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A simple method was developed to isolate the compound middle lamella (CML) in its natural state using wet-beating, sedimentation, and methanol dialysis. The isolated CML fraction was characterised. The CML was isolated in 2.0% yield from the mature wood of Ginkgo biloba. Scanning electron microscopy (SEM) images of the isolated CML fraction showed the presence of flake-like structures. However, some small amounts of secondary wall substance was observed. The lignin content of the CML fraction was approximately 45%. Thioacidolysis reactions with the isolated CML fraction revealed that the lignin β-O-4 structure was scarce in the isolated fraction and that numerous structures with two or more consecutive condensed-type bonds were present. Results from the solid-state carbon-13 nuclear magnetic resonance (13C NMR) experiments indicated that the CML fraction had low crystallinity, indicating low cellulose content. Liquid-state NMR analysis of the lignin from the CML fraction revealed the absence of p-hydroxyphenyl (H) units.

Lignin is thought to be covalently bound to carbohydrates like hemicellulose during biosynthesis to form a lignin-carbohydrate complex (LCC). However, successive polymerisation with monolignols after the formation of LCC has not yet been clarified. To investigate the reaction of LCC, its enzymatic dehydrogenative polymerisation was conducted using deuterium-labelled coniferyl alcohol and model compounds, i.e., a lignin model (β-O-4 dimer model) compound (LM) and an LCC model (benzyl ether type) compound (LCCM). The obtained polymers (DHPs) were methylated and subjected to thioacidolysis, and the degradation products were quantified by gas chromatography-mass spectrometry (GC-MS). The results showed that the amount of coniferyl alcohol connected to the LCCM via β-O-4 binding was almost the same as that bound to the LM. However, the amount of unreacted LCCM was larger than that of LM, suggesting that the LCCM is less likely to form condensed structures, i.e., 5-5, β-5, and 4-O-5 structures. This could be due to the steric hindrance of the carbohydrate at the benzylic position.

To discuss the macromolecular structure and properties of lignin, the complementary use of solid- and liquid-state nuclear magnetic resonance (NMR) combined with a specific 13C-enrichment technique may provide useful information. The 13C-enriched lignin was prepared by administering [guaiacyl ring-5(G5)-13C]-coniferin to a growing Ginkgo biloba L. shoot. The 13C-enriched cellulolytic enzyme lignin (EL) and its acetate prepared from the ginkgo shoot were examined by solid- and liquid-state 13C NMR spectroscopy. The 13C NMR spectrum derived only from the G5 carbon was obtained as a difference spectrum based on the spectra of the G5-13C enriched and un-enriched (UE) samples. The condensed structures, including the enriched G5-carbon, were evaluated using difference spectra. The chemical shifts of the enriched G5 carbon in both the solid- and liquid-state 13C NMR spectra agreed with each other in principle. The quantitative ratio of the condensed and uncondensed structures at G5 was found to be larger by solid-state cross polarization/magic angle spinning (CP/MAS) NMR than by liquid-state NMR.

Summary The woody stems of coniferous gymnosperms produce specialised compression wood to adjust the stem growth orientation in response to gravitropic stimulation. During this process, tracheids develop a compression‐wood‐specific S2L cell wall layer with lignins highly enriched with p‐hydroxyphenyl (H)‐type units derived from H‐type monolignol, whereas lignins produced in the cell walls of normal wood tracheids are exclusively composed of guaiacyl (G)‐type units from G‐type monolignol with a trace amount of H‐type units. We show that laccases, a class of lignin polymerisation enzymes, play a crucial role in the spatially organised polymerisation of H‐type and G‐type monolignols during compression wood formation in Japanese cypress (Chamaecyparis obtusa).We performed a series of chemical‐probe‐aided imaging analysis on C. obtusa compression wood cell walls, together with gene expression, protein localisation and enzymatic assays of C. obtusa laccases.Our data indicated that CoLac1 and CoLac3 with differential oxidation activities towards H‐type and G‐type monolignols were precisely localised to distinct cell wall layers in which H‐type and G‐type lignin units were preferentially produced during the development of compression wood tracheids.We propose that, not only the spatial localisation of laccases, but also their biochemical characteristics dictate the spatial patterning of lignin polymerisation in gymnosperm compression wood.

