Characterization of Secondary Metabolites of Leaf Buds from Some Species and Hybrids of Populus by Gas Chromatography Coupled with Mass Detection and Two-Dimensional High-Performance Thin-Layer Chromatography Methods with Assessment of Their Antioxidant Activity.

Poplars provide medicinal raw plant materials used in pharmacy. Leaf buds are one of the herbal medicinal products collected from poplars, having anti-inflammatory and antiseptic properties, but there are no quality standards for their production and there is a need to determine their botanical sources. Therefore, the chemical compositions of the leaf buds from four species and varieties of poplars, Populus balsamifera, P. × berolinensis, P. × canadensis 'Marilandica', and P. wilsonii were investigated and compared using gas chromatography coupled with mass detection (GC-MS) and two-dimensional high-performance thin-layer chromatography (2D-HPTLC) in order to search for taxa characterized by a high content of biologically active compounds and with a diverse chemical composition that determines their therapeutic effects. The presence of 163 compounds belonging to the groups of flavonoids, phenolic acids derivatives, glycerides, and sesquiterpenes was revealed. Moreover, the conditions for the separation and identification of biologically active compounds occurring in analyzed leaf buds using 2D-HPTLC were optimized and used for metabolomic profiling of the studied poplars, enabling their fast and simple botanical identification. The total phenolic (TPC) and flavonoid (TFC) contents of examined extracts were determined and their antioxidant capacities were estimated by spectrophotometric DPPH, ABTS, and FRAP assays. Based on the analysis of phytochemicals and antioxidant activity, P. × berolinensis buds were selected as the raw plant material for medicinal purposes with the highest content of active compounds and the strongest antioxidant activity.

Chemical Composition of Volatile and Extractive Components of Canary (Tenerife) Propolis.

The vegetation of the Canary Islands is characterized by a large number of endemic species confined to different altitudinal levels. It can be assumed that these circumstances determine the characteristic features of the chemical composition of local beekeeping products, including propolis. We report, for the first time, the chemical composition of propolis from Tenerife (Canary Islands). The volatile emissions of three propolis samples collected from different apiaries are represented by 162 C1-C20 compounds, of which 144 were identified using the HS-SPME/GC-MS technique. The main group of volatiles, consisting of 72 compounds, is formed by terpenoids, which account for 42-68% of the total ion current (TIC) of the chromatograms. The next most numerous groups are formed by C6-C17 alkanes and alkenes (6-32% TIC) and aliphatic C3-C11 carbonyl compounds (7-20% TIC). The volatile emissions also contain C1-C6 aliphatic acids and C2-C8 alcohols, as well as their esters. Peaks of 138 organic C3-C34 compounds were recorded in the chromatograms of the ether extracts of the studied propolis. Terpene compounds form the most numerous group, but their number and content in different samples is within very wide limits (9-63% TIC), which is probably due to the origin of the samples from apiaries located at different altitudes. A peculiarity of the chemical composition of the extractive substances is the almost complete absence of phenylcarboxylic acids and flavonoids, characteristic of Apis mellifera propolis from different regions of Eurasia and North America. Aromatic compounds of propolis from Tenerife are represented by a group of nine isomeric furofuranoid lignans, as well as alkyl- and alkenyl-substituted derivatives of salicylic acid and resorcinol.

Chemical Composition and Biological Activity of Argentinian Propolis of Four Species of Stingless Bees.

The chemical composition of propolis of four species of stingless bees (SLBs) from Argentina was determined, and its antibacterial and anticancer activity was evaluated on selected types of microbes and cancer cell lines. Volatile secretions of all propolis samples are formed by 174 C2-C15 organic compounds, mainly mono- and sesquiterpenes and their derivatives. The chromatograms of ether extracts showed 287 peaks, of which 210 were identified. The most representative groups in the extracts of various propolis samples were diterpenoids (mainly resin acids), triterpenoids and phenolic compounds: long-chain alkenyl phenols, resorcinols and salicylates. The composition of both volatile and extractive compounds turned out to be species-specific; however, in both cases, the pairwise similarity of the propolis of Scaptotrigona postica and Tetragonisca fiebrigi versus that of Tetragona clavipes and Melipona quadrifasciata quadrifasciata was observed, which indicated the similarity of the preferences of the respective species when choosing plant sources of resin. The composition of the studied extracts completely lacked flavonoids and phenolcarboxylic acids, which are usually associated with the biological activity and medicinal properties of propolis. However, tests on selected microbial species and cancer cell lines showed such activity. All propolis samples tested against Paenibacillus larvae, two species of Bacillus and E. coli showed biofilm inhibition unrelated to the inhibition of bacterial growth, leading to a decrease in their pathogenicity. Testing the anticancer activity of ether extracts using five types of cell cultures showed that all four types of propolis studied inhibit the growth of cancer cells in a dose- and time-dependent manner. Propolis harvested by T. clavipes demonstrated the highest cytotoxicity on all tested cell lines.

