Serbian Artemisia species - chemical composition, acute toxicity and larvicidal activity of the essential oils.

The chemical composition and biological activities of the essential oils obtained from Serbian Artemisia species (A. alba, A. absinthium, A. annua, A. vulgaris, and A. scoparia) were analysed. The essential oil was obtained by merging several samples (same plant species, different localities) and the chemical composition was compared with pre-merging results. In the merged A. scoparia sample four components were not found in any pre-merging sample and one of those is present in the highest percentage (capillin 35.7%). The least toxic essential oil in Artemia salina test was A. annua, followed by A. alba (both showing medium toxicity), while A. absinthium, A. vulgaris, and A. scoparia showed strong toxicity. All tested samples showed activity against Drosophila melanogaster larvae in descending order ΣAS > ΣAN > ΣAV > ΣAB > ΣAA. The essential oil of A. scoparia has exceptional larvicidal activity (in concentrations of 2% and 1% causes complete mortality).

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.

Chemometric Approach to the Composition of Flavonoid Compounds and Phenolic Acids and Antioxidant Potential of Artemisia Species from Different Habitats.

Four Artemisia species from Serbia were selected for the study: A. annua L., A. absinthium L., A. vulgaris L. and A. scoparia Waldst. et Kit. because of the proven excellent action in treating certain medical conditions and diseases. A. absinthium L., A. vulgaris L., A. annua L. and A. scoparia Waldst. et Kit. collected from different habitats across Serbia (48 samples in total) were studied from the statistical aspect considering the phenolic and flavonoid contents, compositions, and antioxidant activities of methanol extracts in correlation with the soil type. The components were identified using HPLC (High Performance Liquid Chromatography), while antioxidant activities were determined by seven assays (TP (Total phenolic content), TF (Total flavonoid content), DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-based, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation-based, FRAP (Ferric Reducing Antioxidant Power), TRP (Total Reducing Power), and CUPRAC (Cupric Reducing Antioxidant Capacity). The results were processed using five statistical methods (PCA (Principal Component Analysis), ANOVA (Analysis of Variance), MANOVA (Multivariate analysis of variance), DA (Discriminant analysis), and AHC (Agglomerative Hierarchical Clustering)). Principal component analysis enabled very well separation of the analyzed Artemisia species based on the content of total phenolics, total flavonoids, and the values obtained from antioxidant tests, but not on the individual compounds identified and quantified by HPLC. The MANOVA analyses showed that for A. scoparia and A. annua there was a significant effect of soil type on the total phenolics, total flavonoids, and antioxidant tests. In contrast, for A. vulgaris and A. absinthium, that effect was not significant. Additional MANOVA analyses showed a significant effect of soil type on phenolic and flavonoid compounds in the case of A. vulgaris, A. annua, and A. absinthium. The overall correct classification rate of all samples of four investigated Artemisia species by the discriminant analysis was 81.25 % using the training sample and 72.92 % the cross-validation results based on TP, TF, and antioxidant tests, but a much lower based on selected compounds identified and quantified by HPLC. MANOVA analyses based on particular Artemisia species show that soil is a significant factor affecting the measurable variables.

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.

Trace element content in commercial complementary food formulated for infants and toddlers: Health risk assessment.

The study aimed to analyze trace elements content in baby purees and fruit juices and to evaluate the health risk of young children. The average daily dose, hazard quotient, hazard index and total diet hazard quotient were calculated to assess the potential health risk on per capita and consumers only groups of infants and toddlers. There was no significant health risk for studied groups regarding the intake of trace elements via purees and juices consumption. Health risk for lead was not estimated since the oral reference dose for this metal was not yet established and PTWI value was withdrawn. The average daily dose of lead for infants (0.32 - 0.46 µg/kg bw/day) and toddlers (2.01 - 2.29 µg/kg bw/day) were in accordance with the daily lead exposure intervals estimated by EFSA. Applying statistical analysis, the products were classified into three groups according to the content of trace elements.

