Isopimaric Acids Research Articles

Multivariate GC-MS data analysis of the apolar fraction of brown propolis produced in southern Brazil

In this study, a total of thirty-nine propolis samples from southern Brazil were extracted with diethylether and investigated by Gas Chromatography – Mass Spectrometry (GC – MS). The objective of this study was first to contribute to the elucidation of the chemical composition of brown propolis produced in southern Brazil through investigation of the chemical profile of its apolar fraction and second to investigate if the GC – MS of apolar propolis extracts combine with multivariate analysis may help in origin discrimination of brown propolis. Fifty-three compounds were tentatively identified and were classified into the following classes: alcohols (9), hydrocarbons (9), aliphatic acids (4), aromatic acids (10), esters (1), phenylpropanoids (1), terpenes (15), aldehydes (1) and anthraquinones (1). Resinic terpenes such as pimaric, isopimaric, dehydroabietic and abietic acids were the most abundant and recurrent compounds in the investigated samples. GC – MS based iPLS-DA (Partial Least Squares - Discriminant Analysis) model correctly discriminated propolis produced in different regions of Paraná state. It was concluded that the whole GC – MS profile of apolar extracts of brown propolis combined with multivariate analysis is an auxiliary tool for geographic origin authentication of propolis produced within the state of Paraná.

Trace analysis of resin acids in surface waters by direct injection liquid chromatography time of flight mass spectrometry and triple quadrupole mass spectrometry

A rapid and sensitive liquid chromatography (LC) quadrupole time of flight (QTOF) method has been developed for the determination of resin acid concentrations in aqueous pulp and paper effluent related samples. Calibration R2 of ≥0.995 for twelve resin acids, namely dehydroabietic, 8(14)-abietenic, dihydroisopimaric, levopimaric, neoabietic, pimaric, sandaracopimaric, abietic, isopimaric, palustric, chlorodehydroabietic, and dichlorodehydroabietic acids, was demonstrated in the range 1 µgL−1 to 40 µgL−1. An improved lower limit of quantitation was achieved without use of complex sample extraction and clean-up procedures undertaken by other published methods. Excellent precision and accuracy results were achieved for dehydroabietic, chlorodehydroabietic, dichlorodehydroabietic, isopimaric (integrated inclusive of all C20H30O2 resin acids), dihydroisopimaric and 8(14)-abietenic resin acids, with t-99 percentile detection limits spanning the range 0.05 to 0.07 µgL−1. While measurement for the C20H30O2 resin acids by isopimaric equivalence is considered semi-quantitative and could be an under estimate for the abietic acid component, the developed method demonstrated clear advantage over time consuming, hazardous, and unstable derivatization procedures used for gas chromatography and capillary electrophoresis. The developed LC/QToF method was successfully transferred to an LC triple quadrupole mass spectrometer for routine high throughput trace level analysis. Real world samples, including sea water and estuary water, demonstrated excellent spike recoveries by this procedure, indicating that the method is well suited to the monitoring of industrially derived resin acids in environmental surface waters. While no interferences were observed during routine sample analysis using myristic-1-13C acid and palmitic-1-13C acid internal standards, these were later substituted by myristic-d27 and palmitic-d31 acid in order to improve method robustness for environmental samples where endogenous parent fatty acids could be present.

ECOLOGICAL AND GEOGRAPHICAL FEATURES OF THE ACCUMULATION OF METABOLITES IN THE NEE-DLES OF LARIX CAJANDERI IN THE TERRITORY OF YAKUTIA

The accumulation of primary and secondary metabolites in Larix cajanderi needles in some areas of North (Kobyayskiy, Anabarskiy uluses and Verkhoyanskiy district), Western (Nyurbinskiy district and Vilyuyskiy ulus) and Central (Megino-Kangalasskiy ulus) Yakutia was studied. It was found that the maximum content of dihydroquercetin, rutin, pimaric, isopimaric and dehydroabietic acids in Larix cajanderi needles was recorded in the northern part of Yakutia, which may be associated with the protective functions of flavonoids and diterpenic acids from ultraviolet radiation under conditions of longer daylight hours in the spring and summer. ... A direct relationship was observed between the content of the studied flavonoids and diterpenic acids and the duration of daylight hours in the spring-summer period in the studied regions of Yakutia, which may be a confirmation of their protective functions from UV radiation. It is shown that as a result of processing the metabolomic profiles of Larix cajanderi needles by the method of principal components, three groups were formed, which correspond to the floristic regions of Yakutia. Samples of Larix cajanderi needles collected in Megino-Kangalassky, Vilyuisky ulus and Nyurba region formed a separate group, which is part of the Central Yakutsk floristic region, from the territory of the Verkhoyansk region and Kobyaysky ulus correspond to the Yano-Indigirsky floristic region, and from the Anabar and the ulus are located separately Arctic floristic region.