Secondary metabolites of downy birch buds (Betula pubescens Erch.).

The subject of this study is the composition of low-molecular-weight metabolites in downy birch (Betula pubescens) buds and their participation in protection from various kinds of stress. Using the GC-MS, 640 compounds were detected, of which 314 were identified in downy birch buds for the first time. The volatile components detected using the SPME technique mainly consisted (about 70% of the total ionic current of the chromatogram, TIC) of mixtures of sesquiterpenoids. The exudate covering the buds, along with sesquiterpenoids (approximately 60% of TIC), included flavonoids (25% of TIC). The main part of the material extracted by supercritical carbon dioxide from buds comprised sesquiterpenoids and triterpenoids (47 and 28% of TIC, respectively). Via column chromatography, 25 known compounds (mainly flavonoids and triterpenoids) were isolated, most of which were first discovered in the buds of downy birch. Many compounds of these classes have strong biological activity and probably either directly or indirectly perform a protective function in birch buds. An assumption is made about the biological role of a number of secondary metabolites (such as volatile isomeric megastigmatriens and triterpene seco-acids) as well as about these compounds' possible means of biosynthesis, which were first discovered in the buds of downy birch.

Gas chromatographic-mass spectrometric determination of glycosides without prior hydrolysis.

The article presents for the first time the linear temperature programmed retention indices on a column with stationary phases of 5% phenylpolydimethyl silicone and the mass spectra of trimethylsilyl (TMS) derivatives of 71 glycosides (both commercial preparations and compounds extracted from plant tissues) which were not characterized earlier by these parameters. Converted to their TMS derivatives, the glycosides were thermally stable: they exhibited single peaks on their chromatograms without products of thermal decomposition. Therefore this work demonstrates the suitability of high resolution-high temperature gas chromatography (HR-HT/GC) to analyse different groups of glycosides including compounds with disaccharide moieties without the necessity of their hydrolyses. Since a limited number of commercial and plant-isolated glycosides were available, an attempt was made to assess their retention indices using the known "structure-retention relationships" approach. It was demonstrated that the retention indices of silanised glycosides and their aglycones were characterized by a linear dependence.

Selective Behaviour of Honeybees in Acquiring European Propolis Plant Precursors.

Honey bees harvest resins from various plant species and use them in the hive as propolis. While there have been a number of studies concerning the chemical composition of this antimicrobial product, little is known about selective behavior and bee preference when different potential plant sources of resin are available. The main objective of this paper was to investigate some aspects of behavioral patterns of honeybees in the context of resin acquisition. Samples of propolis originating from temperate zones of Europe and the supposed botanical precursors of the product were analyzed. Taxonomical markers of bud resins of two white birch species, aspen, black poplar, horse-chestnut, black alder, and Scots pine were determined through GC-MS analysis. All these trees have been reported as sources of propolis, but comparisons of the chemical composition of their bud resins with the compositions of propolis samples from seven European countries have demonstrated the presence of taxonomical markers only from black poplar, aspen, and one species of birch. This suggests selective behavior during the collection of bud resins by honeybees. To examine the causes of such selectivity, the antimicrobial properties of bud resins were determined. Horse-chestnut resins had lower antimicrobial activity than the other resins which did not differ significantly.

Rapid GC/MS determination of botanical precursors of Eurasian propolis.