The effects of Artemisia methanol extracts and ferulic acid, rutin, rosmarinic acid, and quercetin on micronucleus distribution on human lymphocytes.

Methanol extracts of A. absinthium, A. annua, A vulgaris, A. scoparia, and A. alba from Serbia at concentrations of 1.00; 2.00 and 4.00 µg/ml, and as pure compounds identified in examined extracts (ferulic acid, rutin, rosmarinic acid, and quercetin) were examined for their effects on micronucleus distribution in human lymphocytes in vitro. All extracts greatly reduced the number of micronuclei in the concentration of 2.00 µg/ml comparing to the control, whereby A. alba extract exhibited the most prominent effect on decreasing frequency of micronucleus (45.2%), followed by extracts of A. vulgaris, A. absinthium, A. scoparia and A. annua. The same declining trend was observed for the extracts at concentrations of 4.00 µg/ml, and 1.00 µg/ml, respectively. Among pure compounds (concentration 2.00 µg/ml), rutin showed the most pronounced effect on decreasing frequency of micronucleus (41.7%), higher than ferulic acid, quercetin, and rosmarinic acid comparing to the control.

Mineral Composition of Herbaceous Species Seseli rigidum and Seseli pallasii: a Chemometric Approach.

Nutrients play an essential role in many metabolic processes whose deficiency or excess can be harmful to the plant itself and through the food chain to both animals and humans. Medicinal plants used in the food and pharmaceutical industries can be contaminated with increased concentrations of heavy metals. The plant species Seseli rigidum and Seseli pallasii from the Balkan Peninsula are used in traditional medicine and spices in the diet, so it was necessary to determine the mineral composition to ensure their safe application. In this work, the mineral composition was determined in medicinal species of the genus Seseli using inductively coupled plasma with optical emission spectrometry (ICP-OES). Two multivariate statistic methods -principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to distinguish samples regarding their mineral composition. The mineral composition of both studied species is following the literature data. The results obtained using multivariate statistics methods agree and distinguish certain parts of the tested plants based on the highest content of micro, macro, or trace elements.

Pseudotsuga menziesii (Pinaceae): Volatile Profiles, Antimicrobial Activity and Toxicological Evaluation of Its Essential Oil.

The present article investigates the chemical composition of volatiles of essential oil (EO) and headspace (HS) fraction, as well as biological activities of EO obtained from needles with twigs of Pseudotsuga menziesii var. menziesii cultivated in Serbia. The major class of compounds was monoterpene hydrocarbons with α-terpinolene, sabinene and ß-pinene (EO), and sabinene, α-terpinolene and ß-pinene (HS) as the dominant volatiles. Tested EO exhibited mostly low antimicrobial potential against investigated strains (ATCC and respiratory isolates), where MICs ranged 1.25-20.00 mg/mL. Nevertheless, based on presented results, where antimicrobial testing was done for the first time on human respiratory system isolates, there is a potential of this EO to be used as an adjuvant in the treatment of human respiratory infections, especially those caused by Pseudomonas aeruginosa or Candida albicans strains. Regarding toxicological evaluation, EO showed moderate toxicity in Artemia salina toxicity bioassay (LC50 =347.41, after 24 h) as well as week toxicity against Drosophila melanogaster with the ability only to moderately delay larval and pupal development.

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 of the essential oils and headspace volatiles of Ferulago sylvatica (Besser) Reichenb. from Serbia.

Chemical composition of the headspace volatiles and essential oils isolated from different parts of Ferulago sylvatica was determined by GC and GC/MS analyses. The results showed that headspace volatiles obtained from the aerial parts and roots were similar regarding the number of identified compounds and main components. However, essential oils obtained from different plant organs showed significant differences in chemical composition. Myrcene was the most abundant component of the inflorescences and shoots volatiles, while α-pinene make up over 50% of the root volatiles. Only three components were identified in the root essential oil with 2,3,6-trimethyl benzaldehyde (92.7%) as the main component. In the shoots sample the terpenoid fractions represented 56% of the oil, unevenly distributed between monoterpenoids and sesquiterpenoids with germacrene D (32.5%) recognized as the main constituent. On the other hand, more than 94% of the inflorescences oils were monoterpenoids with myrcene as the most abundant contributor (29.2%).