High-Throughput 1H-Nuclear Magnetic Resonance-Based Screening for the Identification and Quantification of Heartwood Diterpenic Acids in Four Black Pine (Pinus nigra Arn.) Marginal Provenances in Greece.

A high-throughput quantitative Nuclear Magnetic Resonance 1H-NMR method was developed and applied to screen the quantity of the diterpenic resin acids in the heartwood of black pine, due to the renewed scientific interest in their medicinal properties and use in various diseases treatment. The 260 samples were taken from Pinus nigra clones, selected from four provenances of the Peloponnese (Greece), participating in a 35-year-old clonal seed orchard. Total resin acids per dry heartwood weight (dhw) varied greatly, ranging from 30.05 to 424.70 mg/gdhw (average 219.98 mg/gdhw). Abietic was the predominant acid (76.77 mg/gdhw), followed by palustric acid (47.94 mg/gdhw), neoabietic acid (39.34 mg/gdhw), and pimaric acid (22.54 mg/gdhw). Dehydroabietic acid was at moderate levels (11.69 mg/gdhw), while levopimaric, isopimaric, and sandaracopimaric acids were in lower concentrations. The resin acid fraction accounted for 72.33% of the total acetone extractives. Stilbenes were presented in significant quantities (19.70%). The resin acid content was composed mainly of the abietane type resin acids (83.56%). Peloponnesian Pinus nigra heartwood was found to be the richest source of resin acids identified to date and is considered the best natural source for the production of such bioactive extracts. The results indicate a high potential for effective selection and advanced breeding of pharmaceutical and high economic value bioactive substances from Pinus nigra clones.

Distribution of low-molecular lipophilic extractives beneath the surface of air- and kiln-dried Scots pine sapwood boards.

During industrial wood drying, extractives migrate towards the wood surfaces and make the material more susceptible to photo/biodegradation. The present work provides information about the distribution, quantity and nature of lipophilic substances beneath the surface in air- and kiln-dried Scots pine (Pinus sylvestris L.) sapwood boards. Samples were taken from knot-free sapwood surfaces and the composition of lipophilic extractives, phenols and low-molecular fatty/resin acids layers at different nominal depths below the surface was studied gravimetrically, by UV-spectrometry and by gas chromatography-mass spectrometry (GC-MS). The concentration of total extractives was significantly higher in kiln-dried than in air-dried samples and was higher close to the surface than in the layers beneath. The scatter in the values for the lipophilic extractives was high in both drying types, being highest for linoleic acid and slightly lower for palmitic, oleic and stearic acids. The amount of fatty acids was low in kiln-dried boards, probably due to a stronger degradation due to the high temperature employed. The most abundant resin acid was dehydroabietic acid followed by pimaric, isopimaric, and abietic acids in both drying types. It is concluded that during kiln-drying a migration front is created at a depth of 0.25 mm with a thickness of about 0.5 mm.

Organic tracers in aerosols from the residential combustion of pellets and agro-fuels