In this work, ether extracts of propolises from 11 countries of Europe and Asia together with extracts of the buds of their principal plant precursors were prepared and investigated by gas chromatography-mass spectrometry. For the first time, chemical compositions of the exudates of aspen, white birch and silver birch buds were determined. Basing on the data on the content of individual components and their groups, the method of rapid examination of plant precursors of propolis was developed. This new method is based on the use of a chromatogram for one extracted ion m/z=219, characteristic for a group of similar substances: phenylpropenoids. The I(T) retention indices of silylated derivatives of 449 substances registered in the buds of 6 species of trees and 37 samples of propolis extracts were calculated. These useful analytical parameters (majority of which are absent in the available databases) are attached in Supplementary information.

Gas chromatographic and mass spectrometric characterization of trimethylsilyl derivatives of some terpene alcohol phenylpropenoids.

This paper reports mass spectra and linear temperature programmed retention indices (I(T)) for 31 esters of mono- and sesquiterpene alcohols with hydroxycinnamic acids. In this study, 14 phenylpropenoids were synthesized by esterification of terpenols with p-coumaric, ferulic, caffeic and sinapinic acids. Other phenylpropenoids were semi-quantitatively isolated by column chromatography from exudates covering the buds of two birch species (Betula pubescens and B. litwinowii). Main diagnostic ions in phenylpropenoids mass spectra were determined by GC/MS analysis. The possibility of predicting I(T) values was demonstrated with standard error of prediction between 3 and 11 of retention index units.

HS-SPME/GC-MS analysis of volatile and semi-volatile organic compounds emitted from municipal sewage sludge.

The aim of the research involved identification and semi-quantitative determination of unknown volatile and semi-volatile organic compounds emitted to air by sewage sludge formed in the process of municipal wastewater treatment in a sewage treatment plant. Samples taken directly after completion of the technological process as well as the sludge stored on the premise of the sewage treatment plant were analyzed. A simple method using off-line headspace solid-phase microextraction combined with gas chromatography-mass spectrometry has been proposed for extraction and detection of organic pollutants. For reliable identification of compounds, combination of two independent parameters: mass spectra and linear temperature programmed retention indices were employed. Over 170 compounds of different structure were identified including aliphatic and aromatic hydrocarbons, alcohols, esters, carbonyls, as well as sulfur, nitrogen, and chlorine containing compounds. The prevailing substances included: ethyl ether, n-hexane, p-xylene, o-xylene, mesitylene, m-ethylbenzene, limonene, n-decane, n-undecane, and n-dodecane. A few compounds such as methanetiol, dimethyl polisulfide, octaatomic sulfur, phthalic anhydride, and indoles were identified in the sludge for the first time.

Gas chromatographic retention indices of trimethylsilyl derivatives of terpene alcohols.

This paper presents the experimentally determined retention indices (RI(TMS)) for a set of 75 silylated terpenols (33 monoterpenols and 42 sesquiterpenols). The attempt was made to assess the dependence of RI(TMS) on RI (for non-silylated terpenols) and on RI(Ac) (for acetylated terpenols). Satisfactory linear regression parameters (RI(TMS)=b(0)+b(1)RI) were observed for tertiary substituted monoterpenols and primary or secondary substituted sesquiterpenols. The mass spectra of silylated terpenols that were not found in the available literature are in Supplementary information.

Enhancement of antibacterial effects of extracts from Cirsium species using sodium picolinate and estimation of their toxicity.

In this study, antimicrobial properties and toxicity of extracts from Cirsium spp.: Cirsium arvense, C. oleraceum, C. palustre, C. rivulare and C. vulgare in combination with sodium picolinate (PS) or sodium benzoate (BS), were investigated. Three micro-organisms were used: Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa. Minimum inhibitory concentration (MIC) of extracts was found at 1.56-50.0 mg mL(-1). Unlike the case of BS, adding PS to extracts from flowers of C. palustre and C. arvense enhanced their antimicrobial effect on S. aureus (MIC from 6.25-12.5 mg mL(-1) to 1.25-5.0 mg mL(-1)). An MTT test was used to study toxicity effects. The extracts from C. palustre or C. arvense mixed with PS had a concentration-dependent, slightly cytotoxic or stimulating effect on the viability of normal human skin fibroblasts. The total phenolic content (TPC) of samples varied from 44 to 178 mg gallic acid equivalent per 1 g of extract. The highest TPC was observed in C. palustre (l) and C. oleraceum (f). Our results did not show any correlation between antimicrobial activities and TPC. Cirsium palustre (f) and C. arvense (f) extracts were analysed by gas chromatography/mass spectrometry (GC/MS). About 30 compounds were found to be present in extracts from two Cirsium species in amounts of not less than 0.2% of TIC.

