Pinosylvin Monomethyl Ether Research Articles

Isolation of stilbenoids from fresh knotwood of Scots pine using a high yield method

Abstract Scots pine knotwood contains valuable amount of stilbenoids such as pinosylvin (Ps) and pinosylvin monomethyl ether (PsMME). The stilbenoids have numerous biological activities. Traditional methods of extracting stilbenoids often yield low quantities and involve complex processes. In the present study, a simple and high-yield method was proposed for the extraction of stilbenoids from fresh Scots pine knotwood using ethanol taking a total of three days. In addition, the impact of drying process, extraction time, extraction solvents (acetone and ethanol) and storage time were evaluated on the yield of the stilbenoids. The increased yield and improved extraction efficiency make this method a promising advancement for promoting the utilization of stilbenoids for their valuable bioactive properties.

MYB4 is the best candidate transcription factor involved in pinosylvin stilbene biosynthesis in Pinus strobus L. cells by fungal elicitor treatment

Key messageDihydropinosylvin monomethyl ether (DPME) and pinosylvin monomethyl ether (PME) are pinosylvin derivatives that show high nematicidal activity against pine wood nematodes (PWNs). Here, we found that fungal elicitor treatment boosted the production of DPME and PME in cultured Pinus strobus L. cells and investigated the transcription factors (TFs) regulating the genes in the pinosylvin stilbenoid biosynthesis pathway.ContextThe discovery of TFs involved in the synthesis of DPME and PME provides an important clue to understanding the pinosylvin stilbenoid synthesis in pine plants.AimsWe investigated the best fungal elicitor for the production of DPME and PME and the transcriptional activities of genes involved in PME and DPME biosynthesis in P. strobus L. cells after fungal elicitor treatment.MethodsThe content of DPME and PME in P. strobus cells was examined after treatment with fungal elicitors prepared from seven different species of fungi. Moreover, the role of fungal elicitors in the transcriptional activity of genes involved in DPME and PME biosynthesis was investigated by transcriptome analysis using RNA sequencing.ResultsPenicillium chrysogenum Thorn was the most efficient fungal elicitor for the production of DPME and PME in P. strobus cells among the other fungal species. The accumulation of DPME and PME in P. strobus cells after P. chrysogenum elicitor treatment increased 12.7-fold and 23.7-fold, respectively, compared to the control. Transcriptome analysis revealed that fungal elicitor treatment resulted in enhanced transcription of the PAL, 4CL, STS, PMT, and ACC genes, which are involved in PME and DPME biosynthesis. Some transcription factors belonging to the bHLH, MYB, WRKY, and ERF families showed a high transcription rate after fungal elicitor treatment.ConclusionsWe found that the fungal elicitor is a strong inducer of the accumulation of pinosylvin derivatives in the cells of P. strobus. We selected one unigene (c133966_g3_i1 in the MYB family) as the best candidate TF that regulates the DPME and PME biosynthesis in P. strobus by transcriptome analysis.

Investigating the Effectiveness of Different Porous Nanoparticles as Drug Carriers for Retaining the Photostability of Pinosylvin Derivative.

Pinosylvin monomethyl ether (PsMME) is a natural compound known for its valuable bioactive properties, including antioxidant and anti-inflammatory effects. However, PsMME's susceptibility to photodegradation upon exposure to ultraviolet (UV) radiation poses a significant limitation to its applications in the pharmaceutical field. This study, for the first time, introduces a strategy to enhance the photostability of PsMME by employing various nanoformulations. We utilized mesoporous silica nanoparticles (MSNs) coated with polydopamine via a poly(ethylene imine) layer (PDA-PEI-MSNs), thermally carbonized porous silicon nanoparticles (TCPSi), and pure mesoporous polydopamine nanoparticles (MPDA). All these nanocarriers exhibit unique characteristics, including the potential for shielding the drug from UV light, which makes them promising for enhancing the photostability of loaded drugs. Here, these three nanoparticles were synthesized and their morphological and physicochemical properties, including size and ζ-potential, were characterized. They were subsequently loaded with PsMME, and the release profiles and kinetics of all three nanoformulations were determined. To assess their photoprotection ability, we employed gas chromatography with a flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) to assess the recovery percentage of loaded PsMME before and after UV exposure for each nanoformulation. Our findings reveal that MPDA exhibits the highest protection ability, with a remarkable 90% protection against UV light on average. This positions MPDA as an ideal carrier for PsMME, and by extension, potentially for other photolabile drugs as well. As a final confirmation of its suitability as a drug nanocarrier, we conducted cytotoxicity evaluations of PsMME-loaded MPDA, demonstrating dose-dependent drug toxicity for this formulation.

