Organic residue analysis of Neolithic pottery from North Greece

Organic residues associated with Neolithic pottery from two Late Neolithic sites, Paliambela and Makriyalos (Northern Greece), were analyzed by gas chromatography-mass spectrometry. The study aimed at identifying the origin of the tar used for waterproofing and gluing broken pots. Reference tars were prepared in laboratory conditions by pyrolysis of the bark from three tree species, namely from Betula pendula (silver birch), Ostrya carpinifolia (hop hornbeam) and Carpinus orientalis (Eastern hornbeam). The results suggest that mostly birch bark tar was used as glue for fixing broken pieces of the pots as well as for waterproofing close-shaped vessels. None of the archaeological tars derived from the two hornbeams. The analysis shows certain variability in the composition of birch bark tar, which is related to the production technique and to the re-use of tar. Of particular interest is the presence of characteristic diterpenoid biomarkers in several samples, which indicates that pine pitch was used for gluing and pine resin for waterproofing vessels for liquids. Correspondence: Sophia Mitkidou, Department of Science, School of Technological Applications, Technological Institute of Kavala, Greece     

Investigating the history of prehistoric glues by gas chromatography-mass spectrometry

Although organic materials are very sensitive to biochemical alteration processes, they may be preserved for millennia in various archaeological contexts. Remains of adhesives made during prehistory were discovered at different sites, in the form of residues adhering to flint tools and ceramic vessels or as free lumps in sediment. To characterise the natural substances exploited for adhesive production during late prehistory, we undertook GC and GC/MS analysis of 90 samples from 8 sites dating from the Neolithic to Iron Age periods. This paper discusses our approach to the study of organic adhesives preserved in archaeological contexts, with a particular focus on the presentation of the various categories of organic adhesives that we analysed and the choice of chromatographic conditions adapted to the specificity of such samples. The results obtained show that birch bark tar, a triterpenoid adhesive made by destructive distillation of white birch bark, was predominantly used during the neolithic period even though other materials such as various barks or organic fossil substance were also used. During the Bronze and Iron ages, which follow the Neolithic period, adhesive production is evolving through the expansion of the range of the natural substances used (identification of diterpenoid pine resin) and the addition of beeswax as a plasticiser to birch bark tar. By combining chromatographic analysis and archaeological data, it was thus possible to follow the evolution of adhesive making at the end of prehistory, testifying to the inventiveness of the craftsmen whatever the period considered.     

Analytical pyrolysis with in situ thermally assisted derivatisation, Py(HMDS)-GC/MS, for the chemical characterization of archaeological birch bark tar

An analytical method based on pyrolysis in the presence of hexamethyldisilazane followed by gas chromatographic/mass spectrometric analysis, Py(HMDS)-GC/MS, was used for the first time to study birch bark tar, an adhesive that has been used since the Palaeolithic period. Py(HMDS)-GC/MS enabled us to obtain information simultaneously both on polymerised and solvent soluble fractions of birch bark tar. A very particular chromatographic pattern was obtained, which was mainly characterised by the presence of homologous series of linear saturated and unsaturated monocaboxylic fatty acids, and homologous series of straight-chain alkenes and alkanes. The pattern can be linked to suberin and suberan, which are known to be present in birch barks. In addition, lupane-type triterpenoids were also revealed. Py(HMDS)-GC/MS was initially applied to a reference material, and was then used to study the archaeological adhesives from a wooden spindle recovered in a Gallo-Roman burial (3rd century AD) and from the golden sheath of a Thracian dagger (8th-7th centuries BC) recovered in Belogradets (Bulgaria).     

GC/MS and chemometrics in archaeometry

Summary 100 mg amounts of glue on two copper-age arrowheads have been investigated by means of gas-chromatography/mass spectrometry and chemometrics. The samples have been identified as birch bark derivatives, but the distribution patterns of triterpenoids and steroids show significant differences to similar samples from younger archaeological periods that can be explained by the use of a different species of birch and a different method of preparation.     