The consumption of pellets and alternative biofuels, such as some agricultural residues, has experienced a tremendous growth in Mediterranean countries, which has changed the panorama of biomass burning emissions. To apply source apportionment models, specific chemical signatures for smoke aerosols are necessary. In this study, filter samples of fine particles (PM2.5) from the combustion of four different types of pellets and three agro-fuels (shell of pine nuts, olive pit and almond shell) in a pellet stove were collected. The PM2.5 samples were extracted with dichloromethane/methanol and the dried extracts were silylated before injection into the gas chromatograph-mass spectrometer (GC-MS). The analysis included monosaccharide anhydrides, resin acids, methoxyphenols and sterols. The amounts emitted were highly variable and dependent on both biofuel and combustion temperature. On average, the anhydrosugar particle mass fractions for the seven biofuels were 30 to 70 times lower than the values reported for manually fired systems. The highest levoglucosan emissions were observed for olive pit. Levoglucosan to mannosan ratios ranged from values around 2 for pellets and shell of pine nuts to much higher quotients, similar to those reported for hardwood combustion. Irrespective of biofuel, vanillin and vanillic acid were always present in the smoke samples. Coniferyl alcohol was only detected in samples from the combustion of olive pit and pellets with the highest softwood content. Sinapyl alcohol was exclusively emitted by olive pit. Dehydroabietic and isopimaric acids were represented in emissions whether from all type of pellets or from shell of pine nuts, whilst abietic acid was only quantifiable in PM2.5 from the combustion of pellets made of coniferous wood. Sitosterol was solely detected in smoke particles from shell of pine nuts and olive pit.

Determination of Terpenoid Content in Pine by Organic Solvent Extraction and Fast-GC Analysis

Terpenoids, naturally occurring compounds derived from isoprene units present in pine oleoresin, are a valuable source of chemicals used in solvents, fragrances, flavors and have shown potential use as a biofuel. This paper describes a method to extract and analyze the terpenoids present in loblolly pine saplings and pine lighter wood. Various extraction solvents were tested over different times and temperatures. Samples were analyzed by pyrolysis-molecular beam mass spectrometry before and after extractions to monitor the extraction efficiency. The pyrolysis studies indicated that the optimal extraction method used a 1:1 hexane/acetone solvent system at 22°C for 1 h. Extracts from the hexane/acetone experiments were analyzed using a low thermal mass modular accelerated column heater for fast-GC/FID analysis. The most abundant terpenoids from the pine samples were quantified, using standard curves, and included the monoterpenes, α- and β- pinene, camphene and δ-carene. Sesquiterpenes analyzed included caryophyllene, humulene and α-bisabolene. Diterpenoid resin acids were quantified in derivatized extractions, including pimaric, isopimaric, levopimaric, palustric, dehydroabietic, abietic and neoabietic acids.

Resin acids as the potential growth-affecting component of pine oleoresin

The nonvolatile fraction of the oleoresin of <i>Pinus sihestris</i> L. was found to contain substances which inhibit growth of wheat ceoleoptile and oat mesocotyl sections in standard bioassays. The inhibition is mainly confined to the fraction of resin acids. Among the seven authentic resin acids tested, the effects of dehydroabietic and abietic acids were most sifgnificant. Palustric, pimaric and isopimaric acids were not effective in the wheat coleoptile section straight growth test. None of the substances, in the amounts tested, except for extremely high concentration, exerted an inhibitory effect on natural or IAA-induced elongation of pine hypocotyl sections. Neither was an inhibitory effect discovered in the microbiological test with the <i>Aspergillus niger</i> van Tiegh. The results obtained with pine hypocotyl sections, allow the conclusion that resin acids interfering with the results of standard bioassays are probably not effective as inhibitory factors in the regulation of pine tissue growth.

GC–MS profiling of the phytochemical constituents of the oleoresin from Copaifera langsdorffii Desf. and a preliminary in vivo evaluation of its antipsoriatic effect