A Pinus strobus transcription factor PsbHLH1 activates the production of pinosylvin stilbenoids in transgenic Pinus koraiensis calli and tobacco leaves.

Transcription factors (TFs) play an important role in regulating the biosynthesis of secondary metabolites. In Pinus strobus, the level of methylated derivatives of pinosylvin is significantly increased upon pine wood nematode (PWN) infection, and these compounds are highly toxic to PWNs. In a previous study, we found that the expression of a basic helix-loop-helix TF gene, PsbHLH1, strongly increased in P. strobus plants after infection with PWNs. In this study, we elucidated the regulatory role of the PsbHLH1 gene in the production of methylated derivatives of pinosylvin such as pinosylvin monomethyl ether (PME) and dihydropinoylvin monomethyl ether (DPME). When PsbHLH1 was overexpressed in Pinus koraiensis calli, the production of PME and DPME was significantly increased. Overexpression of the stilbene synthase (PsSTS) and pinosylvin methyl transferase (PsPMT) genes, known as key enzymes for the biosynthesis of methylated pinosylvins, did not change PME or DPME production. Moreover, PME and DPME were not produced in tobacco leaves when the PsSTS and PsPMT genes were transiently coexpressed. However, the transient expression of three genes, PsSTS, PsPMT, and PsbHLH1, resulted in the production of PME and DPME in tobacco leaves. These results prove that PsbHLH1 is an important TF for the pinosylvin stilbene biosynthesis in pine plants and plays a regulatory role in the engineered production of PME and DPME in tobacco plants.

Polyphenol Content, Antioxidant, and Antibiotic Activities of Pinus Caribaea Morelet Forestry Coproducts

Context and objective In the context of valorization of wood waste from the timber industry, this work aims to study the chemical composition and to evaluate the biological capacities of coproducts from Pinus caribaea Morelet. Methods With this mind, sawdust, bark, and knot-wood from P caribaea were analyzed by high-performance liquid chromatography-mass spectrometry. The antioxidant activities of extracts and reference compounds were assessed in vitro using 2 methods: 2.2-diphenyl-1-picrylhydrazyl and 2.2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid). For antimicrobial evaluation, coproduct extracts were assayed against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Candida albicans. Results Despite the variable composition of sawdust, bark, and knot-wood, the composition of P caribaea is typical of the Pinus genus, with the presence of flavonoids, stilbenes, and lignans. Seven compounds were elucidated: 2 lignans (nortrachelogenin and matairesinol), 3 flavonoids (pinocembrin, pinobanksin, pinobanksin 3-acetate), 2 stilbenes (pinosylvin, pinosylvin monomethyl ether), and 1 phenolic acid (caffeic acid). Conclusion The ethanolic extracts of bark and knots showed significant antioxidant activity. The knots showed significant antibacterial properties against S epidermidis.

Phytochemical Analysis of Pinus cembra Heartwood-UHPLC-DAD-ESI-MSn with Focus on Flavonoids, Stilbenes, Bibenzyls and Improved HPLC Separation.