Chemical composition and antimicrobial activity of essential oil of Bupleurum montanum and B. plantagineum.

The hydrodistilled oils from the aerial parts of Bupleurum montanum and B. plantagineum, which are endemic to North Africa, including Algeria, were analyzed by gas chromatography-mass spectrometry (GC-MS). Ninety-eight compounds were detected in the oil of B. montanum, representing 98.6% of the total oil, and 68 in the oil of B. plantagineum, representing 99.8% of the total. Megastigma-4,6-(E),8(2)-triene was the major constituent of B. montana oil (25.3%). Other important compounds were myrcene, alpha-pinene and benzyl tiglate. Conversely, the major constituents of the oil of B. plantagineum were alpha-pinene (31.9%), cis-chrysanthenyl acetate (28.2%), and myrcene (24.8%), followed by the monoterpene hydrocarbon limonene (5.1%). The mutagenic, antiplasmodial and antimicrobial activities of the essential oils were individually evaluated against eleven microorganisms, using the agar diffusion method, by determination of MIC values. The investigated oils exhibited moderate antimicrobial activity. Maximum activity of the oils was observed against Nocardia asteroides, Staphylococcus aureus and Enterococcus faecalis. Fungicidal activity against Candida albicans was also found for both oils.

Gas chromatographic retention indices of biologically and environmentally important organic compounds on capillary columns with low-polar stationary phases.

This work presents linear temperature programmed retention indices on the columns with stationary phases of 5% phenylpolydimethyl silicone of 389 organic compounds, including extractive substances of plant tissues and environmentally important compounds. Certain factors which influence the values and reproducibility of retention indices during gas chromatographic analysis of multicomponent mixtures are discussed.

GC identification of organic compounds based on partition coefficients of their TMS derivatives in a hexane-acetonitrile system and retention indices.

This paper reports the experimentally determined and calculated partition coefficients (Kp) in a heterogeneous system consisting of two partially mixing organic solvents for trimethylsilyl derivatives of 119 compounds including phenols, phenolcarboxylic acids, mono- and dicarboxylic acids, and some carbohydrates and polyols. It was demonstrated that a simple model based on the available physico-chemical properties of molecules can be used to accurately predict the hexane-acetonitrile partition coefficients. Phenols, other organic acids, and carbohydrates form complex mixtures in extracts of plant tissues, in various biological fluids, as well as in waste waters. For group identification of these mixture components, it is desirable to use the parameterj which is a combination of Kp and gas chromatographic retention indices: j = kI - log Kp. This approach increases identification reliability in gas chromatographic analysis. The use of the group parameterj in the analysis of nonvolatile organic components of waste waters is reported.

Experimental determination and calculation of distribution coefficients between air and fiber with polydimethylsiloxane coating for some groups of organic compounds.

This paper reports the results of experimental determination of distribution coefficients K(fa) for five terpene hydrocarbons and five aliphatic ketones between air and polydimethylsiloxane (PDMS) coating for solid-phase microextraction. To estimate the values of K(fa) for compounds of the same classes, which did not undergo experiments, it is proposed to use an empirical two-parameter equation in which various physicochemical and structural characteristics weakly correlated with each other are used as descriptors. It is also shown that for these purposes it is possible to use distribution coefficients of compounds in any other two-phase heterogeneous system, e.g. octanol-water or hexane-acetonitrile. This approach was applied to estimate K(fa) values of 92 volatile organic compounds.

GC-MS analysis of compounds extracted from buds of Populus balsamifera and Populus nigra.

The composition of hexane and ether extracts from buds of two poplar species (Populus balsamifera and P. nigra) was investigated by GC-MS method. In hexane extracts, 54 "neutral" compounds were recorded. The greatest amounts of them are sesquiterpenes and n-alkanes. Among 56 components of ether extracts, many aliphatic acids and hydroxyacids were detected. However, the main fraction consists of phenolcarboxylic acids, substituted cinnamic acids, and their esters. It was established that chemotaxonomic differences between Populus balsamifera and P. nigra are observed in the case of both hexane and ether bud extracts.