Volatiles composition and antioxidant activity Inula oculus-christi L. from Serbia.

The chemical composition of the essential oil and the volatiles obtained by static headspace (HS) of Inula oculus-christi L. is presented. The GC-MS analysis of the hydrodistilled oil resulted in the identification of 90 components, representing 92.7% of the oil. The most abundant compounds were: caryophyllene oxide (9.8%), trans-longipinocarveol (9.2%), eucalyptol (7.3%) and intermedeol (6.2%). The major constituent of I. oculus-christi L. HS volatiles was eucalyptol (87.4%). The antioxidant activity was evaluated by four different methods: 2,2-diphenyl-1-picryl-hydrazylhydrate free radical assay (DPPH), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method, total reducing power (TRP), ferric reducing antioxidant power (FRAP), and cupric reducing antioxidant capacity (CUPRAC). Total phenolic content in (TPC) examined oil was 177.95 µg GAE/mg oil. Radical scavenging potential of the oil was promising RSC-DPPH was 57.4% and RSC-ABTS was 82.7%.[Formula: see text].

First Report about Mineral Content, Fatty Acids Composition and Biological Activities of Four Wild Edible Mushrooms.

The goal of this research was a comprehensive analysis of four wild edible mushroom species, Cantharellus cinereus, Clavariadelphus pistillaris, Clitocybe nebularis and Hygrocybe punicea, which have not been analyzed so far. Extracts of different polarities have been prepared and evaluated for their antioxidant activities by DPPH, ABTS, FRAP, TRP and CUPRAC methods. For all extracts, total phenolic content was determined. Based on the analysis, it was shown that solvent type had a significant effect on the antioxidant capacities of mushroom extracts, so water extracts showed the highest activity. Furthermore, the analysis includes determination of mineral composition, fatty acid profiles and antimicrobial activity. Unsaturated fatty acids, which are very important for human health, are dominant in the studied mushroom species. Linoleic and oleic acid consist of over 50 % of the total fatty acid composition. Seventeen biologically important and toxic elements have been analyzed by ICP-OES and ICP-MS and results showed that the element concentrations were species-dependent. Also, it has been found that analyzed mushrooms did not show any antimicrobial activity. Chemometric analysis was used to understand the connection between the extracts of different polarities.

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.

Comparative Study of Fatty Acids Profile in Eleven Wild Mushrooms of Boletacea and Russulaceae Families.

Eleven species of wild mushrooms which belong to Boletaceae and Russulaceae families were examined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS) analysis for the presence of fatty acids. As far as we know, the fatty acid profiles of B. purpureus and B. rhodoxanthus were described for the first time. Twenty-six fatty acids were determined. Linoleic (19.5 - 72%), oleic (0.11 - 64%), palmitic (5.9 - 22%) and stearic acids (0.81 - 57%) were present in the highest contents. In all samples, unsaturated fatty acids dominate. Agglomerative hierarchical clustering was used to display the correlation between the fatty acids and their relationships with the mushroom species. Based on the fatty acids profile in the samples, the mushrooms can be divided into two families: Boletaceae and Russulaceae families, using cluster analysis.

Essential oil composition of Erica spiculifolia Salisb - first report.

The essential oil isolated from Erica spiculifolia Salisb. by hydrodistillation was analysed by GC-MS. One hundred compounds representing 92.6% of total oil were identified. Among the detected compounds 38 were monoterpenoids (46.2%), 30 were sesquiterpenoids (31.7%), 2 diterpene (0.4%) and 30 compounds (14.3%) were with various non-terpenoid structures. Oxygenated monoerpenes were by far the main class (45.3%) with α-terpineol (7.5%) endo-borneol (7.2%), pinocarveol (5.9%) and thymol (3.7%) as the major compounds. The predominant components of sesquiterpenes were caryophyllene oxide (5.0%), caryophyllene (4.2%), τ -murrolol (3.5%), spathulenol (2.9) and α-cadinol (2.3%).