The heartwood of the Swiss Stone Pine, Pinus cembra L., has been scarcely investigated for secondary metabolites for a long period of time. Considering age and relative simplicity of heartwood investigations dating back to the 1940s to 1960s, we conducted the first investigation of P. cembra heartwood by HPLC, using UHPLC-DAD-ESI-MSn and HPLC-DAD techniques in combination with isolation and NMR spectroscopy, with focus on stilbenes, bibenzyls and flavonoids. Analytical problems in the HPLC analysis of Pinus stilbenes and flavonoids on reversed stationary phases were also challenged, by comparing HPLC on pentafluorophenyl (PFP) and C18 stationary phases. Seven flavonoids (1, 2, 3, 7, 8, 11, 12), four stilbenes (4, 6, 10, 13), two bibenzyls (5, 9), three fatty acids (14, 16, 17) and one diterpenic acid (15) were detected in an ethanolic extract of Pinus cembra heartwood. HPLC comparison of reversed stationary phases in HPLC showed that the antifungal, antibacterial and chemosensitizing dihydropinosylvin monomethyl ether (9) and pinosylvin monomethyl ether (10) can be separated on PFP, but not on C18 material, when eluting with a screening gradient of 20-100% acetonitrile. Flavonoid separation showed additional benefits of combining analyses on different stationary phases, as flavonoids 7 and 8 could only be separated on one of two C18 stationary phases. Earlier phytochemical results for heartwood investigations were shown to be mostly correct, yet expandable. Substances 5 to 12 were found in alignment with these references, proving remarkable phytochemical analyses at the time. Evidence for the described presence of pinobanksin could not be found. Substances 1 to 4 and 13 have to our knowledge not yet been described for P. cembra.

Stilbenoid compounds inhibit NF-κB-mediated inflammatory responses in the Drosophila intestine.

Stilbenoid compounds have been described to have anti-inflammatory properties in animal models in vivo, and have been shown to inhibit Ca2+-influx through the transient receptor potential ankyrin 1 (TrpA1). To study how stilbenoid compounds affect inflammatory signaling in vivo, we have utilized the fruit fly, Drosophila melanogaster, as a model system. To induce intestinal inflammation in the fly, we have fed flies with the intestinal irritant dextran sodium sulphate (DSS). We found that DSS induces severe changes in the bacteriome of the Drosophila intestine, and that this dysbiosis causes activation of the NF-κB transcription factor Relish. We have taken advantage of the DSS-model to study the anti-inflammatory properties of the stilbenoid compounds pinosylvin (PS) and pinosylvin monomethyl ether (PSMME). With the help of in vivo approaches, we have identified PS and PSMME to be transient receptor ankyrin 1 (TrpA1)-dependent antagonists of NF-κB-mediated intestinal immune responses in Drosophila. We have also computationally predicted the putative antagonist binding sites of these compounds at Drosophila TrpA1. Taken together, we show that the stilbenoids PS and PSMME have anti-inflammatory properties in vivo in the intestine and can be used to alleviate chemically induced intestinal inflammation in Drosophila.

Investigation of pitch deposits throughout the fiber line of softwood pulp mill

The formation of detrimental pitch deposits can cause various problems at pulp and paper mills, as they can contribute to unscheduled shutdowns and even paralyze the operation of the entire mills. To understand, avoid and control of pitch deposits formation in pulp mills, we investigated the overall chemical composition of 16 pitch deposit samples collected from the fiber line of a softwood kraft pulp mill. Sodium salts were tentatively speculated as the dominant fractions of the pitch deposits in addition to the minor fractions of the extractives and carbohydrates. Low molar mass extractives from dichloromethane, acetone and water extracts were analysed by gas chromatography–mass spectrometry. The extractable fraction is chemically heterogeneous, representing the degradation products of carbohydrates, lignin, and woody extractives. Furthermore, new chlorinated stilbenes (monochloropinosylvin, dichloropinosylvin, trichloropinosylvin dimethyl ether, and dichlorinated pinosylvin monomethyl ether) and other chlorinated components (chloro-manoyl/epimanoyl oxide, 12- and 14-chlorodehydroabietic acids) were for the first time identified as part of pitch deposits in the fiber line. Moreover, significant amount of 3-hydroxy fatty acids was found from the drying stage, also representing novel pulp mill deposit compounds. Pinostilbene and pterostilbene are rarely identified from the industrial woody chips of Scotch pine. Overall, the investigation of extractive profile from pitch deposits may provide us with a new perspective to understand the formation mechanism of pitch deposits and study the binding behavior between the extractive and surfaces of the process equipment. The new findings may provide guidance for the pulp mills to design optimal strategies to avoid and control pitch deposits.

