Secretory Tissues and Volatile Components of Disc Florets in Several Wild Helianthus L. Species.

Although flower pollinator interactions are known to be mediated by floral traits, not enough attention has been paid to the research of secretory tissues and volatile components of sunflower disc florets as potentially important parameters in breeding programs. (1) To our knowledge, this is the first integrated study aimed at better understanding the attractiveness of sunflower capitula to insects. In the study, we have made a very detailed comparative analysis of secretory tissues and the characterization of the volatile components (VOCs) of disc florets in 10 wild perennial Helianthus species. (2) For anatomical analyses, cross-sections were obtained from the nectary zone of disc florets using a cryotechnique procedure. Micromorphological observation and morphological and anatomical analysis of disc florets were performed using light and scanning electron microscopy. For VOCs, we applied headspace, GC-FID, and GC/MS analyses. (3) The obtained results indicate that there is a difference between the analyzed traits among studied species. H. eggertii, H. hirsutus, H. mollis, H. resinosus, and H. tuberosus had high disc diameter values, a high cross-section area and disc floret corolla length, as well as the largest cross-section area and thickness of the disc florets nectary. In the analyzed VOCs, 30 different compounds were detected. The highest yield and quantity of α-Pinene was observed in H. mollis. (4) Inflorescence features, such as receptacle diameter, corolla and secretory tissue properties, and floret VOCs production and characterization, provided valuable information that can be used as guidelines in sunflower breeding programs to maximize pollinator attractiveness and increase seed yield.

Clinopodium L. Taxa from the Balkans-Are There Unique Leaf Micromorphological and Phytochemical Patterns?

The concept of the genus Clinopodium L. has changed considerably since its first description. Most of the currently accepted species of the genus have traditionally been treated as separate genera in the group Satureja sensu lato: Clinopodium L., Calamintha sensu Miller or Moench, and Acinos sensu Miller or Moench. This study aimed to gain a better insight into the species diversity of Clinopodium L. from the Balkans by analyzing the taxa that have traditionally been placed in separate genera. The alkane profile and the micromorphological characteristics of the leaves are analyzed. The leaves are visualized using scanning electron microscopy, and alkanes are isolated using n-hexane as a solvent and analyzed using gas chromatography/mass spectrometry. The alkane profile showed the differentiation of the Acinos-group from the other taxa based on the dominant n-C31, while most of the other taxa contained n-C33 as the dominant alkane. The micromorphological features also showed clear differences between the previously recognized genera, especially in the capitate trichomes. The results showed that micromorphological patterns are highly variable in certain groups of the genus Clinopodium.

Phytochemistry and Micromorphology of Epicuticular Waxes in Four Juniper Species With Fragmented Distribution in the Balkans.

The cuticle is important in the interaction between the plant and its environment, especially in the dry areas. Four species of junipers from the section Sabina wild growing in the Balkans were selected to study leaf wax composition using GC/MS and GC-FID and its surface morphology under SEM to understand the correlation between the distribution and/or habitat of these species and their cuticles. SEM micrographs showed continuous, smooth epicuticular layers with crusts in all species but with a species-specific distribution of different 3D crystalloid types and different cuticle thickness. n-C33 alkane was the most abundant compound, followed by n-C29, n-C31, and n-C35, depending on the species and the site. The average chain length (N) was the lowest in J. phoenicea, but with the greatest dispersion around it. At the same time, the two most continental species (J. foetidissima and J. excelsa) show the N with the lowest dispersion around it. The statistical analyses confirmed the significance of climate on the evolution of the specific epicuticular wax composition in studied junipers.

Interactions between Natural Products-A Review.

Plant-based natural products have been used as a source for therapeutics since the dawn of civilization. According to the World Health Organization (WHO), more than 80% of the world's population relies on traditional medicine for their primary healthcare. Numerous natural extracts, widely known in Traditional Chinese Medicine, Indian Ayurveda medicine and other practices, have led to the modern discovery and development of new drugs. Plants continuously interact with their environment, producing new compounds and ever-changing combinations of existing ones. Interestingly, some of the compounds have shown lower therapeutic activity in comparison to the extract they were isolated from. These findings suggest that the higher therapeutic activity of the source extract was due to the synergistic effect of several compounds. In other words, the total therapeutic potential of the extract cannot be explained only by the sum of its parts alone. In traditional medicine, most herbal remedies are based on a mixture of plants, and it is the interaction between different constituents that amplifies their therapeutic potential. Considering the significant influence traditional medicine has on human healthcare, knowing and studying the synergistic effect of compounds is paramount in designing smart therapeutic agents.