Phytochemical analysis of Saponaria officinalis L. shoots and flowers essential oils.

Phytochemical analysis by GC and GC/MS of the essential oil samples obtained from fresh shoots and flowers of Saponaria officinalis L. allowed the identification of 96 components in total, comprising 94.7% and 86.0% of the total oils compositions, respectively. Regarding the shoots essential oil, the major of 87 identified volatile compounds were phytol (14.1%), tricosane-6,8-dione (13.4%), patchouli alcohol (7.9%) and tricosane (7.2%), whereas patchouli alcohol (20.0%), heneicosane (11.5%) and tricosane (8.4%) were dominant among the 66 volatiles in the flower oil. Nonterpenoid compounds had the highest contribution in S. officinalis shoots essential oil (53.7%), while in the flower oil, constituents were almost evenly distributed between the oxygenated sesquiterpenoid (41.2%) and nonterpenoid compounds (39.5%).

The effect of multiple contamination of soil on LMW and MMW PAHs accumulation in the roots of Rubus fruticosus L. naturally growing near The Copper Mining and Smelting Complex Bor (East Serbia).

Samples of roots and spatial soils of native Rubus fruticosus L. were collected from the spots positioned at different distances from the copper smelter and city heating plants in the industrial zone of the town of Bor (Serbia) and subjected to chemical analyses in order to determine the content of several heavy metals, and 15 priority polycyclic aromatic hydrocarbons (PAHs). In this study, the results for 9 low and medium molecular weight PAHs (LMW and MMW PAHs) are represented and processed using the calculation of bio-concentration factors and statistical methods such as hierarchical cluster analysis and Pearson's correlation study with the aim of investigating the plant capabilities for their uptake from the soil and later accumulation into the root tissue, under the hostile circumstances of multiple contamination. The obtained data revealed different accumulation rates for the investigated PAHs and showed that in several cases, the contents of root PAHs were under the strong influence of present contaminants such as soil copper and some soil PAHs, indicating at the same time that R. fruticosus can regulate the processes of LMW and MMW PAHs extraction/accumulation using different mechanisms, depending on the existing environmental circumstances. The used mechanisms could be exploited in phytoremediation methods based not only on the extraction and concentration of PAHs in plant roots but also on PAH degradation or stabilization in the soil. Also, the results of this study confirmed that, except in the case of naphthalene and fluoranthene, there was no PAH pollution, which originated solely from the industrial zone.

Phenolic profiles and metal ions analyses of pulp and peel of fruits and seeds of quince (Cydonia oblonga Mill.).

Six hydroxycinnamic acids were identified and determined quantitatively in methanol and acetone extracts from quince peel and pulp, namely 3-O-caffeoylquinic acid (3-CQA), 4-p-coumaroylquinic acid (HC1), 4-O-caffeoylquinic acid (4-CQA), 5-O-caffeoylquinic acid (5-CQA), derivative of p-coumaroylquinic acid (HC2) and 3,5-dicaffeoylquinic acid (3,5-diCQA). The most abundant hydroxycinnamic acid was 5-CQA (neochlorogenic acid) with 259.12-481.4mg/kgf.w. in peel and 97.33-217.36mg/kg in quince pulp. Six flavonols were determined in the extracts from quince, quercetin-3-galactoside (Q-Ga), quercetin-3-rutinoside (Q-Ru), quercetin-3-glucoside (Q-Glu), kaempferol-3-rutinoside (K-Ru), kaempferol-3-glucoside (K-Glu) and derivative of quercetin produced in the reaction between quercetin-glucoside and p-coumaric acid (Q-Glu-p-CouA). Elemental analysis of quince seeds has not been performed previously. Also, using principal component and cluster analyses, we determined a strong negative relationship between total phenols and flavonoids, and Ni and Pb, specifically higher concentrations of these compounds were associated with lower concentrations of these metals.

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.