Seasonal Variability of Volatile Components in Calypogeia integristipula.

Liverworts contain a large number of biologically active compounds that are synthesised and stored in their oil bodies. However, knowledge about the chemical composition of individual species is still incomplete. The subject of the study was Calypogeia integristipula, a species representing leafy liverworts. Plant material for chemotaxonomic studies was collected from various locations in Poland. The chemical composition was determined in 74 samples collected from the natural environment in 2021 and 2022 in three growing seasons: spring, summer and autumn, and for comparison with samples originating from in vitro culture. The plants were classified as Calypogeia integristipula on the basis of morphological characteristics, oil bodies, and DNA markers. The volatile organic compounds (VOCs) from the biological material were extracted by headspace solid phase microextraction (HS-SPME). The samples were then analysed by gas chromatography-mass spectrometry (GC-MS). A total of 79 compounds were detected, of which 44 compounds were identified. The remaining compounds were described using the MS fragmentation spectrum. Cyclical changes in the composition of compounds associated with the growing season of Calypogeia integristipula were observed. Moreover, samples from in vitro culture and samples taken from the natural environment were shown to differ in the composition of chemical compounds. In terms of quantity, among the volatile compounds, compounds belonging to the sesquiterpene group (46.54-71.19%) and sesqiuterpenoid (8.12-22.11%) dominate. A smaller number of compounds belong to aromatic compounds (2.30-10.96%), monoterpenes (0.01-0.07%), monoterpenoids (0.02-0.33%), and aliphatic hydrocarbons (1.11-6.12%). The dominant compounds in the analysed liverworts were: anastreptene (15.27-31.14%); bicyclogermacrene (6.99-18.09%), 4,5,9,10-dehydro-isolongifolene (2.00-8.72%), palustrol (4.95-9.94%), spathulenol (0.44-5.11%).

Comparison of Extraction Techniques for the Determination of Volatile Organic Compounds in Liverwort Samples.

This article focuses on the comparison of four popular techniques for the extraction of volatile organic compounds (VOCs) from liverworts of the Calypogeia azurea species. Since extraction is the most important step in the sample analysis of ingredients present in botanical preparations, their strengths, and weaknesses are discussed. In order to determine the VOCs present in plants, selecting the appropriate one is a key step of the extraction technique. Extraction should ensure the isolation of all components present in the oily bodies of Calypogeia azurea without the formation of any artifacts during treatment. The best extraction method should yield the determined compounds in detectable amounts. Hydrodistillation (HD), applying Deryng apparatus and solid-liquid extraction (SLE), microwave-assisted extraction (MAE), and headspace solid-phase microextraction (HS-SPME) were used for volatile extraction. The extracts obtained were analysed by gas chromatography coupled to mass spectrometry (GC-MS) to determine the compounds.

Seasonal Variability and Effect of Sample Storage on Volatile Components in Calypogeia azurea.

A change in the composition of specialized metabolites is often observed in stressed plants. Phytochemicals play an important role in adapting plants to the environment, particularly overcoming stress conditions such as temperature, humidity, and light intensity. In this study, seasonal variations in the concentrations of volatile organic compounds (VOCs) were analysed in species of Calypogeia azurea. The article presents the effect of sample storage on volatile organic compounds present in Calypogeia liverwort cells and whether the collection habitats of the sample affect the content of phytochemicals. The VOCs of the species within the liverwort Calypogeia azurea were analysed by GC-MS. Compounds were isolated from plant material using the HS-SPME technique. The samples were collected over several years (2019-2021). Of the several dozen samples collected, 79 compounds were isolated, of which 47 were identified.

Chemical Fingerprinting of Cryptic Species and Genetic Lineages of Aneura pinguis (L.) Dumort. (Marchantiophyta, Metzgeriidae).

Aneura pinguis (L.) Dumort. is a representative of the simple thalloid liverworts, one of the three main types of liverwort gametophytes. According to classical taxonomy, A. pinguis represents one morphologically variable species; however, genetic data reveal that this species is a complex consisting of 10 cryptic species (named by letters from A to J), of which four are further subdivided into two or three evolutionary lineages. The objective of this work was to develop an efficient method for the characterisation of plant material using marker compounds. The volatile chemical constituents of cryptic species within the liverwort A. pinguis were analysed by GC-MS. The compounds were isolated from plant material using the HS-SPME technique. Of the 66 compounds examined, 40 were identified. Of these 40 compounds, nine were selected for use as marker compounds of individual cryptic species of A. pinguis. A guide was then developed that clarified how these markers could be used for the rapid identification of the genetic lineages of A. pinguis. Multivariate statistical analyses (principal component and cluster analysis) revealed that the chemical compounds in A. pinguis made it possible to distinguish individual cryptic species (including genetic lineages), with the exception of cryptic species G and H. The classification of samples based on the volatile compounds by cluster analysis reflected phylogenetic relationships between cryptic species and genetic lineages of A. pinguis revealed based on molecular data.

