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.

In Silico Investigation of Selected Pesticides and Their Determination in Agricultural Products Using QuEChERS Methodology and HPLC-DAD.

In this study, we considered some pesticides as active substances within formulations for the protection of plant-based food in the Republic of Serbia in silico, because these pesticides have not often been investigated in this way previously, and in an analytical way, because there are not very many available fast, cheap, and easy methods for their determination in real agricultural samples. Seven pesticides were detected in selected agricultural products (tomatoes, cucumbers, peppers, and grapes) using the QuEChERS methodology and HPLC-DAD. Standard curves for the investigated pesticides (chlorantraniliprole, methomyl, metalaxyl, thiacloprid, acetamiprid, emamectin benzoate, and cymoxanil) show good linearity, with R2 values from 0.9785 to 0.9996. The HPLC-DAD method is fast, and these pesticides can be determined in real spiked samples in less than 15 min. We further characterized the pesticides we found in food based on physicochemical properties and molecular descriptors to predict the absorption, distribution, metabolism, elimination, and toxicity (ADMET) of the compounds. We summarized the data supporting their effects on humans using various computational tools to determine their potential adverse effects. The results of our prediction study show that all of the selected pesticides considered in this study have good oral bioavailability, and those with high toxicity, therefore, could be harmful to human health. Chlorantraniliprole was shown in a molecular docking study as a good starting point for a new Alzheimer's disease drug candidate.

Selected fungi of the genus Lactarius - screening of antioxidant capacity, antimicrobial activity, and genotoxicity.

It is well-known that mushrooms of the genus Lactarius constitute a natural food resource providing health benefits as a nutritient. This genus contains 4 mushrooms identified as L. deliciosus, L. volemus, L. sanguifluus, L. semisanguifluus and L. piperatus which were collected in Serbia. The aim of this study aimed was to identify and characterize the content of phenolic compounds and examine the antioxidant potential of 5 wild edible mushrooms. L. sanguifluus contained the highest content of total phenol compounds (75.25 mg gallic acid equivalents (GAE) per g dry extract weight (GAE/g DE) and exhibited the greatest antioxidant activity through the ability to remove radicals as evidenced by ABTS assay (8.99 mg of trolox equivalents (TE) per g dry extract weight (mg TE/g DE); total reducing power (TRP) assay mg ascorbic acid equivalents per mg of dry extract weight (0.42 mg AAE/g DE) and CUPRAC (14.23 mg TE /g DE). L. deliciosus methanolic extract produced greatest scavenging of the DPPH radical (46%). The methanol mushroom extracts were screened for in vitro antimicrobial activity against a panel of pathogenic bacterial strains using the microdilution method. Of all the extracts tested, L. sanguifluus extract showed the best antibacterial properties. The cytokinesis block micronucleus assay results for the examined mushrooms demonstrated that extracts at a concentration of 3 µg/ml decreased the number of micronuclei (MN) in the range of 19-49% which is significant bearing in mind that radioprotectant amifostine reduced the frequency of MN by only 16.3%. Data thus demonstrate that the 5 wild edible mushrooms of genus Lactarius contain constituents that are beneficial not only as nutrients but also have the potential as antioxidants, antibacterial and antigenotoxic properties.

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.

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.

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.

Dispersive micro-solid phase extraction of 16 priority polycyclic aromatic hydrocarbons from water by using thermally treated clinoptilolite, and their quantification by GC-MS.

The authors report on a novel sorbent (thermally treated natural zeolite; clinoptilolite) for use in dispersive micro-solid phase extraction (D-µ-SPE) of polycyclic aromatic hydrocarbons (PAHs) from water samples. The method was applied to the D-µ-SPE of 16 priority PAHs which then were quantified by gas chromatography with mass spectrometric detection (GC-MS). The method was validated in terms of specificity and selectivity, linearity and linear range, accuracy, precision, uncertainty, limits of detection and quantification. Figures of merit include (a) linear analytical ranges between 2.08 and 208 ppb, and (b) detection limits in the range from 0.01 to 0.92 ppb. The method was successfully applied to the determination of PAHs in river waters. Graphical abstract Schematic representation of dispersive micro-solid phase extraction (D-µ-SPE) of trace levels of PAHs in water samples by using thermally treated clinoptilolite as sorbent prior to gas chromatography-mass spectrometry analysis (GC-MS).

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.

Synthesis, Antimicrobial Activity and in silico Studies on Thymol Esters.

Derivatisation of parent structure in terpenoids often results in enhancement of biological activity of newly obtained compounds. Thymol, a naturally occurring phenol biosynthesized through the terpene pathway, is a well known biocide with strong antimicrobial attributes and diverse therapeutic activities. We have aimed our study on a single modification of phenolic functionality in thymol in order to obtain a small focused library of twenty thymyl esters, ten of which were new compounds. All compounds were involved in in vitro antimicrobial testing. Another important aspect of current study was implementation of in silico calculation of physico-chemical, pharmacokinetic and toxicological properties, which could be helpful by giving an additional guidance in further research.

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.

Dittrichia graveolens (L.) Greuter Essential Oil: Chemical Composition, Multivariate Analysis, and Antimicrobial Activity.

The chemical composition and in vitro antimicrobial activities of Dittrichia graveolens (L.) Greuter essential oil was studied. Moreover, using agglomerative hierarchical cluster (AHC) and principal component analyses (PCA), the interrelationships of the D. graveolens essential-oil profiles characterized so far (including the sample from this study) were investigated. To evaluate the chemical composition of the essential oil, GC-FID and GC/MS analyses were performed. Altogether, 54 compounds were identified, accounting for 92.9% of the total oil composition. The D. graveolens oil belongs to the monoterpenoid chemotype, with monoterpenoids comprising 87.4% of the totally identified compounds. The major components were borneol (43.6%) and bornyl acetate (38.3%). Multivariate analysis showed that the compounds borneol and bornyl acetate exerted the greatest influence on the spatial differences in the composition of the reported oils. The antimicrobial activity against five bacterial and one fungal strain was determined using a disk-diffusion assay. The studied essential oil was active only against Gram-positive bacteria.

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.