identified. The main O‐aglycones were eugenol ( 19 ; 24.15%), 2‐methoxy‐4‐vinylphenol ( 11 ; 11.50%), and benzyl alcohol ( 20 ; 9.49%), and the main free volatiles were (9Z,12Z)‐octa‐9,12‐dienic acid (38.35%), hexadecanoic acid (22.64%), and phytol (5.80%). The H₂O‐soluble volatile fraction obtained by HDA, containing mostly glucosinolate degradation products and 3,4,5‐trimethylpyrazole ( 17 ), was evaluated for antimicrobial activity by determining inhibition zones with the diffusion method as well as minimal inhibitory concentrations (MIC) and minimal microbicidal concentrations (MMC) with the micro‐dilution method. The fraction expressed activity against the tested Gram‐positive and Gram‐negative bacteria as well as against yeast, with MIC values equal to or lower than 16.7 μg/ml.     

Variability of the Needle Essential Oils of Juniperus deltoides R.P.Adams from Different Populations in Serbia and Croatia

The essential‐oil compositions of one Croatian and three Serbian populations of Juniperus deltoides R.P.Adams have been determined by GC/MS analysis. In total, 147 compounds were identified, representing 97.3–98.3% of the oil composition. The oils were dominated by monoterpenes, which are characteristic components for the species of the section Juniperus. Two monoterpenes, α‐pinene and limonene, were the dominant constituents, with a summed‐up average content of 49.45%. Statistical methods were used to determine the diversity of the terpene classes and the common terpenes between the newly described J. deltoides populations from Serbia and Croatia. Only reports on several specimens from this species have been reported so far, and there are no studies that treat population diversity. Cluster analysis of the oil contents of 21 terpenes showed high correlation with the geographical distribution of the populations, separating the Croatian from the Serbian populations. The comparison of the essential‐oil compositions obtained in the present study with literature data, showed the separation of J. deltoides and J. oxycedrus ssp. oxycedrus from the western Mediterranean region.     

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 β‐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.     

Variability in the Essential‐Oil Composition of Sideritis scardica Griseb. from Native Bulgarian Populations

The essential‐oil composition of six native populations of Sideritis scardica from Bulgaria was studied by GC‐FID and GC/MS analyses. Altogether, 37 components, representing 73.1 to 79.2% of the total oil content were identified. Among them, α‐pinene (4.4–25.1%), β‐pinene (2.8–18.0%), oct‐1‐en‐3‐ol (2.3–8.0%), phenylacetaldehyde (0.5–9.5%), β‐bisabolene (1.3–11.0%), benzyl benzoate (1.1–14.3%), and m‐camphorene ( 1 ; 0.3–12.4%) were the main compounds. All samples were characterized by low contents of oxygenated mono‐ and sesquiterpenes (≤1.6 and 2.3%, resp.). Principal component analysis (PCA) and cluster analysis (CA) showed a significant variability in the chemical composition of the studied samples as well as a correlation between the oil profiles and the ecological conditions of the natural habitats of S. scardica.     

Chemosystematic comparison of essential oils from eight species of four genera of the New Zealand Apiaceae

The composition of the essential oils from the seeds of eight species and the leaves of two species of the New Zealand Apiaceae was determined. Gingidia and Scandia species provided seed oils in high yield (>6%) that contained high proportions of phenylpropanoids. Anisotome haastii provided an oil in moderate yield (1.7%), over half of which was comprised of monoterpenoids and no phenylpropanoids. The four Aciphylla species afforded seed essential oils in low yield (0.5%). Three of these contained neither terpenoids nor phenylpropanoids, but contained fatty acids instead. The oil from Aciphylla squarrosa was different as it contained an abundance of phenylpropanoids. The composition of the leaf essential oils of Scandia rosifolia and Gingidia montana differed significantly from those of the corresponding seed oils. Outstandingly, the seed essential oil of G. montana contained 62% estragole and that of Scandia geniculata contained 79% dill apiole. The results of this present work support the conclusions of Mitchell et al. (1998), about the taxonomy of these genera that were drawn from their work with ribosomal DNA sequences viz., that the Gingidia and Scandia genera were closely related to each other and that the Anisotome and Aciphylla genera were not closely related to each other, but were weakly related to the Gingidia and Scandia genera.     

Essential Oil Variation among Natural Populations of Lavandula multifida L. (Lamiaceae)

Volatiles from twelve wild Tunisian populations of Lavandula multifida L. growing in different bioclimatic zones were assessed by GC (RI) and GC/MS. Thirty‐six constituents, representing 83.48% of the total oil were identified. The major components at the species level were carvacrol (31.81%), β‐bisabolene (14.89%), and acrylic acid dodecyl ester (11.43%). These volatiles, together with α‐pinene, were also the main compounds discriminating the populations. According to these dominant compounds, one chemotype was revealed, a carvacrol/β‐bisabolene/acrylic acid dodecyl ester chemotype. However, a significant variation among the populations was observed for the majority of the constituents. A high chemical‐population structure, estimated both by principal component analysis (PCA) and unweighted pair group method with averaging (UPGMA) cluster analysis based on Euclidean distances, was observed. Both methods allowed separation of the populations in three groups defined rather by minor than by major compounds. The population groups were not strictly concordant with their bioclimatic or geographic location. Conservation strategies should concern all populations, because of their low size and their high level of destruction. Populations exhibiting particular compounds other than the major ones should be protected first.     