Copaiba is the oleoresin (OR) obtained from Copaifera (Fabaceae), a neotropical tree which grows in Amazon regions. The balsam, constituted by an essential oil and a resinous fraction is used as folkloristic remedy in the treatment of several inflammatory diseases and for its antioxidant and antibacterial properties. Aim of this work was (a) to carry out a characterization by GC-MS of the volatile and nonvolatile constituents of Copaifera langsdorffii Desf. oleoresin (OR); (b) to investigate the mechanism of its anti-inflammatory activity; (c) to evaluate its antipsoriatic effect after oral intake/topical application. The volatile fraction (yield: 22.51%, w/w) shows: α-bergamotene (48.38%), α-himachalene (11.17%), β-selinene (5.00%) and β-caryophyllene (5.47%). The OR residue (77.49%, w/w), after derivatization, showed as main constituents the following compounds: copalic, abietic, daniellic, lambertinic, labd-7-en-15-oic, pimaric, isopimaric acids and kaur16-en18-oic acid. Preincubation of LPS-stimulated human THP-1 monocytes with increasing concentrations of the OR purified fraction (OR-PF), containing diterpene acids, diterpenes and sesquiterpenes, reduced the release of pro-inflammatory cytokines (IL-1β, IL-6, TNFα) in a dose-range of 0.1-10 μM. In addition, in cell culture system of human THP-1 monocytes, 1 μM OR-PF counteracts LPS-driven NF-κB nuclear translocation. In a preliminary clinical trial three patients affected by chronic psoriasis, treated with oral intake or topical application of the OR, exhibited a significant improvement of the typical signs of this disease, i.e. erythema, skin thickness, and scaliness. In conclusion, the results of this work, beside an extensive analytical characterization of the OR chemical composition, provide strong evidences that its anti-inflammatory activity is related to the inhibition of the NF-κB nuclear translocation, and consequently of proinflammatory cytokines secretion.

Dissolution and spatial distribution of resin acids and retene in sediments contaminated by pulp and paper industry

Wood extractives in sediments originating from the wood industry may interfere with benthic biota in aquatic environments. The research area was the Aanekoski watercourse in Central Finland, which has been affected by the chemical wood industry for over a century. The goal was to determine the dissolution potency of resin acids (RAs) and their derivative, retene, in the sediment, and their current vertical and spatial stratification to assess the load due to potential erosion. Sediments were collected from two upstream reference sites and three lake-like basins, located as far as 33 km downstream from the mills. The dissolution potency was studied by two different agitation times and temperatures from sediment-water (1+4 v/v) elutriates. The vertical distribution of extractives was determined from the uppermost 20 cm of sediment. Using spatial interpolation, the distribution of extractives was estimated from two upper sediment layers (0–2 and 2–5 cm) downstream from the source. According to the interpolation, the total amount of dehydroabietic (DHAA) and isopimaric acids (IPA) were calculated as kg/ha in the whole sediment area. The total concentration of RAs in the surface sediment reached up to 168 µg/g dw, and they were found to desorb to water up to 77 µg/l. The concentrations of retene were low both in the sediment (<51 µg/g dw) and elutriate (<0.53 µg/l). Spatial interpolation showed that the highest calculated amounts of the most abundant RAs were in Kuhnamo basin, in the sediment layer 2–5 cm; the estimated amount of DHAA and IPA were approximately 440 and 85 kg/ha, respectively. Disturbances may change the exposure situation, causing desorption of sediment-associated compounds in levels that may be harmful to aquatic animals. The amount of desorption varies depending on the concentration of contaminants in sediment, the nature of disturbance, and the sediment organic carbon content. Low retene concentrations can be explained by oxic conditions and low abundance of RAs in the sediments.

Elimination of resin acids by advanced oxidation processes and their impact on subsequent biodegradation

Wood pulping and paper production generate a considerable amount of wastewater, containing many pollutants among them resin and fatty acids. Resin acids contribute substantially to effluent toxicity and were identified to be detrimental to microorganisms of activated sludge and in particular to bacteria in anaerobic wastewater treatment system and other forms of aquatic life. The objective of the present study was to check the applicability of the ozone and advanced oxidation processes (AOPs) to eliminate resin acids from aqueous solutions and to determine the ozone dose required. Furthermore, an investigation of the influence of the oxidation methods on subsequent biological destruction of the byproducts was performed. Aqueous solution of the resin acids: abietic, dehydroabietic and isopimaric acids with different initial composition were subjected to ozonation or AOP processes at different ozone doses. After ozonation or advanced oxidation pretreatment the model solutions were biodegraded in aerated vessels containing activated sludge. During ozonation of the resin acids aqueous solutions the resin acids were almost eliminated, however the reduction of chemical oxygen demand (COD) was rather low. The ozone dose required to obtain reduction of resin acids >90% was in the range of 0.1–0.7 mgO 3/mgCOD, depending on the composition and concentration of model solutions. The toxicity of ozonated resin acids solutions decreased with increasing applied ozone dose up to about 0.3–0.5 mgO 3/mgCOD, thereafter increased. Ozonation and other AOP processes did not increase the rate of biodegradation of resin acids model solutions in aerated activated sludge systems compared to not pretreated solutions.

