Search

L-Glutamic acid decarboxylase (GAD) is an enzyme that ensures the balance between the levels of two neurotransmitters, γ-aminobutyric acid (GABA) and L-glutamic acid (L-Glu), necessary for proper brain functioning. A reduction in the concentrations of GABA and/or GAD activity has been implicated in the symptoms associated with epilepsy, which could be plausibly alleviated by the application of GAD activators. As any unnecessary interference in GAD catalytic activity could be detrimental, it is important to study whether CNS (or other) drug candidates act on GAD or not. The ability to identify and reduce this risk early could significantly improve the process of drug development. Although many methods for measuring GAD activity in various biological samples have been described, only few (such as manometric and radiometric) were adopted as in vitro assays for the screening of potential GAD inhibitors/activators. However, these methods require specialized equipment and/or an expensive radiolabeled substrate, and may have sensitivity and/or reliability issues. Therefore, this study aimed to develop an HPLC-DAD-based assay that would allow a simple and more accurate measurement of GAD inhibition or activation using unpurified mice or rat brain homogenates. This assay is based on the quantification of GABA, formed during the enzymatic reaction, after its derivatization with dansyl chloride. Various parameters were evaluated to optimize the assay procedure (e.g. homogenate volume, incubation time, DMSO content, GAD, GABA, and dansyl-GABA stabilities). This assay was validated for pharmacological screenings using 3-mercaptopropionic acid and gallic acid and GAD obtained from different experimental animals.

Herein n-, iso- and anteiso-series of very-long-chained (VLC) alkanes (C21 -C35 ), fatty acid benzyl esters (FABEs; C20 -C32 ), and 2-alkanones (C23 -C35 ) were identified in the wax of Primula veris L. and P. acaulis (L.) L. (Primulaceae). For the very first time in a sample of natural origin, the presence of iso- and anteiso-VLC FABEs and 2-alkanones was unequivocally confirmed by synthetic work, derivatization, and NMR. It should be noted that the studied species produced unusually high amounts of branched wax constituents (e. g., >50 % of 2-alkanones were branched isomers). The domination of iso-isomers, probably biosynthesized from leucine-derived starters, is a unique feature in the Plant Kingdom. The plant organ distribution of these VLC compounds in P. acaulis samples (different habitats and phenological phases) pointed to their possible ecological value. This was supported by a eutectic behavior of binary blends of FABEs and alkanes, as well as by high UV-C absorption by FABEs.

Helichrysum italicum (immortelle) is a dwarf aromatic shrub native to the Mediterranean region. The typical subspecies (italicum) produces an essential oil rich in neryl acetate and characteristic β-diketones, italidiones, highly valued in the perfume industry. As esters are an important group of aroma-active volatiles, herein the composition of the ester fraction of this immortelle chemotype essential oil was studied in detail. Chromatographic separation of Corsican immortelle essential oil enabled the discovery of numerous potentially olfactory-interesting esters of nerol and/or angelic acid, undetectable by direct GC-MS analyses of the unfractioned oil. Four esters of nerol and medium-chain branched fatty acids represent new natural products, while several other esters have a rather restricted occurrence in the Plant Kingdom.

Two novel β-diketones, 2,6,9-trimethyl-8-decene-3,5-dione (A) and 3,7,10-trimethyl-9-undecene-4,6-dione (B), were identified from the renowned medicinal plant Hypericum perforatum L. The structures of β-diketones A and B were corroborated by syntheses (4 steps starting from methyl acetoacetate, overall yields 30% and 23%, respectively). In solution, these β-diketones predominantly exist as two rapidly interconverting β-keto-enol tautomers. The structures of A and B show some common fragments with the molecules of hyperforin and adhyperforin, respectively, the acknowledged multi-target secondary metabolites from St. John's wort. It is therefore not surprising that A displayed a noteworthy biological activity profile as well (including brine shrimp toxicity, antinociceptive, antidepressant and acetylcholinesterase inhibitory activity). β-Diketone A manifested the most outstanding potency as an acetylcholinesterase inhibitor with IC50 value of 1.51 μM pointing again to the β-keto-enol moiety as a promising lead structure for the development of drugs that could lessen symptoms of Alzheimer's disease (such as dementia, depression and pain).