Phlomis fruticosa L. exerts in vitro antineurodegenerative and antioxidant activities and induces prooxidant effect in glioblastoma cell line.

Despite the significant advances in drug development we are witnessing the inability of health systems to combat both neurodegenerative diseases and cancers, especially glioblastoma. Hence, natural products are comprehensively studied in order to provide novel therapeutic options. This study aimed to explore anti-neurodegenerative and anti-glioblastoma potential of extract of Phlomis fruticosa L. using in vitro model systems. It was found that the methanol extract of P. fruticosa was able to efficiently reduce activities of enzymes linked to neurodegenerative disease including acetylcholinesterase, butyrylcholinesterase and tyrosinase. Furthermore, P. fruticosa extract has shown excellent antioxidant potential, as evidenced by six different methods. Analysis of cytotoxic effect of P. fruticosa extract on A172 glioblastoma cell line revealed that the concentration of the extract necessary for 50 % inhibition of A172 growth (IC50) was 710 µg/mL. The extract did not induce changes in proliferation and morphology of A172 glioblastoma cells. On the other side, production of ROS was increased in A172 cells treated with the extract. Observed cytotoxic effect of P. fruticosa extract might be based on increase in ROS generation upon treatment. Quantitative chemical analysis revealed the presence of twelve different polyphenols with the cis 3-O-caffeoylquinic acid being the most abundant. This study provided scientific evidence for further exploration of P. fruticosa as a promising natural anti-neurodegenerative therapeutic option.

Essential Oil Composition and Micromorphological Traits of Satureja montana L., S. subspicata Bartel ex Vis., and S. kitaibelii Wierzb. Ex Heuff. Plant Organs.

The essential oil (EO) composition of Satureja plants is highly variable. Recent studies suggest that there is an even difference in the EO composition from different plant organs within the same plant. This study aims to examine the chemical profile of EOs and the micromorphological characteristics of different organs of three Satureja species. The relationship between the number of glandular trichomes and EOs profile and relative yield is also investigated. Individuals from five populations were visualized using a scanning electron microscope, while EOs of leaves, calyces, corollas, and whole aerial parts were isolated using simultaneous distillation and extraction and analyzed using gas chromatography/mass spectrometry. Three types of glandular trichomes were detected. Peltate trichomes were present on all plant organs of studied species, while two types of capitate trichomes show different organ and species preferences. The EOs profiles differed across the plant parts, but showed a species specific composition. Univariate and multivariate statistics were used to show a correlation between the peltate trichomes and EO yield, and chemophenetic significance of EO profiles.

Chemical profiling, antimicrobial, anti-enzymatic, and cytotoxic properties of Phlomis fruticosa L.

Structural diversity of biologically active compounds identified in plants after many years of storage is rarely reported in literature. Herein, we studied chemical profile and biological activities of Phlomis fruticosa L. after plant material storage for 20 years. Chemical analyzes were performed by UHPLC-LTQ-Orbitrap/MS, and revealed presence of 44 compounds: including 13 phenolic acids, 9 phenylethanoids, 20 flavonoids and 2 phenolic related compounds (a phenolic acid derivative and an aliphatic alcohol). The extract showed antimicrobial activity, being the most potent against Aspergillus fumigatus with minimum inhibitory concentration of 0.31 mg/mL. Also, the extract was able to inhibit biofilm formed by Candida species and to inhibit biofilm formation by Staphylococcus aureus. Obtained results revealed that the extract has potential to interfere with the cell membrane permeability of Candida albicans and to suppress production of virulence factor staphyloxanthin in S. aureus. Furthermore, the extract inhibited the activity of α-amylase which is one of the therapeutic targets for diabetes type II. Also, the antiproliferative effect of the extract was demonstrated on human cancer cell lines, while the extract did not exhibit any cytotoxic effect on primary human cells. Based on the obtained results, P. fruticosa could be an interesting source of biologically active compounds even after long term storage.