Selected resin acids in effluent and receiving waters derived from a bleached and unbleached kraft pulp and paper mill

Water samples were collected on three dates at 24 sites influenced by effluent from Georgia-Pacific's Palatka Pulp and Paper Mill Operation, a bleached and unbleached kraft mill near Palatka, Florida, USA. The sampling sites were located within the mill retention ponds, Rice Creek, and the St. John's River. Samples were analyzed by gas chromatography-mass spectrometry for abietic, dehydroabietic, and isopimaric acids, all of which are potentially toxic by-products of pulp production. Isopimaric acid concentrations greater than 12 mg/L were measured at the mill's effluent outfall but were less than 20 microg/L at the end of Rice Creek. This result indicates that the waters of Rice Creek provide dilution or conditions conducive for degradation or sorption of these compounds. Large differences in resin acid concentrations were observed between sampling events. In two sampling events, the maximum observed concentrations were less than 2 mg/L for each analyte. In a third sampling event, all of the compounds were detected at concentrations greater than 10 mg/L. Data from the three sample dates showed that resin acid concentrations were below 20 microg/L before the confluence of Rice Creek and the St. John's River in all cases.

Evaluation of liquid chromatography–negative ion electrospray mass spectrometry for the determination of selected resin acids in river water

A liquid chromatography–negative ion electrospray mass spectrometric (LC–ESI-MS) method was evaluated for detection of four prevalent softwood-derived resin acids in natural water. Method detection limits based on a signal-to-noise ratio of 3:1 in river water samples of 0.40, 0.40, 0.30 and 0.25 μg l −1 for abietic, dehydroabietic, isopimaric and pimaric acids, respectively, are comparable or lower than reported GC methods. Unlike the majority of GC methods, however, the three structural resin acid isomers (abietic, isopimaric and pimaric acids) do not separate sufficiently under the various LC conditions evaluated in this work. Therefore, LC–ESI-MS may not be suitable for instances where measurement of individual isomeric resin acids is required. However, the method is suitable for trace analysis of resin acids in natural waters where isomeric speciation is not required.

ChemInform Abstract: Microbial Transformations of Diterpene Acids.

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Microbial transformations of diterpene acids

The ability of Rhodococcus bacteria to transform dehydroabietic and isopimaric acids with a high degree of conversion (up to 95%) was detected.

Degradation of paper mill water components in laboratory tests with pure cultures of bacteria.

The degradation of dissolved and colloidal substances from thermomechanical pulp (TMP) by bacteria isolated from a paper mill was studied in a laboratory slide culture system. Burkholderia cepacia strains hydrolysed triglycerides to free fatty acids, and the liberated unsaturated fatty acids were then degraded to some extent. Saturated fatty acids were not notably degraded. However, the branched anteiso-heptadecanoic fatty acid was degraded almost like the unsaturated fatty acids. About 30% of the steryl esters were degraded during 11 days, increasing the concentrations of free sterols. Approximately 25% of the dehydroabietic, and 45% of the abietic and isopimaric resin acids were degraded during 11 days. The degree of unsaturation seemed to be of greater importance for the degradation of fatty acids than the molar mass. No degradation of dissolved hemicelluloses could be observed with any of the nine bacterial strains studied. Burkholderia cepacia strains and one Bacillus coagulans strain degraded monomeric fructose and glucose in winter TMP water, but in summer TMP water, with much lower sugar concentrations, also other Bacillus strains degraded monomeric sugars.

Retene and resin acid concentrations in sediment profiles of a lake recovering from exposure to pulp mill effluents

The stratigraphy of resin acids (pimaric, isopimaric, sandaracopimaric, dehydroabietic and abietic acids) and base neutral compounds (retene, tetrahydroretene, dehydroabietin and fichtelite) in sediments of a lake recovering after the mill closure from exposure to effluents from sulphite pulp mill was studied. The highest concentrations of these compounds were detected 1.5 km downstream from the effluent pipe in sediment depth of 7–11 cm. This sediment layer is approximately 30 yrs old. The highest concentration of resin acids was 1100 and of retene 3300 μg/g d.w. The retene concentration observed is the highest reported in the literature so far. The results imply that high concentrations of retene may be common in sediments of Finnish lakes polluted by pulp mill effluents.