Immortelle (Helichrysum italicum, Asteraceae) essential oil has been widely used in alternative medicine to accelerate wound healing, as well as in cosmetic products to stimulate skin regeneration and to reduce the appearance of wrinkles. It is also considered a natural and safe culinary spice that could also be applied in the food industry as a preservative in the future. The therapeutic efficacy of this oil changes with the natural variability of the composition. Herein we tested and mutually compared the antimicrobial and anti-inflammatory activities of four commercial immortelle oils differing in the relative amounts of marker compounds, i.e. neryl esters, α-pinene, γ- and ar-curcumenes, and β-diketones. The anti-inflammatory effect of selected chromatographic fractions, enriched in the aforementioned constituents, was evaluated by studying toxicity toward rat peritoneal macrophages, their nitric oxide production, myeloperoxidase, and arginase activities. Subsequently, the compositional and activity data were subjected to a multivariate statistical treatment to reveal the possible correlation(s) between the percentage of essential-oil constituents and the observed activities. The obtained results imply that immortelle oil efficiency as an antimicrobial and/or anti-inflammatory agent is most plausibly a result of a synergistic action between its constituents, and/or, rather unexpectedly, the presence of some minor constituents.

Neurotoxic thujones (α- and β-diastereoisomers) are common constituents of plant essential oils. In this study, we employed a statistical approach to determine the contribution of thujones to the overall observed behaviour-modulating and toxic effects of essential oils (Salvia officinalis L., Artemisia absinthium L., Thuja occidentalis L. and Tanacetum vulgare L.) containing these monoterpene ketones. The data from three in vivo neuropharmacological tests on rats (open field, light-dark, and diazepam-induced sleep), and toxicity assays (brine shrimp, and antimicrobial activity against a panel of microorganisms), together with the data from detailed chemical analyses, were subjected to a multivariate statistical treatment to reveal the possible correlation(s) between the content of essential-oil constituents and the observed effects. The results strongly imply that the toxic and behaviour-modulating activity of the oils (hundreds of constituents) should not be associated exclusively with thujones. The statistical analyses pinpointed to a number of essential-oil constituents other than thujones that demonstrated a clear correlation with either the toxicity, antimicrobial effect or the activity on CNS. Thus, in addition to the thujone content, the amount and toxicity of other constituents should be taken into consideration when making risk assessment and determining the regulatory status of plants in food and medicines.

Reported are the synthesis, spectral and structural characteristics of new quinoxaline-related regioisomeric ligands L1–L4 (1,x,11,11-tetramethyl-1,2,3,4-tetrahydro-1,4-methanophenazine, x = 7, 8, 9 and 6, respectively) and their mononuclear Au(III) complexes (1–4). Fusion of the camphor moiety to the quinoxaline core made two N-atoms of quinoxaline nonequivalent while the introduction of a methyl-substituent at positions 6–9 enabled a tuning of coordination properties of L1–L4. Gold(III) complexes 1–4 and ligands L1–L4 have been studied in detailed by 1D and 2D NMR and the structures of 1–4 have been determined by X-ray crystallography. The results of these analyses revealed a regiospecific coordination of Au(III) to the sterically less hindered N-5 atom (spatially close to the non-substituted bridgehead carbon) of L1–L3, and to N-10 (spatially close to the methyl-substituted bridgehead carbon) of L4. The results of DFT calculations shed light on disparate coordination modes of L1–L4 toward the AuCl3 fragment and explain formation of single coordination products in high yield.

Herein, the results of the first study of the volatile and alkane profiles of Cephalaria ambrosioides Roem. & Schult. (Caprifoliaceae, subfamily Dipsacaceae) were reported. The GC-FID and GC/MS analyses of the essential oils hydrodistilled from leaves and stems (CA1) and flowers (CA2) of C. ambrosioides allowed the identification of 284 different components. The main compounds of the studied oil samples were palmitic acid (24.3 and 32.5% for CA1 and CA2, resp.), hexahydrofarnesyl acetone (1.4 and 10.8% for CA1 and CA2, resp.), (Z)-hex-3-en-1-ol (7.0 and

Motivated by the ethnopharmacological application of Inula helenium, in the present study, the anticandidal potential of hydrodistilled root I. helenium essential oil was investigated by a microdilution method and the obtained minimal inhibitory concentrations (MICs) were further used to explore its synergistic potential. Additionally, the mode of action (sorbitol and cholesterol assays) and anti-virulence effects (anti-biofilm, germ-tube reducing and phospholipase-inhibitory activities) were also studied. GC and GC/MS analyses showed that the isolated essential oil contained alantolactone and isoalantolactone as the dominant compounds (65.8 % and 25.5 %, respectively). The obtained MICs (0.009-0.312 mg/ml) varied for the strains generally demonstrating a medium-to-high anticandidal action. More importantly, the oil exhibited a high synergistic effect in combination with the antifungal agent nystatin. Experiments on the mode of action demonstrated that the oil affected the cell-membrane function. Considering virulence factors, the oil showed an antibiofilm activity, as well as a very high germ-tube reducing potential. The results demonstrated a complete inhibition of the enzyme phospholipase in the presence of the tested I. helenium root essential oil. Based on the present results, I. helenium oil could be considered a natural agent that deserves to be further explored as a crop for the treatment of candididases.

