Marija S. Denić

A novel toxic alkaloid from poison hemlock (Conium maculatum L., Apiaceae): identification, synthesis and antinociceptive activity.

2-Pentylpiperidine, named conmaculatin, a novel volatile alkaloid related to coniine was identified from the renowned toxic weed Conium maculatum L. (Apiaceae). The structure of conmaculatin was corroborated by synthesis (8 steps starting from cyclohexanol, overall yield 12%). Conmaculatins strong peripheral and central antinociceptive activity in mice was observed in a narrow dose range (10-20mg/kg). It was found to be lethal in doses higher than 20mg/kg.

Antimicrobial phenolic abietane diterpene from Lycopus europaeus L. (Lamiaceae)

A new acetylated highly oxygenated abietane-type diterpenoid named euroabienol was isolated in pure state from Lycopus europaeus L. (Lamiaceae) fruits and its structure elucidated through both extensive spectral and chemical means. The presence of a phenolic C ring with a rare substitution pattern in euroabienol and its high relative amount in the fruits (1%, based on the weight of the fruits) urged us to try to establish its possible biological role. Thus, it was screened for its in vitro antimicrobial activity against fifteen strains of bacteria and six fungal strains. Euroabienol showed a broad spectrum of activity and probably is a first line defense plant metabolite against pathogen attack. This is the first report on the occurrence of abietanes in the genus Lycopus.

Essential Oils from the Roots of Echinops bannaticus Rochel ex Schrad. and Echinops sphaerocephalus L. (Asteraceae): Chemotaxonomic and Biosynthetic Aspects

Herein, we report, for the first time, the results of detailed chemical and statistical analyses of the essential oils from the roots of two Echinops species (Asteraceae), E. bannaticus Rochel ex Schrad., and E. sphaerocephalus L., from the weed flora of Serbia. Among 106 and 81 constituents, respectively, S‐containing polyacetylene compounds and triquinane sesquiterpenoids made up ca. 80% of the oils. Several of these compounds are reported here as new metabolites for the two species or even for the genus Echinops. A multivariate statistical comparison of the essential‐oil composition data for these two and additional six taxa of this genus available from the literature permitted an examination of the mutual relationships of the taxa within this morphologically highly uniform genus. Principal component analysis (PCA) and agglomerative hierarchical clustering revealed a grouping of E. bannaticus and E. sphaerocephalus (both belonging to the section Echinops), and their close relationship with E. grijsii, suggesting a circumscription of this Chinese taxon to the section Echinops. PCA Correlation matrix offered valuable insight into the biosynthetic links between essential‐oil constituents, and these agreed excellently with the currently proposed ones for the polyacetylene S‐containing compounds, triquinanes, and monoterpenes.

Synthesis of small combinatorial libraries of natural products: identification and quantification of new long-chain 3-methyl-2-alkanones from the root essential oil of Inula helenium L. (Asteraceae).

INTRODUCTION Recently, a potent anti-staphylococcal activity of Inula helenium L. (Asteraceae) root essential oil was reported. Also, bioassay guided fractionation of the oil pointed to eudesmane sesquiterpene lactones and a series of unidentified constituents as the main carriers of the observed activity. OBJECTIVE To identify nine new constituents (long-chain 3-methyl-2-alkanones) from a fraction of this root essential oil with a low minimum inhibitory concentration value (0.8 µg/mL) by employing a synthetic methodology that leads to the formation of a small combinatorial library of these compounds. METHODS The identity of these constituents was inferred from mass spectral fragmentation patterns and GC retention data. A library of 3-methyl-2-alkanones (C11 -C19 homologous series) was synthesised in three steps starting from methyl acetoacetate and the corresponding alkyl halides. The synthetic library was also screened for in vitro anti-microbial activity. RESULTS Gas chromatographic analyses of I. helenium essential oil samples with spiked compounds from the synthesised library corroborated the tentative identifications of the long-chain 3-methyl-2-alkanones. The availability of these anti-microbial compounds from this library made it possible to construct GC/FID calibration curves and determine their content in the plant material: 0.08 - 24.2 mg/100 g of dry roots. CONCLUSION The small combinatorial library approach enabled the first unequivocal identification of long-chain 3-methyl-2-alkanones as plant secondary metabolites, and, also, allowed determination of not only a single compound and biological properties, but those of a group of structurally related compounds.

RP-HPLC assay of doxycycline in human saliva and gingival crevicular fluid in patients with chronic periodontal disease

A reversed-phased HPLC method with fluorescence detection was optimized and validated for determination of DOXY in human saliva and gingival crevicular fluid (GCF) with tetracycline as internal standard. Single step extraction with acetonitrile for both types of samples was performed. The separation was achieved at Zorbax Extend-C18 analytical column at 30°C. Mobile phase was consisted of an aqueous phase containing magnesium acetate, ammonium acetate, Na₂EDTA, triethyl-ammonium acetate buffered to pH 7.5 with ammonium hydroxide solution and acetonitrile. The volume ratio of the buffered water mixture of salts and acetonitrile was 86:14. Fluorescence detector was set at λex=380 nm and λem=520 nm. Under the optimized experimental conditions, good linearity was found in the range of 5.0-250.0 ng/mL for GCF with LOD of 1.63 ng/mL and LOQ of 4.93 ng/mL and 20.0-500.0 ng/mL for saliva with LOD of 6.36 ng/mL and LOQ of 19.28 ng/mL. This method was successfully applied for determination of DOXY in saliva and GCF obtained from patients with chronic periodontal disease.

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.