Anticancer and chemosensitizing activities of stilbenoids from three orchid species.

Recently, we have isolated and identified several bioactive flavonoids and stilbenoids with potential anticancer activity from Thai orchids. In this study, we further investigated the cytotoxic and chemosensitizing activities of these phytochemicals (namely, pinocembrin, cardamonin, isalpinin, galangin, pinosylvin monomethyl ether, 2,3'-dihydroxy-5'-methoxystilbene, (E)-2,5'-dihydroxy-2'-(4-hydroxybenzyl)-3'-methoxystilbene, 2,3-dihydroxy-3',5'-dimethoxystilbene, 2,3'-dihydroxy-5,5'-dimethoxystilbene, 3,4'-dihydroxy-5-methoxystilbene and batatasin III) against breast cancer MCF7 cells and its two multidrug resistant (MDR) sublines (MCF7/DOX and MCF7/MX). Cytotoxicity was determined with MTT assay for the estimation of the half maximal cytotoxic concentrations (IC50). Effects of the test compounds on activities of efflux transporters (BCRP, P-gp, MRP1, and MRP2) were evaluated with substrate accumulation assays using fluorometry and flow cytometry analysis. Out of these 11 test compounds, the stilbene pinosylvin monomethyl ether displayed its cytotoxicity specifically toward MCF7 cells (IC50 = 6.2 ± 1.2μM, 72-h incubation) with 4.96 folds higher than normal fibroblast. Its potency decreased in MCF7/DOX and MCF7/MX cells by 3.94 and 7.38 folds, respectively. Our transporter assay indicated that this stilbene significantly reduced the activities of P-gp, MRP1, and MRP2, but not BCRP. After 48-h co-incubation, this stilbene (at 2μM) synergistically increased doxorubicin- and mitoxantrone-mediated cytotoxicity in MCF7, MCF7/DOX, and MCF7/MX cells potentially by increasing the intracellular level of cytotoxic drug. Pinosylvin monomethyl ether could sensitize breast cancer cells to chemotherapy and overcome MDR, in part, via the inhibition of drug efflux transporters.

Inhibitory Mechanism of Pinosylvin Monomethyl Ether against Aspergillus flavus.

Control of Aspergillus flavus is beneficial for the agricultural economy and food safety. Stilbenes exhibit antifungal properties through an unknown mechanism. Here, six stilbenes isolated from Cajanus cajan were screened for anti-A. flavus activity. Among them, pinosylvin monomethyl ether (PME) showed the strongest anti-A. flavus activity and has a broad antifungal spectrum with negligible hemolysis within the concentration range measured. PME inhibited the spore germination of A. flavus and the accumulation of aflatoxin B1. Mechanistic studies showed that PME could bind the cell membrane phospholipids, resulting in increased permeability and decreased fluidity. Further metabolic analysis showed that PME caused the lysis of cell membranes and subsequent collapse of spores, which resulted in a cell wall autolysis-like phenotype. Structure-activity relationship analysis revealed the importance of maintaining amphiphilicity harmony by substituent groups for the antifungal activity of stilbenes. Together, natural stilbenes are promising antifungal lead compounds worthy of further exploration and research for potential application in the food, pharmaceutical, and agricultural industries.