The effects of basil (Ocimum basilicum) and sage (Salvia officinalis) essential oils on selected virulence factors (biofilm formation, mature biofilm resistance, motility, and pyocyanin production) of Pseudomonas aeruginosa clinical isolates were evaluated in the present study for the first time. The two essential oils were chemically characterized by GC and GC-MS analyses. Linalool and (E)-anethole were found to be the main components of the investigated basil oil, while α-thujone and camphor were the major constituents of the studied sage essential oil. The oils inhibited biofilm formation up to 99.9% vs control, and significant reductions (74.7-99.9%) were also noted when the oils were applied to mature biofilms. Likewise, swimming, swarming, and twitching motility patterns were highly affected by both oils. The basil and sage oils reduced pyocyanin production by 13.32-55.6% and 5.0-58.7%, respectively. Thus, basil and sage essential oils are potentially highly efficient antipseudomonal agents that could be used against both acute and chronic infections.

The effects on breast milk composition of advanced maternal age and maternal dietary habits during pregnancy and lactation have not, to our knowledge, been investigated in southeastern Europe and the Balkans. The aim of this study was to compare the content of retinol and β-carotene in colostrum and mature milk samples obtained from different maternal age (MA) groups and to assess the potential relationship with maternal and demographic characteristics, dietary patterns, and lifestyle habits during pregnancy and lactation.Forty-three nursing mothers were divided in two groups according to MA: ≥35 y of age (n = 22) and35 y of age (n = 21). Total lipid concentrations were determined by gravimetric method, whereas retinol and β-carotene contents were assessed by high-performance liquid chromatography method. Dietary patterns during pregnancy and lactation were assessed using food frequency questionnaires, and principal component analysis (PCA) statistical analysis was performed.Except for retinol levels in mature milk, significantly higher levels of total fats, retinol, and β-carotene were found in the older group. Results of PCA analysis showed that MA was strongly correlated with fat content, retinol, and β-carotene levels in colostrum samples, whereas in mature milk samples MA was highly correlated with fat content and moderately with β-carotene. In terms of dietary patterns, retinol contents in milk samples from both groups were weakly to moderately correlated with consumption frequency of eggs, meat, milk and dairy products, whereas β-carotene contents were weakly to strongly associated with consumption frequency of fruits and vegetables.PCA analysis used in the study clearly confirmed that MA, total lipids, retinol, and β-carotene levels might serve as a good criterion for delimitation of breast milk samples collected in different stages of lactation from mothers of various ages. The present findings could represent key basis for further investigations.

Streptomyces sp. NP10 was previously shown to synthesize large amounts of free fatty acids (FFAs). In this work, we report the first insights into the biosynthesis of these fatty acids (FAs) gained after genome sequencing and identification of the genes involved. Analysis of the Streptomyces sp. NP10 draft genome revealed that it is closely related to several strains of Streptomyces griseus. Comparative analyses of secondary metabolite biosynthetic gene clusters, as well as those presumably involved in FA biosynthesis, allowed identification of an unusual cluster C12-2, which could be identified in only one other S. griseus-related streptomycete. To prove the involvement of identified cluster in FFA biosynthesis, one of its three ketosynthase genes was insertionally inactivated to generate mutant strain mNP10. Accumulation of FFAs in mNP10 was almost completely abolished, reaching less than 0.01% compared to the wild-type strain. Cloning and transfer of the C12-2 cluster to the mNP10 mutant partially restored FFA production, albeit to a low level. The discovery of this rare FFA biosynthesis cluster opens possibilities for detailed characterization of the roles of individual genes and their products in the biosynthesis of FFAs in NP10. Copyright © 2018 Schneider, Ilic-Tomic, Rückert, Kalinowski, Genčić, Živković, Stankovic, Radulović, Vasiljevic, Nikodinovic-Runic and Zotchev. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