Extract of Herba Anthrisci cerefolii: Chemical Profiling and Insights into Its Anti-Glioblastoma and Antimicrobial Mechanism of Actions.

Anthriscus cerefolium (L.) Hoffm. is a plant traditionally used around the globe since antiquity. Although widely used in many traditional medicines in different cultures, from the scientific point of view it is poorly investigated. Glioblastoma, a tumor type with poor prognosis, is the most common and lethal brain tumor in adults. Current therapeutic strategies for glioblastoma include surgery, radiation and chemotherapy. On the other hand, it has been revealed that patients with cancers are highly susceptible to microbial infections due to the invasive nature of cancer treatment approaches. This study was designed to investigate the chemical profile of herba Anthriscii cerefoli methanolic extract by applying UHPLC-LTQ OrbiTrap MS4 analysis and to analyze its anti-glioblastoma and antimicrobial activities. This study revealed that methanolic extract of herba Anthrisc cerefolii contained phenolic acids and flavonoids, with 32 compounds being identified. Anti-glioblastoma activity was investigated in vitro using A172 glioblastoma cell line. The cytotoxic effects of the extract on A172 cells were compared to the same effect on primary human gingival fibroblast (HGF-1) cells. Decreased rate of proliferation and changes in cell morphology were detected upon treatment of A172 cells with the extract. The antimicrobial activity of extract was tested against Staphylococcus aureus and Candida species. The extract was active against the tested bacterium and yeasts, inhibiting free floating cells and microbial biofilms. This study is the first one to provide a detailed description of the chemical profile of A. cerefolium extract dealing with scientific insights into its anti-glioblastoma and antimicrobial activities.

Ononis spinosa L., an edible and medicinal plant: UHPLC-LTQ-Orbitrap/MS chemical profiling and biological activities of the herbal extract.

This study explored the chemical profile of the aerial parts of Ononis spinosa and further investigated its biological activities. Chemical profiling of the extract revealed the presence of 63 different compounds: phenolic acids, flavonoid glycosides and aglycones, isoflavonoid glycosides and aglycones, and other related compounds. Our results revealed that the extract was active against 8 strains of free floating bacteria. It showed anti-biofilm potential against Staphylococcus aureus and was able to supress the production of staphyloxanthin in S. aureus at sub-minimal inhibitory concentrations. Its antioxidant activity was evaluated by using several assays (phosphomolybdenum, DPPH, ABTS, CUPRAC, FRAP, and metal chelating assay), which showed that the extract exhibited a dose dependent activity. Inhibition of AChE, BChE, amylase, glucosidase and tyrosinase was achieved by the extract, demonstrating its anti-enzymatic activity. The antiproliferative potential of the extract towards human cancer cell lines (HepG2, MCF-7, SiHa and A172) was determined by using the crystal violet assay. Ki67, a marker of proliferation was downregulated in the A172 glioblastoma cell line.

Juniperus phoenicea var. turbinata (Guss.) Parl. Leaf Essential Oil Variability in the Balkans.

In the present work, the leaf essential oil from 97 individuals of Juniperus phoenicea var. turbinata (Guss.) Parl. from the Balkan Peninsula was analyzed. The essential oil was dominated by monoterpene hydrocarbons (45.5 - 71.8%), of which α-pinene was the most abundant in almost all of the samples (38.2 - 55.8%). Several other monoterpenes and sesquiterpenes were also present in relatively high abundances in samples such as myrcene, δ-3-carene, ß-phellandrene, α-terpinyl acetate, (E)-caryophyllene and germacrene D. Multivariate statistical analysis suggested the existence of three possible chemotypes based on the abundance of the four components. Even though the intrapopulation variability was high, discriminant analysis (DA) was able to separate populations. DA showed high separation between western and eastern populations but also grouped geographically closer populations along the west Balkan shoreline. The potential influence of the climate on the composition of the essential oil was also studied.

Chemodiversity of Epicuticular n-Alkanes and Morphological Traits of Natural Populations of Satureja subspicata Bartl. ex Vis. along Dinaric Alps - Ecological and Taxonomic Aspects.