Production of Nematicidal Pinosylvin Stilbenes in Cell Suspension Cultures of Pinus koraiensis by Fungal Elicitation.

Pinosylvin stilbenes are natural phenolic compounds found in the Pinaceae family and act as phytoalexins. Some pinosylvin stilbenes have strong nematicidal activity against pine wood nematodes (PWNs: Bursaphelenchus xylophilus). Here, we established the efficient production of two pinosylvin stilbenes, dihydropinosylvin monomethylether (DPME) and pinosylvin monomethylether (PME), by cell suspension culture of Pinus koraiensis after fungal elicitation. DPME and PME were found in small amounts (less than 40 µg/g DW) in the stem bark and leaves of P. koraiensis plants. Cell suspension cultures were established from the cultures of calli derived from mature zygotic embryos of P. koraiensis in 1/2 Litvay medium containing 2.2 μM 2,4-D and 2.2 μM BA. Two types of fungal elicitors, fungal cell extract (CE) and fungal medium filtrate (MF), were prepared from three species of fungi (Penicillium chrysogenum, P. pinophilum, and P. roquefortii). CE and MF treatments strongly stimulated the production of PME and DPME in cultured cells. The production of PME in suspension cells of P. chrysogenum, P. pinophilum, and P. roquefortii MF treatments after 3 days was 5734 µg/g DW, 4051 µg/g DW, and 6724 µg/g DW, respectively. Pinosylvin synthase (PkSTS) and pinosylvin O-methyltransferase (PkPMT) are key genes in DPME and PME biosynthesis. qPCR analysis revealed that the expression of the PkSTS and PkPMT in cultured cells was highly enhanced after fungal elicitor treatment. The cell extracts after MF treatment resulted in 92.5 ± 7.8% immobilization of the adult PWNs and 63.7 ± 3.5% immobilization of the juvenile PWNs within 24 h. However, control cell extracts without MF treatment showed 11.3 ± 1.4% nematicidal activity against adult PWNs. Our results suggest that pinosylvin stilbenes can be produced from the cell culture of P. koraiensis after fungal elicitor treatment and can be used as nematicidal compounds against PWNs.

Impact of steam-sterilization and oven drying on the thermal stability of phenolic extractives from pine and black locust wood

The main goal of the investigation was to measure and estimate the thermal stability of pinosylvin, pinosylvin monomethyl ether, robinetin, and dihydrorobinetin, i.e., characteristic compounds of wood of Scots pine and black locust, respectively. The pure compounds were analyzed with chromatography before and after they were steam-sterilized and oven-dried. Detailed thermogravimetric analysis was followed to check the thermal decomposition of the stilbenes and flavonoids of wood. The tested phenolic compounds were susceptible to thermal degradation. After the steam-sterilization and oven drying, the amount of investigated phenolic extractives decreased by more than a half. The thermogravimetric analysis showed that stilbenes decompose differently than flavonoids, which can be attributed to the different chemical structures. Twenty minutes of steam sterilization followed by 24 h of oven drying decreased the amount of phenolic compounds in the vials; however, after the applied thermal treatments, the stilbenes and robinetins stayed available in such quantities that they can still provide sufficient bioactivity.

Enhanced production of pinosylvin stilbene with aging of Pinus strobus callus and nematicidal activity of callus extracts against pinewood nematodes