The amount of β-1 structures in lignin is small; however, they are assumed to significantly affect the reactivity of lignin because they form dienone structures. A method employing thioacidolysis and subsequent desulfurization yields products that can be analyzed via gas chromatography-mass spectrometry (GC-MS) to quantify these β-1 structures. However, the retention times and response factors of the reaction products have not been accurately determined thus far. Here, 12 standard compounds combined with p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units were synthesized, and their retention times and response factors were determined through GC-MS, using selective ions. Based on these data, we also investigated the β-1 structures of lignocellulosic lignin samples. Our results clarified that the successful formation of the β-1 structure was dependent on the type of aromatic rings present; there were very few β-1 structures containing H units; and the amount of G-G type was higher and that of the heterotype, i.e., G-S type, was lower than the stochastic value.

International audience; Root-knot nematodes (RKNs; genus Meloidogyne) are a class of plant parasites that infect the roots of many plant species. It is believed that RKNs target certain signaling molecules derived from plants to locate their hosts; however, currently, no plant compound has been unambiguously identified as a universal RKN attractant. To address this question, we screened a chemical library of synthetic compounds for Meloidogyne incognita attractants. The breakdown product of aminopropylamino-anthraquinone, 1,3-diaminopropane, as well as its related compounds, putrescine and cadaverine, were found to attract M. incognita. After examining various polyamines, M. incognita were found to be attracted specifically by natural compounds that possess three to five methylene groups between two terminal amino groups. Using cryo-TOF-SIMS/SEM, cadaverine was indeed detected in soybean root cortex cells and the surrounding rhizosphere, establishing a chemical gradient. In addition to cadaverine, putrescine and 1,3-diaminopropane were also detected in root exudate by HPLC-MS/MS. Furthermore, exogenously applied cadaverine is sufficient to enhance M. incognita infection of Arabidopsis seedlings. These results suggest that M. incognita is likely attracted by polyamines to locate the appropriate host plants, and the naturally occurring polyamines have potential applications in agriculture in developing protection strategies for crops from RKN infection.

Non‐vitamin K dependent oral anticoagulants (NOAC) are now widely used in patients with nonvalvular atrial fibrillation (NVAF) for stroke prevention and in patients with venous thromboembolism (VTE) for the treatment and secondary prevention of the disease. Among NOAC, edoxaban demonstrated noninferiority to warfarin for stroke prevention in NVAF and for VTE treatment, with superior safety. EMIT‐AF/VTE (Edoxaban Management in Diagnostic and Therapeutic Procedures) ({"type":"clinical-trial","attrs":{"text":"NCT02950168","term_id":"NCT02950168"}}NCT02950168) is a multicenter, prospective, and noninterventional registry study designed to collect detailed information on the periprocedural management of patients with NVAF and VTE receiving edoxaban. The primary objective of EMIT‐AF/VTE is to document the periprocedural management of patients receiving edoxaban and to collect data on safety and other outcomes in these patients. The primary safety outcome is the rate of major bleeding. Other assessments include the evaluation of efficacy outcomes, periprocedural dosing, and timing of edoxaban. The observation period will start 5 days prior to the procedure and end 30 days post‐procedure. EMIT‐AF/VTE will aim to prospectively enroll up to approximately 1400 procedures from Europe. Enrollment commenced in December 2016 and will be completed in July 2018. As of July 2018, before database lock and with several procedure forms still temporarily inserted, a preliminary number of 1204 patients have been enrolled, who underwent a total of 1453 procedures. The prospective EMIT‐AF/VTE registry program will expand the knowledge of periprocedural management of patients with NVAF and VTE receiving edoxaban in clinical practice.