Straightforward and rapid determination of acrylamide in coffee beans by means of HS-SPME/GC-MS.

A straightforward and rapid preparation procedure for the extraction of the acrylamide from coffee beans, by means of the volatile silylated derivatives of acrylamide (AA) and headspace solid phase microextraction (HS-SPME) is described. Commercially available SPME fibre coated with polydimethylsiloxane (PDMS) was used. The silylation reactions of the AA were executed with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The deuterium labelled d3-acrylamide was used as an internal standard. The acrylamide level was quantified using gas chromatography coupled with the mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. The limit of quantification (LOQ) for measuring acrylamide was 3 µg/kg. The described method demonstrates satisfactory repeatability (RSD = 2.6%), with an intermediate precision (RSD = 9.4%) and recovery (99-105%).

High correlation of chemical composition with genotype in cryptic species of the liverwort Aneura pinguis.

Chemical constituents of cryptic species detected within the liverwort Aneura pinguis were identified using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). Fibre coating with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) was used. A total of 48 samples of A. pinguis were analysed. The studied plants were identified genetically based on barcode DNA sequences and represented three cryptic species (A, B and F) of A. pinguis. Cryptic species A and B are genetically diverse; both represent three evolutionary lineages: A1, A2, A3 and B1, B2, B3, respectively. The cryptic species F that was recently detected is not diverse. The most characteristic compounds in analysed samples were sesquiterpene hydrocarbons (up to 17.7% for A1; 15.7% for A2; 20.6% for A3; 7.7% for B1; 2.0% for B2; 3.7% for B3; 10.2% for F), oxygenated sesquiterpenoids (up to 68.0% for A1; 54.7% for A2; 52.6% for A3; 63.5% for B1; 88.7% for B2; 82.7% for B3; 78.8% for F), and linear aliphatic hydrocarbons (up to 14.8% for A1; 1.1% for A2; 12.1% for A3; 6.9 for B1; 5.2% for B2; 1.1% for B3; 7.0% for F). The dominant compound in the studied samples was pinguisone. The second dominant compound present in the tested plant material was deoxopinguisone, except for lineage B2, where only a small relative concentration of this compound was found. A high content of deoxopinguisone in cryptic species A (lineages A1, A2 and A3) was accompanied by the presence of isopinguisone and methyl norpinguisonate, whereas these two compounds were not detected in cryptic species B (lineages B1 and B3) and F. The chemical compounds detected in the studied samples of A. pinguis were subjected to multivariate statistical analysis. The results showed that the chemical composition depends mainly on the genotype of the plant and slightly on the habitat. However, there was no clear correlation between the volatile compounds and the date of collection of the studied plants.

Antioxidant properties of thio-caffeine derivatives: Identification of the newly synthesized 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine as antioxidant and highly potent cytoprotective agent.

A series of nine thio-caffeine analogues were synthesized and characterised by NMR, FT-IR and MS spectroscopic methods. Molecular structures of four of them were determined using single crystal X-ray diffraction methods. The antioxidant properties of all compounds, at concentration ranges from 0.025 to 0.1mg/mL, were evaluated by various chemical- and cell-based antioxidant assays. Human erythrocytes were used to examine in vitro haemolytic activity of all compounds and their protective effect against oxidative haemolysis induced by AAPH, one of the commonly used free radical generator. All compounds studied showed no effect on the human erythrocytes membrane structure and permeability with the exception of 8-(phenylsulfanyl)caffeine. Among the nine caffeine thio-analogues tested, the newly synthesized 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine possessed exceptionally high antioxidant properties. Moreover, it protects human erythrocytes against AAPH-induced oxidative damage as efficiently as the standard antioxidant Trolox. Therefore, 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine may have a significant cytoprotective potential caused by its antioxidant activity.

Volatile compounds in cryptic species of the Aneura pinguis complex and Aneura maxima (Marchantiophyta, Metzgeriidae).