Pinosylvin stilbenes are phenolic compounds mainly occurring in the Pinaceae family. We previously reported that the accumulation of two pinosylvin stilbene compounds, dihydropinosylvin methyl ether (DPME) and pinosylvin monomethyl ether (PME), in Pinus strobus trees was highly enhanced by infection with pine wood nematodes (PWNs: Bursaphelenchus xylophilus), and these two compounds showed strong nematicidal activity against PWNs. In this work, we established a system of pinosylvin stilbene (DPME and PME) production via the in vitro culture of P. strobus calli, and we examined the nematicidal activity of callus extracts. Calli were induced from the culture of mature zygotic embryos of P. strobus. Optimized growth of calli was obtained in 1/2 Litvay medium with 1.0 mg/L 2,4-D and 0.5 mg/L BA. DPME and PME accumulation did not occur in nonaged (one-month-old) calli but increased greatly with prolonged callus culture. The concentrations of DPME and PME in three-month-old dark-brown calli were 6.4 mg/g DW and 0.28 mg/g DW, respectively. The effect of methyl jasmonate treatment on the accumulation of DPME and PME was evaluated in cell suspension culture of P. strobus. However, the treatment appeared to show slight increase of DPME accumulation compared to callus browning. A test solution prepared from crude ethanol extracts from aged calli (three months old) containing 120 µg/ml DPME and 5.16 µg/ml PME treated with PWNs resulted in 100% immobilization of the adult PWNs and 66.7% immobilization of the juvenile PWNs within 24 h. However, nonaged callus extracts did not show any nematicidal activity against juvenile PWNs and showed less than 20% nematicidal activity against adult PWNs. These results indicate that pinosylvin stilbenes can be effectively produced by prolonged culture of P. strobus calli, can be isolated using simple ethanolic extraction, and are applicable as beneficial eco-friendly compounds with nematicidal activity against PWNs.

Enhanced accumulation of pinosylvin stilbenes and related gene expression in Pinus strobus after infection of pine wood nematode.

Pine wood nematodes (PWNs; Bursaphelenchus xylophilus) infect pine trees and cause serious pine wilt disease. Eastern white pine (Pinus strobus) has resistance to PWN. However, the detailed defense mechanisms of P. strobus against PWN are not well known. When P. strobus plants were infected with PWNs, the accumulation of stilbenoids, dihydropinosylvin monomethyl ether (DPME) and pinosylvin monomethyl ether (PME) was increased remarkably. Both DPME and PME had high nematicidal activity. The nematicidal activity of the two compounds was resulted in a developmental stage-dependent manner. Pinosylvin monomethyl ether was more toxic to adult PWNs than juveniles, whereas DPME was found more toxic to juvenile PWNs than the adults. The genes involved in PME and DPME biosynthesis such as phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), pinosylvin synthase (STS) and pinosylvin O-methyltransferase (PMT) were isolated using de novo sequencing of the transcriptome in P. strobus. In addition, transcription factors (TFs; bHLH, MYB and WRKY) related to stilbene biosynthesis were isolated. qPCR analyses of the selected genes (PAL, 4CL, STS and PMT) including TFs (bHLH, MYB and WRKY) revealed that the expression level of the selected genes highly enhanced after PWN infection. Our results suggest that pinosylvin-type stilbenoid biosynthesis is highly responsive to PWN infection and plays an important role in PWN resistance of P. strobus trees.

Extractives of Stemwood and Sawmill Residues of Scots Pine (Pinus sylvestris L.) for Biorefining in Four Climatic Regions in Finland—Phenolic and Resin Acid Compounds

This study aimed to identify and quantify phenolic and resin acid extractive compounds in Scots pine stemwood and sawmill residues in four climatic regions of Finland to evaluate their most optimal sources for bio-based chemical biorefining and bioenergy products. The sample consisted of 140 trees from 28 stands, and sawdust lots from 11 log stands. NMR for the overall extractive analysis and HPLC for the quantitative estimation of phenolic and resin acid compounds were employed. Correlation analysis, multivariate factor analysis, principle component analysis and multiple linear regression modelling were applied for statistical analysis. HPLC identified 12 extractive compounds and NMR five more resin acids. Pinosylvin (PS), pinosylvin monomethyl ether (PSMME), and partly neolignans/lignans occurred in the largest concentrations. Wood type caused the most variation, heartwood having larger concentrations than sapwood (sawdust between them). Regional differences in the concentrations were smaller, but factor analysis distinguished the northern and the southern regions into their own groups. The results indicated higher concentrations of PS, PSMME, and vanillic acid in southern regions and those of, e.g., PSMME glycoside, lignan 2, and neolignan 1 in northern regions. The rather low concentrations of extractives in stemwood and sawdust imply value-added products, efficient sorting and/or large raw material volumes.