Rapid Determination of Betulin in Betula platyphylla Outer Bark Using Near-Infrared Spectroscopy

A simple, rapid, and nondestructive method for the determination of betulin in the outer birch bark was developed using near infrared spectroscopy (NIRS). NIRS data of the outer birch bark collected throughout the year was preprocessed and analyzed by principal component analysis, which led to clear discrimination of the samples according to their harvest times. The reference content of betulin, a major constituent of the outer birch bark, was determined using ultra performance liquid chromatography with a diode array detector (UPLC-DAD). The optimized and validated analytical conditions of UPLC-DAD provided better separation and faster analysis time compared to a conventional HPLC method. Betulin content also showed seasonal variation and was higher in the samples collected during the summer season. Partial least squares calibration techniques were employed to estimate the relationship between the NIRS data and betulin contents. The spectral data showed high correlation coefficients (over 0.700) with betulin content. These results indicate that NIRS combined with UPLC can be used to determine the quality and to quantify the betulin content of the outer birch bark.     

Studies in organic archaeometry VII--differentiation of wood and bark pitches by pyrolysis capillary gas chromatography (PY-CGC)

Tars and pitches were prepared from wood and bark of various deciduous trees and conifers and analysed with the intention to distinguish between various starting materials. All this was done before the background of an archaeometrical problem: to provide a basis for the chemical identification of those pitches which are frequently found in archaeological excavations. Intending to extend the already existing possibilities for such analyses the present paper is dealing with the application of pyrolysis coupled with capillary gas chromatography and by subsequent chemometric studies. It could be shown that by this technique indeed not only tars and pitches prepared from deciduous trees and from conifers could clearly be differentiated, but that even respective species could be identified. Thus a fundament is provided for future studies aiming to identify the starting material of real archaeological finds by a novel method which requires by far less quantities of sample material as compared to the methods hitherto used by us.     

Sorption of methane and ethane high-molecular tars at supercritical temperatures in the Henry region

A theoretical and experimental study of the absorption of methane and ethane by high-molecular tars occurring in oil-gas-condensate deposits is carried out. An original approach to determining the physicochemical parameters of high-molecular tars and to constructing isotherms of absorption of the main components of natural gas by such tars was proposed. The model of the process was developed by analogy with the theory of dissolution of gases in liquids. The sorption of gases by the tar at supercritical temperatures was described within the framework of the van’t Hoff theory of dilute solutions. The proposed approach made it possible to develop a new effective thermodynamic method for calculating the solubility of the component of natural gas in tar at supercritical temperatures and pressure typical of the conditions of natural gas recovery.     

Phenyl-substituted polycyclic aromatic compounds as intermediate products during pyrolytic reactions involving coal tars, pitches and related materials

Summary Several so far unreported phenyl-substituted polycyclic, aromatic compounds were detected in coal tars, coal tar pitches and ring furnace flue gases by their gas chromatographic retention and mass spectra. In the case of thiophene-derived compounds, the elemental sulfur traces obtained by GC-AED supplied further evidence of their identity. Pure authentic substances were synthesised and analysed by GC-MS and GC-AED to prove the identity of the unknown compounds. Concentration profiles of phenyldibenzofurans and phenyldibenzothiophenes in different coal tars, coal tar pitches and ring furnace flue gases are reported. Reaction mechanisms are proposed which suggest that during oxidative pyrolysis phenyldibenzofurans and phenyldibenthiophene are the intermediate products on the path from dibenzofuran and dibenzothiophene to triphenyleno[1,12-bcd]furan and benzobisbenzofurans, and the respective thiophenes.     

Study of the composition of tars produced from blends of coal and polyethylene wastes using high-performance liquid chromatography