No clear picture has yet been elaborated concerning the mechanism of lignin growth, and thus this topic is the focus of the present paper. Namely, the enzymatic dehydrogenative polymerization (DHP formation) of coniferyl alcohol (CA, as a monolignol) and three dilignols and their reaction kinetics were investigated. The dilignols [guaiacylglycerol-β-coniferyl ether (IβO4 ), dehydrodiconiferyl alcohol (IIβ5 ), and pinoresinol (IIIββ )] and CA as a monolignol [(3-OCD3)-coniferyl alcohol (CAOCD3 )] were synthesized and subjected to enzymatic DHP formation. The dilignol derived from CAOCD3 could be identified by its higher molecular weight in comparison with the starting dilignols (IβO4 , IIβ5 , and IIIββ ). Based on the observed consumption rate of the CA and its dilignols, it was proposed that a radical transfer system exists between the dilignols, which is generated from the CA and the starting substrates.

Glycol lignin possessing thermoplastic and fusible properties can be a biorefinery platform material; however, glycol lignin is obtained as very small particles in suspension and the low dewatering efficiency is a major difficulty for the mass production. To improve the productivity of glycol lignin in the refinery plant, glycol lignin-based flocculants were prepared and their flocculation performance was examined. The flocculants (cationized glycol lignins) were synthesized by a reaction with glycidyltrimethylammonium chloride (GTA) in various reaction conditions. The prepared flocculants exhibited high flocculation performance and successfully economized the production cost of glycol lignin. Their flocculation efficiency was affected by the precipitation temperature, the length of the glycol chain, the molecular weight and the functional group of the glycol lignin. The flocculation behavior was interpreted by phase separation theory at a lower critical solution temperature (LCST).

In many flowering plants, floral scents are a significant trait for visitors, playing an important role in attracting pollinators and/or detracting herbivores. The evolution of flowering plants from hermaphroditism to dioecy is often accompanied by sexual dimorphism in floral scent. In this study, floral scents emitted by different sexual morphs of the subdioecious shrub Eurya japonica Thunb. were collected using a dynamic headspace method, and sexual and temporal variations were evaluated by gas chromatography–mass spectrometry (GC–MS). Two volatiles, α‐pinene and linalool, were identified as the major components of floral scents in females, hermaphrodites, and males. The males emit higher amounts of floral scents, particularly α‐pinene, compared to females or hermaphrodites. Floral scents emitted by males generally decrease as flowers enter senescence, whereas those from females or hermaphrodites do not significantly differ. Intraspecific variations in floral scents of subdioecious species provided by this study would contribute to better understanding of sexual dimorphism in floral scent.

Lignin is the second most abundant natural polymer on Earth and is a major cell wall component in vascular plants. Lignin biosynthesis has three stages: biosynthesis, transport, and polymerization of its precursors. However, there is limited knowledge on lignin precursor transport, especially in monocots. In the present study, we aimed to elucidate the transport mode of lignin monomers in the lignifying tissues of bamboo (Phyllostachys pubescens). The growth manners and lignification processes of bamboo shoots were elucidated, which enabled us to obtain the lignifying tissues reproducibly. Microsomal membrane fractions were prepared from tissues undergoing vigorous lignification to analyze the transport activities of lignin precursors in order to show the ATP-dependent transport of coniferin and p-glucocoumaryl alcohol. The transport activities for both precursors depend on vacuolar type H+-ATPase and a H+ gradient across the membrane, suggesting that the electrochemical potential is the driving force of the transport of both substrates. These findings are similar to the transport properties of these lignin precursors in the differentiating xylem of poplar and Japanese cypress. Our findings suggest that transport of coniferin and p-glucocoumaryl alcohol is mediated by secondary active transporters energized partly by the vacuolar type H+-ATPase, which is common in lignifying tissues. The loading of these lignin precursors into endomembrane compartments may contribute to lignification in vascular plants.