In vitro inhibition of extractives from knotwood of Scots pine (Pinus sylvestris) and black pine (Pinus nigra) on growth of Schizophyllum commune, Trametes versicolor, Gloeophyllum trabeum and Fibroporia vaillantii

The main goal of the study was to examine and compare the inhibition effect of knotwood extractives of Scots (Pinus sylvestris) and black pine (Pinus nigra) against the two white-rot fungi and two brown-rot fungi. Knotwood was extracted in a Soxhlet apparatus. Extracts were chemically analyzed, and the inhibitory effect of purified pinosylvins and crude hydrophilic extracts was tested against growth of the white-rot fungi Schizophyllum commune and Trametes versicolor and the brown-rot fungi Fibroporia vaillantii and Gloeophyllum trabeum with the in vitro antifungal assay. Knotwood of Scots pine and black pine contained comparable amounts of total extractives. Pinosylvin, pinosylvin monomethyl ether and nortrachelogenin were characteristic compounds in knotwood extracts of both pines. Scots pine knotwood contained larger amounts of pinosylvin than black pine. The ratio between pinosylvin monomethyl ether and pinosylvin was higher in black pine knotwood. Purified pinosylvins and crude hydrophilic extracts inhibited the growth of all the white-rot and brown-rot fungi tested. Pure pinosylvins better inhibited fungal growth than crude knotwood extracts, whereas the efficiency was not unambiguous and relates to fungi species. Crude hydrophilic extracts of Scots pine more efficiently inhibited fungal growth than extracts of black pine. With the present investigation, hydrophilic extracts of pine knotwood were demonstrated as formulations of natural compounds with good antifungal properties. Broken tops of Scots pine, which can lie on forest grounds for a month or even more, still contain high amounts of phenolic extractives and are therefore a potential raw material for recovery of bioactive compounds.

Utilization of wood industrial wastes for production of chemicals and pharmaceuticals

Objectives: This study aims to isolate and identify valuable chemicals and pharmaceuticals from the waste of wood (Pinus sylvestris L.) industry and to determine their biological value. Methods: Chromatographic technique was used to isolate compounds from the waste. The chemical identity of the isolated compounds were evidenced by different spectroscopic analyses as well as comparative studies of reported data. The microbial transformed product of pinosylvin mono methyl ether was quantitatively determined using fluorimetric method. All the isolated compounds were tested for their antitoxoplasmosis activity (in vitro) against Toxoplasma gondii. Findings/ Novelty: Nine compounds were isolated and identified including three diterpenes : (1b -hydroxy dehydroabietic acid, 15-hydroxy dehydroabietic acid and Methyl-15-hydroxy dehydroabiet-18-ate) ; two aromatic aldehydes (coniferaldhyde and 3-hydroxy, 5-methoxy benzaldhyde) ; two stilbene derivatives; (Pinosylvin and Pinosylvin mono methyl ether) ; a flavan: (Pinocembrin) and a phytosterol (b -sitosterol).For the first time, the fluorimetric method was used for quantitative determination of the novel metabolite produced, E-Pinosylvin mono methyl ether 5-O-b -D-glucopyranoside, from the microbial biotransformation technique. All isolated compounds showed variable antitoxopasmosis activity, carried out for the first time, against T. gondii according to their % mortality and recorded EC50. Keywords: Pinus sylvestris L.; saw dust; antitoxoplasmosis; fluorimetry; microbial transformation

Separation and isolation of major polyphenols from maritime pine (Pinus pinaster) knots by two-step centrifugal partition chromatography monitored by LC-MS and NMR spectroscopy.