Tars produced at semi-industrial scale in a coke oven of 6 x 10(3) kg capacity were used to investigate the effect of using polyethylene waste as an additive in the carbonization process with coal. The polyethylene wastes used were low-density polyethylene from the agriculture greenhouses and high-density polyethylene from domestic sources. The high-performance liquid chromatography analysis of the soluble fractions in toluene and carbon disulfide, using two polystyrene-divinylbenzene columns and a mixture of dichloromethane-methanol as a mobile phase, provides useful information on the composition of tars and their derived pitches in terms of the substitution and molecular topology of polynuclear aromatic compounds (PACs). Differences in composition of tars produced with polyethylene waste at 1% (w/w) have been found to be negligible, while a higher amount of the waste (3%, w/w) promoted the formation of peri-condensed PACs at the expense of the substituted cata-condensed PACs. This behaviour is due to more extensive secondary reactions of tar precursors via dealkylation and aromatic condensation taking place during the carbonization process as a consequence of a more viscous co-carbonizing system. Changes in tar composition caused by this amount of polyethylene waste addition were comparable to those promoted by an increase in the carbonization temperature at semi-industrial and industrial ovens and by the coal preheating before the carbonization process. The characteristic features in tar composition were also found for the derived pitches from tars obtained with the polyethylene waste addition.     

Evaluation of Antioxidative Mechanisms In Vitro and Triterpenes Composition of Extracts from Silver Birch (Betula pendula Roth) and Black Birch (Betula obscura Kotula) Barks by FT-IR and HPLC-PDA

Silver birch, Betula pendula Roth, is one of the most common trees in Europe. Due to its content of many biologically active substances, it has long been used in medicine and cosmetics, unlike the rare black birch, Betula obscura Kotula. The aim of the study was therefore to compare the antioxidant properties of extracts from the inner and outer bark layers of both birch trees towards the L929 line treated with acetaldehyde. Based on the lactate dehydrogenase test and the MTT test, 10 and 25% concentrations of extracts were selected for the antioxidant evaluation. All extracts at tested concentrations reduced the production of hydrogen peroxide, superoxide anion radical, and 25% extract decreased malonic aldehyde formation in acetaldehyde-treated cells. The chemical composition of bark extracts was accessed by IR and HPLC-PDA methods and surprisingly, revealed a high content of betulin and lupeol in the inner bark extract of B. obscura. Furthermore, IR analysis revealed differences in the chemical composition of the outer bark between black and silver birch extracts, indicating that black birch may be a valuable source of numerous biologically active substances. Further experiments are required to evaluate their potential against neuroinflammation, cancer, viral infections, as well as their usefulness in cosmetology.     

Sesquiterpenes from the inner bark of the silver birch and the paper birch

The compositions of the mixtures of sesquiterpenoids, largely hydrocarbons that were found in the inner bark of the silver birch, Betula pendula Roth and the paper birch, Betulapapyrifera Marshall, grown in New Zealand were analyzed by SPME-GCMS. The major components of the volatile oil from the inner bark of B. pendula were trans alpha-bergamotene (31%) and alpha-santalene (19%). This composition was quite different from that of the oil from the branches, buds and leaves of the same species from Turkey, but was very similar to that of the oil from the bark of B. pubescens from Russia. The major components of the oil from the inner bark of B. papyrifera were trans alpha-bergamotene (18%), ar-curcumene (12%), E-beta-farnesene (12%), Z-beta-farnesene (10%) and cis-alpha-bergamotene (8%).     

神木煤显微组分热解和加氢热解的焦油组成

利用色谱-质谱联用技术测定了神木煤镜质组和惰质组在不同热解条件下焦油的组成,考察了显微组分类型和反应气氛对苯类、酚类、萘类、含氧杂环和多环芳烃类化合物产率的影响。结果表明,惰质组和镜质组焦油在组成和长链烃类、芳烃、含氧杂环和多环芳烃的相对质量分数方面存在很大差异。镜质组焦油中长链烃类的种类和相对质量分数较高,惰质组焦油中芳烃、含氧杂环和多环芳烃的种类和相对质量分数较高,反映了镜质组显微组分芳香度较低以及烷基侧链长和多以及惰质组稠环芳烃结构多和芳环缩聚程度高的特点。加氢热解比热解有较高的焦油收率,随氢气压力的增加,焦油的收率大幅度增加。镜质组和惰质组热解和加氢热解焦油组成和相对质量分数的差异反映了热解过程中氢气的加氢稳定化作用和加氢裂解作用。     

Characterization by gas chromatography-mass spectrometry of diterpenoid resinous materials in Roman-age amphorae from northern Greece