The stability and chemical reactions of lignans during alkaline cooking is not yet fully clarified although their degradation products may have a considerable effect on the pulp and its bleaching, such as spotting and color contamination. To fill this gap, a phenolic lignan (pinoresinol) and four non-phenolic furofuran lignans (gmelinol, paulownin, eudesmin and sesamin) were submitted to alkaline cooking at various conditions. The effect of cooking duration shows that non-phenolic lignans are more stable during a 3-h reaction time than pinoresinol. After a 24-h reaction, most of the lignans were decomposed or polymerized. The effect of cooking temperature shows that pinoresinol, eudesmin and sesamin are more susceptible to degradation with increasing temperature. The concentration of the alkaline liquors only affect significantly to pinoresinol because of its acidity. It was also observed that pinoresinol, gmelinol and eudesmin give rise to small amounts of vanillin and acetovanillone, while paulownin and sesamin were mainly polymerized or degraded. To confirm the reaction mechanism of the occurrence of acetovanillone in the reaction, 13C-labeled pinoresinol was also submitted to alkaline cooking. It was revealed that the cleavage at β-β′ position occurred during alkaline reaction. The stability of these compounds during alkaline cooking are not affected by the hydroxyl group in the C-β′ position.

Bamboos are among the largest woody grasses and grow very rapidly. Although lignin is a crucial factor for the utilization of bamboo biomass, the lignification mechanism of bamboo shoots is poorly understood. We studied lignification in the bamboo Sinobambusa tootsik during culm development. Elongation growth began in May and ended in late-June, when the lignin content was approximately half that in mature culms. Thioacidolysis analysis indicated that p-hydroxyphenyl units in lignin formed even at late stages of lignification. The syringyl/guaiacyl ratio varied during culm development. Various lignin precursors were detected in developing culms by liquid chromatography–mass spectrometry. The ferulic acid content decreased from May to June, indicating that ferulic acid was utilized in early stages of cell wall formation. Monolignol glucosides were detected at early stages of lignification, whereas the contents of monolignols, coniferaldehyde, sinapaldehyde, p-coumaric acid, and ferulic acid peaked at later stages of lignification. Therefore, lignin precursors may be supplied differentially during the lignification process. In August, the rate of lignification decreased, although the contents of various lignin precursors peaked, implying that the rate-limiting step in the cessation of lignification in bamboo is transport or polymerization of lignin precursors, rather than their biosynthesis.

Alkaloids are basic nitrogen-containing chemicals that have important physiological and pharmacological characteristics. Many vascular plant species contain alkaloids, and their roles in planta are of interest. However, the detailed distribution of alkaloids remains unclear because of their low water solubility and low concentrations in plants. In this study, we visualized the distribution of salicifoline, a water-soluble quaternary ammonium alkaloid, in the freeze-fixed stems of Magnolia kobus by cryo time-of-flight secondary ion mass spectrometry. Most of the salicifoline was distributed in living phloem tissues. In the xylem, salicifoline was detected in ray cells, lignifying wood fibres, and in vessels in the latest annual ring. The salicifoline distribution in the xylem varied with the cell wall formation stage. These results provide new insights into the storage, transportation, and role of the alkaloid salicifoline in M. kobus.

To investigate the possibility of decontaminating 137Cs-contaminated Cryptomeria japonica wood, kraft pulping was conducted and the Cs behavior in the reaction process was examined. 133Cs-treated or 137Cs-contaminated bark, sapwood, and heartwood chips of Cryptomeria japonica were digested using an aqueous solution of NaOH and Na2S. The pulp was washed with ultrapure water and filtered, after which the filtrate (black liquor) was collected. The black liquor was acidified to separate the supernatant and precipitation. The Cs (133Cs and 137Cs) concentrations in the chip and reaction products were measured. As for wood samples, the majority of Cs was present in black liquor, while only a minor amount of Cs was retained in the pulp (