Pine knots are a rich source of lignans, flavonoids, and stilbenes. These bioactive compounds are widely known for their roles to combat human disorders but also to protect plants against pathogens. In order to gain knowledge inside their potential activities, a suitable isolation and purification of these high-added value compounds is required. To this end, centrifugal partition chromatography, as a rapid and useful methodology of separation, was employed and developed. The coefficient partition values (KD ) of six major compounds in nine biphasic solvent systems were determined to evaluate the most appropriate system. Two-step centrifugal partition chromatography was required to separate lignans using ARIZONA system K (n-heptane/ethyl acetate/methanol/water 1:2:1:2, v:v) and to isolate stilbenes and flavonoids using ARIZONA system P (n-heptane/ethyl acetate/methanol/water 6:5:6:5, v:v). Eight one-compound enriched-fractions were obtained as follows: nortrachelogenin (70.1%), secoisolariciresinol (53.7%), isolariciresinol (61.1%), taxifolin (48.4%), pinocembrin (91.3%), pinobanksin (91.1%), pinosylvin (91.4%), and pinosylvin monomethyl ether (91.1%). Additionally, the centrifugal partition chromatography allowed to unravel the composition of pine knot owing to the several fractions generated. Twenty-two compounds were characterized by liquid chromatography-mass spectrometry and NMR spectroscopy, some of which are described for the first time in literature.

Chemical profile of organic residues from ancient amphoras found in Pyrgi and Castrum Novum, Tyrrhenian Sea (Italy)

The organic residues in the form of black layer spots inside amphoras found on the sites of the former ancient ports of Pyrgi and Castrum Novum on the coast of the Tyrrhenian Sea were investigated using GC–MS and HPLC with fluorimetric detection. According to our hypothesis, the residues could be from waterproofing materials from pine trees, which ensured that amphoras could be used for transportation and the storage of liquids. Samples of the residues were removed, extracted with hexane and separated into three parts. The first part served directly for GC–MS analysis of hydrocarbons. The second part after the evaporation of hexane and silylation of the residue with MSTFA was used for the analysis of fatty and resin acids. Finally, the third part after evaporation of hexane and dilution of the residue with methanol was used for HPLC analysis of pinosylvin mono methyl ether and confirmation of retene presence using a fluorimetric detector. 18-Norabieta-8,11,13-triene, 19-norabieta-8,11,13-triene and 1,2,3,4-tetrahydroretene were found in the hexane extract, which are intermediates from the tar/pitch production process. The most abundant peak in the chromatogram of the hexane extract was dehydroabietic acid and retene. The latter is considered to be a marker for pine pitch. The most abundant compound in the derivatized hexane extract was also dehydroabietic acid, which occurs only as a minor component in fresh resins. This molecule is formed during the oxidative dehydrogenation of abietic acid, which predominates in original resins. Pinosylvin mono methyl ether was found in the methanolic extract using HPLC with fluorimetric detection and, to the best of our knowledge, it is the first finding of this substance in archaeological samples. Together with the abovementioned compounds, it supports the hypothesis of the resin's origin from trees of the Pinaceae family.

Isolation of pure pinosylvins from industrial knotwood residue with non-chlorinated solvents

Abstract The aim of the study was to find appropriate non-chlorinated solvent systems for the extraction and separation of pure pinosylvin (PS) and pinosylvin monomethylether (PSMME) from Scots pine/Norway spruce wood residues. The extraction was performed in a pressurized solvent extractor and the purification and isolation of pure substances was done by flash chromatography. More precisely, pure PS was isolated via sequential flash chromatography in two repetitions in the gradient mode of the mobile phase, with cylcohexane (CX) and ethylacetate (EtOAc), beginning with 100% CX and ending up with 100% EtOAc. The extractives were qualitatively and quantitatively analyzed in terms of PS and PSMME contents by gas chromatography-flame ionization detector (GC-FID), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC) and proton nuclear magnetic resonance (1H-NMR) spectroscopy.