A combined gas chromatography-mass spectrometry approach has been used for the characterization of two lumps of resin and 17 adsorbed residues on Roman-age vessels, mainly amphorae, from northern Greece. The data show that a diterpenic resin from plants of the Pinacae family is the main component of the tarry material associated with the analyzed archaeological samples. The identification and mass spectrometric fragmentation of several characteristic diterpenoid biomarkers is discussed. The abundance of secondary products identified in the archaeological samples suggests that the oxidative degradation of abietic acid and dehydroabietic acid to aromatic products was the main pathway. Of particular interest is the presence of characteristic saturated abietane hydrocarbons in one sample, which indicate that a reductive process also occurred on a small scale. The overall similarity in the composition of the residues suggests the common use of pine tar as a waterproofing and sealing agent at different sites in northern Greece during the Roman period.     

Degradation effects in the extraction of antioxidants from birch bark using water at elevated temperature and pressure

Experiments with birch bark samples have been carried to enable a distinction between extraction and degradation effects during pressurised hot water extraction. Two samples, E80 and E180, contained birch bark extracts obtained after extraction at 80 and 180 °C for up to 45 min, respectively. Two other samples, P80 and P180, were only extracted for 5 min at the two temperatures and were thereafter filtered and hydrothermally treated at 80 and 180 °C, respectively. During the latter treatment, samples were collected at different times to assess the stability of the extracted compounds. An offline DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, as well as a high performance liquid chromatographic separation coupled to an electrochemical detector, were used to determine the antioxidant capacity of the processed samples. The results obtained with the different techniques were compared to assess the yield of the extraction and degradation processes. In addition, an online hyphenated system comprising high performance liquid chromatography coupled to diode-array; electrochemical; and tandem mass spectrometric detection (HPLC-DAD-ECD-MS/MS) was used to study the compositions of the extracts in more detail. The results for the samples processed at 80 °C showed that the extraction reached a steady-state already after 5 min, and that the extracted compounds were stable throughout the entire extraction process. Processing at 180 °C, on the other hand, gave rise to partly degraded extracts with a multitude of peaks in both the diode array and electrochemical detectors, and a higher antioxidant capacity compared to for the extracts obtained at 80 °C. It is concluded that HPLC-DAD-ECD is a more appropriate technique for the determination of antioxidants than the DPPH assay. The mass spectrometric results indicate that one of the extracted antioxidants, catechin, was isomerised to its diastereoisomers; (+)-catechin, (−)-catechin, (+)-epicatechin, and (−)-epicatechin.     

Birch bark research and development

This review will detail progress made in the previous decade on the chemistry and bioactivity of birch bark extractive products. Current and future applications of birch bark natural products in pharmaceuticals, cosmetics, and dietary supplements for the prevention and treatment of cancer, HIV,and other human pathogens are reviewed. Current developments in the technology of birch bark processing are discussed. New approaches for the synthesis of potentially valuable birch bark triterpenoid derivatives are also reviewed.     

Olive oil or lard?: Distinguishing plant oils from animal fats in the archeological record of the eastern Mediterranean using gas chromatography/combustion/isotope ratio mass spectrometry

Distinguishing animal fats from plant oils in archaeological residues is not straightforward. Characteristic plant sterols, such as β-sitosterol, are often missing in archaeological samples and specific biomarkers do not exist for most plant fats. Identification is usually based on a range of characteristics such as fatty acid ratios, all of which indicate that a plant oil may be present, none of which uniquely distinguish plant oils from other fats. Degradation and dissolution during burial alter fatty acid ratios and remove short-chain fatty acids, resulting in degraded plant oils with similar fatty acid profiles to other degraded fats. Compound-specific stable isotope analysis of δ(13)C(18:0) and δ(13)C(16:0), carried out by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), has provided a means of distinguishing fish oils, dairy fats, ruminant and non-ruminant adipose fats, but plant oils are rarely included in these analyses. For modern plant oils where C(18:1) is abundant, δ(13)C(18:1) and δ(13)C(16:0) are usually measured. These results cannot be compared with archaeological data or data from other modern reference fats where δ(13)C(18:0) and δ(13)C(16:0) are measured, as C(18:0) and C(18:1) are formed by different processes resulting in different isotopic values. Eight samples of six modern plant oils were saponified, releasing sufficient C(18:0) to measure the isotopic values, which were plotted against δ(13)C(16:0). The isotopic values for these oils, with one exception, formed a tight cluster between ruminant and non-ruminant animal fats. This result complicates the interpretation of mixed fatty residues in geographical areas where both animal fats and plant oils were in use.     