Volatile Composition Screening of Salix spp. Nectar Honey: Benzenecarboxylic Acids,Norisoprenoids, Terpenes,and Others

Salix spp. nectar honey volatiles of Croatian origin were analyzed by headspace solid‐phase microextraction (HS‐SPME) and ultrasonic solvent extraction (USE), followed by gas chromatography and mass spectrometry (GC, GC/MS). Isolated volatiles were found in the honey headspace and extracts with almost exclusive distribution of several abundant compounds (e.g., phenylacetic acid, pinocembrin, 8‐hydroxy‐4,7‐dimethylcoumarin, and 3‐hydroxy‐trans‐β‐damascone in the extracts, or safranal and lilac alcohols in the headspace). Comparison with Croatian Salix spp. honeydew honey revealed similarities regarding distribution of important shikimate pathway derivatives (e.g., high percentage of phenylacetic acid) and several norisoprenoids (α‐isophorone and 4‐oxoisophorone). On the other hand, distinct features of this honey were occurrence of compounds such as pinocembrin, 8‐hydroxy‐4,7‐dimethylcoumarin, phenylacetonitrile, norisoprenoids (major ones: 3‐hydroxy‐trans‐β‐damascenone and trans‐β‐damascone), more pronounced variability of linalool‐derived compounds, as well as the abundance of 3‐methylpropanoic acid, 3‐methylbutanoic acid, 2‐methylpentanoic acid, and 3‐methylpentan‐1‐ol.     

Volatile‐Oils Composition,and Bioactivity of the Essential Oils of Plectranthus barbatus,P. neochilus,and P. ornatus Grown in Portugal

Volatile‐oils chemical composition and bioactivity of the essentail oils from Plectranthus barbatus, P. neochilus, and P. ornatus (Lamiaceae) were assessed. Aerial parts from these three related Plectranthus species were collected from cultivated plants grown in Portugal, during vegetative and flowering phases. Volatiles, isolated by distillation? extraction, were analyzed by GC and GC/MS. Monoterpene hydrocarbons (12–74%) and sesquiterpene hydrocarbons (4–45%) constituted the main fractions in all volatiles. α‐Pinene ( 3 ; 12–67%), oct‐1‐en‐3‐ol ( 6 ; traces–28%), β‐pinene ( 7 ; 0.1–22%), and β‐caryophyllene ( 50 ; 7–12%) dominated P. barbatus volatiles. P. neochilus major volatile components were α‐terpenyl acetate ( 41 ; traces–48%), α‐thujone ( 2 ; 2–28%), β‐caryophyllene ( 50 ; 2–28%), β‐pinene ( 7 ; 1–25%), and α‐pinene ( 3 ; 1–19%). Oct‐1‐en‐3‐ol ( 6 ; 13–31%), β‐pinene ( 7 ; 11–24%), α‐pinene ( 3 ; 11–19%), and β‐caryophyllene ( 50 ; traces–11%) were the main constituents from P. ornatus volatiles. These chemical compositions were rather different from those previously found for specimens harvested in Africa and Brazil. Moreover, the volatiles from the flowers are herewith reported for the first time. Essential oils, isolated by hydrodistillation from leaves and stems, showed a yellowish color and unpleasant odor, with yields ranging from 0.08% to 0.84% (v/dry weight). Antioxidant and antimicrobial activities of the essential oils were evaluated by DPPH^. and TBARS assays, and agar disc‐diffusion method, respectively. Results showed low or moderate antioxidant capacity and significant antimicrobial activity against Gram‐positive bacteria.     

Leaf n‐Alkanes as Characters Differentiating Coastal and Continental Juniperus deltoides Populations from the Balkan Peninsula

The composition of the cuticular n‐alkanes isolated from the leaves of nine populations of Juniperus deltoides R.P.Adams from continental and coastal areas of the Balkan Peninsula was characterized by GC‐FID and GC/MS analyses. In the leaf waxes, 14 n‐alkane homologues with chain‐lengths ranging from C₂₂ to C₃₅ were identified. n‐Tritriacontane (C₃₃) was dominant in the waxes of all populations, but variations between the populations in the contents of all n‐alkanes were observed. Several statistical methods (ANOVA, principal component, discriminant, and cluster analyses) were used to investigate the diversity and variability of the cuticular‐leaf‐n‐alkane patterns of the nine J. deltoides populations. This is the first report on the n‐alkane composition for this species. The multivariate statistical analyses evidenced a high correlation of the leaf‐n‐alkane pattern with the geographical distribution of the investigated samples, differentiating the coastal from the continental populations of this taxon.