Morphological characters and the composition of epicuticular leaf n-alkanes of two Satureja subspicata Bartl. ex Vis. subspecies (subsp. liburnica Silic and subsp. subspicata) from nine natural populations along Dinaric Alps range were studied. Morphological characters were chosen based on Silic's subspecies separation. Seventeen n-alkane homologues (C19 - C35 ) were identified using gas chromatography/mass spectrometry (GC/MS) and GC/flame ionisation detector (FID). The most abundant n-alkane in all populations was n-nonacosane (C29 ), followed by n-hentriacontane (C31 ), with the exception of Divaca population where these two alkanes were co-dominant. Diversity and variability of n-alkane patterns and morphological characters and their relation to different geographic and bioclimatic parameters, including exposure, were analysed by several statistical multivariate methods (PCA, HCA, Discriminant Analysis, Mantel test). These tests showed clear separation of subsp. liburnica from subsp. subspicata, even though population Velebit showed separation from other subsp. liburnica populations based on phytochemical characters. Mantel test showed high correlation with geographical distribution in both investigated data sets. High correlation between morphological and phytochemical characters was also established. However, exposure can influence n-alkane profile, suggesting precaution while taking samples from natural habitats.

Essential Oil Composition of Centaurea atropurpurea and Centaurea orientalis Inflorescences from the Central Balkans - Ecological Significance and Taxonomic Implications.

The essential oil composition of Centaurea atropurpurea and Centaurea orientalis flowering heads (capitula) from Central Balkans have been determined by GC-FID and GC/MS analyses. In total, 121 compounds were identified, representing on average 97.7% of the oil composition. In all samples, sesquiterpenes were most abundant group, representing 53.9 - 74.0% of the total oil. Sesquiterpene hydrocarbons dominated in all studied populations of C. orientalis and C. atropurpurea, except C. atropurpurea f. flava in which essential oil was characterized with high level of oxygenated sesquiterpenes. The dominant components differed between species, and also between typical C. atropurpurea and C. atropurpurea f. flava. The most abundant compounds of essential oil of C. orientalis were germacrene D and α-cadinol. In C. atropuruprea, germacrene D and ß-caryophyllene were the most abundant, while caryophyllene oxide and ß-caryophyllene were dominant in C. atropurpurea f. flava oil. Taxonomical and ecological implications are further discussed.

Essential-oil variability of Juniperus deltoides R.P.Adams along the east Adriatic coast - how many chemotypes are there?

The composition of the essential oils isolated from twigs of ten Juniperus deltoides R.P. Adams populations from the east Adriatic coast was determined by GC-FID and GC/MS analyses. Altogether, 169 compounds were identified, representing 95.6-98.4% of the total oil composition. The oils were dominated by monoterpenes (average content of 61.6%), which are characteristic oil components of species of the Juniperus section. Two monoterpenes, α-pinene and limonene, were the dominant constituents, comprising on average 46.78% of the essential oils. Statistical methods were deployed to determine the diversity of the terpene classes and the common terpenes between the investigated populations. These statistical analyses revealed the existence of three chemotypes within all populations, i.e., a α-pinene, limonene, and limonene/α-pinene type.

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.) Silic, 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.

Biogeographic variation of foliar n-alkanes of Juniperus communis var. saxatilis Pallas from the Balkans.

The composition of the epicuticular n-alkanes isolated from the leaves of ten populations of Juniperus communis L. var. saxatilis Pallas from central (continental) and western (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. All samples were dominated by n-tritriacontane (C33 ), but differences in two other dominant n-alkanes allowed separating the coastal from the continental populations. Several statistical methods (ANOVA, principal component, discriminant, and cluster analyses as well as the Mantel test) were deployed to analyze the diversity and variability of the epicuticular-leaf-n-alkane patterns of the ten natural populations of J. communis var. saxatilis and their relation to different geographic and bioclimatic parameters. Cluster analysis showed a high correlation of the leaf-n-alkane patterns with the geographical distribution of the investigated samples, differentiating the coastal from the continental populations of this taxon. Several bioclimatic parameters related to aridity were highly correlated with this differentiation.

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.

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.