Aneura pinguis is one of the liverwort species complexes that consist of several cryptic species. Ten samples collected from different regions in Poland are in the focus of our research. Eight of the A. pinguis complex belonging to four cryptic species (A, B, C, E) and two samples of closely related species Aneura maxima were tested for the composition of volatile compounds. The HS-SPME technique coupled to GC/FID and GC/MS analysis has been applied. The fiber coated with DVB/CAR/PDMS has been used. The results of the present study, revealed the qualitative and quantitative differences in the composition of the volatile compounds between the studied species. Mainly they are from the group of sesquiterpenoids, oxygenated sesquiterpenoids and aliphatic hydrocarbons. The statistical methods (CA and PCA) showed that detected volatile compounds allow to distinguish cryptic species of A. pinguis. All examined cryptic species of the A. pinguis complex differ from A. maxima. Species A and E of A. pinguis, in CA and PCA, form separate clusters remote from two remaining cryptic species of A. pinguis (B and C) and A. maxima. Relationship between the cryptic species appeared from the chemical studies are in accordance with that revealed on the basis of DNA sequences.

Characterization of surface of trifluorophenylketoiminepropyl stationary phase coated porous layer open tubular columns.

A series of 3 fluorinated adsorbents were prepared by bonding 4,4,4-trifluoro-1-phenyl-1,3-ketoiminepropyl silane onto 5µm Nucleosil silica. The first one contained sole fluoroketoimine groups, while the other two adsorbents were derivatives of the first one with copper(II) chloride and copper(II) acetylacetonate bound through the fluoroketoimine groups. A description of syntheses of these adsorbents is given and the course of each stage of the syntheses is characterized by elemental analysis, surface area determination, UV-vis, EPR, ICP-OES, IR, (29)Si NMR, (13)C NMR and thermogravimetric analysis. For the columns coated with the adsorbents obtained the donor-acceptor character of the adsorption layer was established and the enthalpy of adsorption was determined for a selected group of hydrocarbons. The evenness of capillary column walls coating and mechanical stability of the stationary phases was evaluated on the basis of the flow restriction factor and SEM images. Efficiency of the columns obtained and their comparison with commercial counterparts are presented.

Quantitative relationship and application of 3-benzylketoimine metal dichlorides in the analysis of volatile hydrocarbons.

The paper concerns the analysis of volatile hydrocarbons on newly designed and produced capillary columns. According to the type of coating used, the columns are of the PLOT type, and according to the adsorbate-adsorbent type interactions they belong to the category of complexation GC. The adsorbent used was silica, whose surface was modified with bonded 2-(3-triethoxysilylpropylimino)-3-(benzyl)-pentanone-4 to change the adsorption characteristics. The presence of the ketoimine group of strongly electron-accepting properties permitted the binding of transition metals. The metal ions are capable of interacting selectively with nucleophilic compounds including olefins. The transition metal ions used for sorbent modification were copper(II) chlorides and nickel(II) chlorides. The supported adsorbent was immobilised by bonding the modified silica to the column walls by a film of poly(dimethylsiloxane). Due to this strategy, the coating was very stable, no sorbent loss from the column was observed and the column could be washed with solvent. Besides exemplary separations, the paper quotes retention parameters of the group of compounds analysed and presents a discussion of the specific interactions between the adsorbate and adsorbent.

Determination of Acylglycerols in Diesel Oils by GC.

In many EU countries and outside the EU, besides the addition of pure methyl ester B-100 to diesel oil, mixtures of methyl esters are also added to fuel. To be used as fuel, methyl esters must meet certain requirements, one of which is a certain level of acylglycerols. The paper presents results of determination of acylglycerols in diesel oil dotted with fatty acid methyl esters. The compounds were determined by gas chromatography using a high-temperature capillary column DB-5HT, made by J&W, and 1,2,3-tricaproylglycerol as internal standard. The analytical method proposed permits not only determination of acylglycerols, but also differentiation if the FAME added originated from pure vegetation oil or used cooking oil.

Ketoimine modified silica as an adsorbent for gas chromatographic analysis of olefins.

New adsorbents for capillary complexation GC have been proposed. The adsorbents contain free ketoimine groups which enable binding copper(II) and chromium(III) chlorides. The adsorbents obtained have been characterised by elemental analysis, differential scanning calorimetry and electron spin resonance. The specific interactions between the adsorbent and adsorbate molecules have been characterised by retention parameters (retention index - I, specific retention volume - Vg, molecular retention index - deltaMe). Attempts were also made at separating mixtures of olefins.