Main conclusion: The screening of rice mutants with improved cellulose to glucose saccharification efficiency (SE) identifies reduced xylan and/or ferulic acid, and a qualitative change of lignin to impact SE. To ensure the availability of sustainable energy, considerable effort is underway to utilize lignocellulosic plant biomass as feedstock for the production of biofuels. However, the high cost of degrading plant cell wall components to fermentable sugars (saccharification) has been problematic. One way to overcome this barrier is to develop plants possessing cell walls that are amenable to saccharification. In this study, we aimed to identify new molecular factors that influence saccharification efficiency (SE) in rice. By screening 22 rice mutants, we identified two lines, 122 and 108, with improved SE. Reduced xylan and ferulic acid within the cell wall of line 122 were probable reasons of improved SE. Line 108 showed reduced levels of thioglycolic-released lignin; however, the amount of Klason lignin was comparable to the wild-type, indicating that structural changes had occurred in the 108 lignin polymer which resulted in improved SE. Positional cloning revealed that the genes responsible for improved SE in 122 and 108 were rice CONSTITUTIVE PHOTOMORPHOGENIC 1 (OsCOP1) and GOLD HULL AND INTERNODE 1 (GH1), respectively, which have not been previously reported to influence SE. The screening of mutants for improved SE is an efficient approach to identify novel genes that affect SE, which is relevant in the development of crops as biofuel sources.

In order to understand the kraft pulp decolouring mechanism on using a nonionic detergent, the pulp washing process and the resulting pulp handsheets were investigated by examining the brightness, kappa number, thioacidolysis product yield, and dewatering efficiency in the pressing sheet making process. The pulp decolouring could be attributed to a decrease in the lignin content and an improvement in the dewatering efficiency. Furthermore, the detergent distribution in the aqueous pulp suspension obtained during the pulp washing process was visualised using cryo-time-of-flight secondary ion mass spectrometry/scanning electron microscopy (cryo-TOF-SIMS/SEM). The detergent was clearly observed at the transverse surface of the pulp fibre cell wall and was also detected in the lumen of the fibres, suggesting the permeation of the detergent into the pulp fibre cell wall. Based on these results, the pulp decolouring mechanism can be proposed as follows: the detergent permeates into the pulp fibre cell wall and promotes the solution-exchange between the inside and the outside parts of the fibre cell wall, finally washing away the chromophoric substances such as lignin and its degradation products owing to the enhanced dewatering efficiency.

The aim of the present study was to investigate the extractives of Gmelina arborea stem from the sapwood (sW) to heartwood (hW) including the transition zone (tZ) between them by means of quantitative GC-MS, while the spatial distribution of four typical compounds was analysed by TOF-SIMS. The focus was on gmelinol, paulownin, 7′-O-ethyl arboreol, and β-sitosterol, which were isolated and purified from hW. The four compounds revealed a characteristic distribution pattern corresponding to their key role in biosynthesis and depending on their susceptibility to secondary reactions in the hW.

Monolignols are precursors of lignin, and their glucosides are often found in plants. Glucosylation creates water‐soluble and chemically stable monolignols by protecting the phenolic hydroxyl group. To discuss the role of sinapyl alcohol glucoside, syringin, in planta, the cellular distribution of syringin in the transverse and radial surfaces of quick‐frozen stems of Syringa vulgaris L. (lilac) was visualized by cryo‐time‐of‐flight secondary ion mass spectrometry and scanning electron microscopy (cryo‐TOF‐SIMS/SEM) analyses. The amount and rough distribution of syringin were confirmed by high‐performance liquid chromatography measurements using serial tangential sections of freeze‐fixed lilac stems. The syringin distribution was also discussed with reference to the tissue classification from microscopic observations. Syringin was mainly found in the phloem region. In the xylem region, syringin was evenly distributed irrespective of the cell type from the cambial zone to the early differentiating stage region and selectively distributed in vessels in the later differentiating stage region. After the lignification of wood fibers, syringin was found in rays and some vessels in the initial part of the annual rings. Previously, artificially administered isotope‐labeled syringin was shown to be assimilated into lignin in the differentiating xylem region. Based on this, our present data showing syringin storage in the differentiating xylem region and its variation depending on the lignification stage suggest that syringin works as a lignin precursor. Additionally, detection of syringin in vessels and rays indicates intercellular transportation of syringin in lilac stems.