A predominantly odd-numbered, Gaussian-like distribution of the relative amounts of 3-methyl-2-alkanones, from I. helenium root essential oil, was observed. This distribution pattern indicates that their biosynthesis is related to that of fatty acids and related compounds. Simple (non-branched) 2-alkanones also show an odd carbon number prevalence in plants and other organisms, and it was shown that their biosynthesis indeed proceeds via the acetate pathway. In this paper, we propose three possible biosynthetic pathways by which 3-methyl-2-alkanones could be formed in the plant tissues. The essential distinction between them lies in the way the branching methyl group is introduced. The Gaussian parameter σ for the observed distribution of these ketones could be interpreted as the error introduced by the first elongase enzyme system involved in the biosynthesis of fatty acid-derived compounds. BIOSINTEZA 3-METIL-2-ALKANONA DUGOG UGLJOVODONICNOG LANCA IZ ETARSKOG ULJA KORENA BILJNE VRSTE INULA HELENIUM L. (ASTERACEAE) 3-Metil-2-alkanoni dugog ugljovodonicnog lanca su nađeni u etarskom ulju korena biljne vrste I. helenium L. Uocena je Gausova raspodela njihovih relativnih kolicina, pri cemu su homolozi sa neparnim brojem C-atoma bili zastupljeniji. Ovakva raspodela ukazuje na to da njihova biosinteza protice veoma slicno kao i biosinteza masnih kiselina i srodnih jedinjenja. 2-Alkanoni sa neparnim brojem C-atoma su, takođe, zastupljeniji kod biljaka i ostalih organizama, a za njih je dokazano da zaista nastaju po gore pomenutom acetatnom biosintetskom putu. Imajuci sve ovo u vidu, predložili smo tri moguca biosintetska puta kojim bi 3-metil-2-alkanoni mogli nastati u biljnim tkivima. Osnovna razlika između predloženih puteva je u nacinu na koji se uvodi metil racva. Gausov parametar σ uocenih raspodela kolicina alkana bi se mogao posmatrati kao greska prvog enzimskog sistema elongaze koji ucestvuje u biosintezi masnih kiselina i jedinjenja koja se iz njih izvode. HIGHLIGHTS Homologous 3-methyl-2-alkanones (C 11 –C 19 ) detected in Inula helenium root essential oil had a predominantly odd-numbered Gaussian-like distribution. This specific distribution pattern indicates that their biosynthesis most likely proceeds via the acetate pathway. There are three possible biosynthetic pathways, with a different mode of introducing the methyl branch, by which 3-methyl-2-alkanones could be produced. The observed Gaussian parameter σ could be interpreted as the error of the first elongase enzyme system involved in the biosynthesis of fatty acid-derived compounds.

The use of lanthanide complexes for resolving intricate NMR signals and, in the case of chiral ligands, for determining enantiomeric excess has progressively decreased in the last 30 years. Recently, a sesquiterpene aldehyde from Inula helenium with a possible potent antistaphylococcal activity remained unidentified due to the impossibility of separating the compound from its complex matrix available in very low amounts (ca. 5 mg). Detailed analyses of 1D and 2D NMR spectra of this original complex sample allowed access to a very limited amount of structural data for the unknown aldehyde. We decided to investigate the potential usefulness of lanthanide-induced shift reagents for the resolution and assigning of overlapped 1H NMR signals originating from different components of this complex mixture (i.e. for a qualitative analysis). The incremental addition of tris(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato)europium(III) (Eu(fod)3) led to a simplification of the NMR spectra in terms of signal overlap and removal of chemical shift degeneracy, allowing the mining of crucial data from the shifted NMR spectra. 2D NMR spectra (1H–1H–COSY, NOESY, HSQC and HMBC) of the sample mixed with Eu(fod)3 proved to be particularly valuable in this respect. The obtained additional information revealed that the compound in question was a rare sesquiterpene – elemenal (elema-1,3,11(13)-trien-12-al). Therefore, herein we report on a new chromatography-free methodology that could be of value in structure elucidation of unknown compounds even if they are not available in a pure state.

Herein, a novel soil bacterium Streptomyces sp. NP10 able to grow outside usual streptomycetes optimum conditions (e.g., at 4 A degrees C, pH 9 and high NaCl concentration), exhibiting atypical hemolytic, DNAse, and cellulolytic activities, is described. This strain produces and excretes into the growth medium large amounts of free long-chain fatty acids (FAs). A concurrent lipidomics study revealed a large structural diversity of FAs with over 50 different n- and branched-chain, (un)saturated, and cyclopropane FAs (C-7-C-30) produced by this strain. Two of these, i-17:0cy9-10 and a-18:0cy9-10, represent new natural products and the first ever identified branched cyclopropane FAs. Both free and bound lipid profiles of Streptomyces sp. NP10 were dominated by saturated branched chain FAs (i-14:0, a-15:0, and i-16:0). Although these free FAs showed only a moderate antimicrobial activity, our results suggest that they could have an ecophysiological role in interspecies signaling with another soil microorganism Pseudomonas aeruginosa. This work represents the first comprehensive report on the structural diversity and complexity of the free FA pool in Streptomyces. A naturally occurring streptomycete, such as Streptomyces sp. NP10, which secretes significant amounts of free long-chain FAs (non-cytotoxic) into the medium, could be useful in microbial biodiesel production. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3430]