Chemical profiles of birch and alder bark by ambient mass spectrometry

Desorption atmospheric pressure photoionization (DAPPI) is an ambient mass spectrometry (MS) technique that allows the analysis of both polar and nonpolar compounds directly from the surfaces of various sample types. Here, DAPPI was used to study the chemical profiles in different parts of birch and alder tree barks. Four distinct fractions of Betula pendula (silver birch) bark were collected from three different developmental stages of the stem, after which the chemical profiles of the different tissue types were measured. Of special interest were triterpenoids, a class of important defensive substances, which are found in the bark of the silver birch. Additionally, the chemical profiles of lenticels and the surrounding surfaces in the phellem of B. pendula (silver birch), Alnus glutinosa (black alder), and Alnus incana (gray alder) were screened with DAPPI. Another ambient MS technique, laser ablation atmospheric pressure photoionization (LAAPPI), was further used for the mass spectrometry imaging of lenticels on the B. pendula phellem. All the studied birch bark fractions showed individual chemical profiles in DAPPI. The mass spectra from the young apical stem and the transition zone resembled each other more than the mature stem. Instead, the phellem was found to contain a high amount of triterpenoids in all the developmental stages of the stem. The most intense peaks in the DAPPI mass spectra of the birch bark fractions were those of betulin and lupeol. Betulinic and betulonic acid peaks were intense as well, and these compounds were detected especially in the lenticels of the tree samples.

Graphical abstract

     

Physico-chemical comparison of betulinic acid, betulin and birch bark extract and in vitro investigation of their cytotoxic effects towards skin epidermoid carcinoma (A431), breast carcinoma (MCF7) and cervix adenocarcinoma (HeLa) cell lines

Betulin and betulinic acid are pentacyclic triterpenes present in the bark of the birch tree and other vegetal sources. Quantitatively, in birch bark betulin is more significant than betulinic acid; therefore, birch can be a large and feasible source of raw material for betulin extraction. Betulin can be used as extracted or, after chemical modification, as a starting compound for its acid, betulinic acid, with both substances possessing various interesting pharmacological properties. The purpose of this study is to analyse the betulin and betulinic acid content of a birch tree bark extract, as well as its cytotoxic activity. The extraction was done using a Soxhlet extractor and chloroform/dichlormethane/methanol (1?:?1?:?1) as solvent. The betulin and betulinic acid content of the extract was estimated using standards of pure betulin and betulinic acid, by thermal analysis as opposed to pure substance (thermogravimetric and differential thermal analysis). The extract and the main compounds were also analysed by NMR. The results indicated a high amount of betulin in the final extract (up to 50%), and an important quantity of betulinic acid: over 3%. The cytotoxic activity indicated a high proliferation inhibition for the birch tree extract but was still comparable with betulinic acid and betulin.     

The influence of tissue phosphate on plant flammability: A kinetic study

The concentration of phosphate (P) in leaves is an important indicator of the ecological strategy of a plant species. P is also a fire retardant, which if effective at the low concentrations typical of plant tissue may influence wildfire behaviour, but the size of such effects is unknown.We studied two mechanisms of P fire retardance, gas-phase and solid-phase, using samples of pure cellulose with controlled quantities of added P. The gas-phase mechanism was not detectable at P concentrations found in plants. However, significant solid-phase effects led to increased charring and reduced supply of flammable tars to the flame. Activation energies were not significantly altered, but pre-exponential constants for charring and tar production were significantly increased and decreased, respectively. These data provide a basis for mechanistically modelling the influence of ecological variation in P on the behaviour of wildfires around individual plants and across vegetation boundaries.