Apparent contradiction: psychrotolerant bacteria from hydrocarbon-contaminated arctic tundra soils that degrade diterpenoids synthesized by trees.

Resin acids are tricyclic terpenoids occurring naturally in trees. We investigated the occurrence of resin acid-degrading bacteria on the Arctic tundra near the northern coast of Ellesmere Island (82 degrees N, 62 degrees W). According to most-probable-number assays, resin acid degraders were abundant (10(3) to 10(4) propagules/g of soil) in hydrocarbon-contaminated soils, but they were undetectable (<3 propagules/g of soil) in pristine soils from the nearby tundra. Plate counts indicated that the contaminated and the pristine soils had similar populations of heterotrophs (10(6) to 10(7) propagules/g of soil). Eleven resin acid-degrading bacteria belonging to four phylogenetically distinct groups were enriched and isolated from the contaminated soils, and representative isolates of each group were further characterized. Strains DhA-91, IpA-92, and IpA-93 are members of the genus Pseudomonas. Strain DhA-95 is a member of the genus Sphingomonas. All four strains are psychrotolerant, with growth temperature ranges of 4 degrees C to 30 degrees C (DhA-91 and DhA-95) or 4 degrees C to 22 degrees C (IpA-92 and IpA-93) and with optimum temperatures of 15 to 22 degrees C. Strains DhA-91 and DhA-95 grew on the abietanes, dehydroabietic and abietic acids, but not on the pimaranes, isopimaric and pimaric acids. Strains IpA-92 and IpA-93 grew on the pimaranes but not the abietanes. All four strains grew on either aliphatic or aromatic hydrocarbons, which is unusual for described resin acid degraders. Eleven mesophilic resin acid degraders did not use hydrocarbons, with the exception of two Mycobacterium sp. strains that used aliphatic hydrocarbons. We conclude that hydrocarbon contamination in Arctic tundra soil indirectly selected for resin acid degraders, selecting for hydrocarbon degraders that coincidentally use resin acids. Psychrotolerant resin acid degraders are likely important in the global carbon cycle and may have applications in biotreatment of pulp and paper mill effluents.

Molecular characterization of smoke from campfire burning of pine wood (Pinus elliottii)

Although campfires are typically enjoyable events, people are exposed to high concentrations of gaseous and particulate pollutants. The combustion conditions of wood burned in campfires are different from those of indoor wood burning in stoves or fireplaces. Typically, wood logs, twigs, and branches with their leaves or needles are burned in campfires, whereas mostly wood logs are burned in indoor settings. Consequently, the molecular source profiles for organic particulate matter emissions may be different from a campfire. For human exposure assessment, there is a need to fingerprint the organic compound compositions in campfire wood smoke. Here we present the detailed biomarker composition of conifer wood smoke from a campfire. The major biomarkers emitted are dehydroabietic acid, the resin acids (pimaric, iso-pimaric, sandaracopimaric and abietic acids), retene, pimanthrene, methylcyclopentenophenanthrene and β-sitosterol, with levoglucosan and lignin phenolics such as vanillic acid in the polar fraction. PAH are minor components. A marker and PAH profile has been generated which can be utilized for tracing emissions from campfire burning and evaluating personal exposure risk.

Diterpenes from the berries of Juniperus excelsa.

From the hexane extract of berries of Juniperus excelsa, one new and four known diterpenes were isolated besides a known sesquiterpene. The structures of the known diterpenes were identified as isopimaric, isocommunic, (-)ent-trans communic and sandracopimaric acids, along with the sesquiterpene 4a-hydroxycedrol and the new compound which was elucidated as 3 alpha-acetoxylabda-8(17),13(16),14-trien-19-oic acid (juniperexcelsic acid). Cytotoxic activity of the hexane extract was investigated against a panel of cell line and found highly active against LNCaP, KB-V (+VLB) and KB-V (-VLB) cell lines. Furthermore, the hexane and methanol extracts, and the new compound were found to be moderately active against Mycobacterium tuberculosis.