Lignin is a biopolymer consisted of monolignols but the growth mechanism of lignin has not yet been elucidated. In this study, enzymatic dehydrogenative polymerization of dilignols, guaiacylglycerol-β-coniferyl ether (I), dehydrodiconiferyl alcohol (II), and pinoresinol (III), was conducted using crude enzyme obtained from Japanese cypress (Chamaecyparis obtusa) in order to investigate the step following the initial radical coupling between monolignols. The results suggested the occurrence of radical transfer during the reaction, similar to the reaction using horseradish peroxidase (HRP). The behavior of erythro and threo of I during the reaction was analyzed, and it was found that the crude enzyme has a substrate specificity toward erythro rather than threo forms, but HRP shows negligible specificity. And that erythro of I shows a slightly greater tendency to form a radical by radical transfer from II or III than the threo form.

Sulfuric acid lignin (SAL) is a byproduct of the acid saccharification of lignocellulosic materials for bio-fuel generation. The recalcitrant and highly complex structure of SAL significantly limits its industrial use, making it a useless industrial waste. In a previous study, we proposed a hydrothermal reaction for SAL that can convert it into water-soluble compounds (HSALs), which considerably expanded its industrial use scenario. In this study, we converted this water-soluble HSAL into two ionic modifications with applications as flocculants and dispersants. Firstly, the introduction of a carboxy group was carried out with bromoacetic acid. In the carboxymethylated HSAL (C-HSAL), the carboxymethyl groups content was as high as 3.66 mmol/g. Dispersibility tests using gypsum paste showed that C-HSAL exhibited a dispersibility almost equivalent to that of commercial lignosulfonate. Furthermore, another investigation was conducted synthesizing cationized HSAL derivatives by the reaction with glycidyl trimethylammonium chloride (GTA). Dye removal tests were performed with the obtained lignin-based flocculants using three organic dyes. The results showed ideal dye removal ratios for the GTA-HSALs (higher than 90% for all dyes tested), thereby suggesting that they have a huge potential as core molecules to develop high-efficient dye flocculants.

Lignin is a cell wall component of vascular plants crucial for survival in terrestrial environments. While p-hydroxyphenyl lignin is minor, it is considered to be localised in the outermost part of the cell wall providing strong adhesion between cells, which determines cell shape. Transport of the lignin precursor from the cytosol to the cell wall is critical to regulate temporal and spatial lignin deposition; however, little information on the transport step is available. Here, we report transport activity of p-glucocoumaryl alcohol, a precursor of p-hydroxyphenyl lignin, in a broad-leaved tree (hybrid poplar, Populus sieboldii × P. grandidentata) and a coniferous tree (Japanese cypress, Chamaecyparis obtusa). Membrane vesicles of both trees were prepared from differentiating xylem with vigorous lignification and used for transport assays. Several inhibition assays indicated that not ABC transporters but the proton gradient and V-ATPase are involved in p-glucocoumaryl alcohol transport depending on ATP. These results support the hypothesis that p-glucocoumaryl alcohol is loaded into the secretory vesicles and delivered to the cell wall by exocytosis. Furthermore, this transport mechanism was common in both poplar and Japanese cypress, strongly suggesting that p-glucocoumaryl alcohol transport in the differentiating xylem is conserved within woody plants.

Electropolymerization was carried out in order to obtain a bio-based polymer by a simple procedure. Ferulic acid, a component of gramineous plants, was selected for this purpose. A thin polymer film was produced in an organic solvent medium (i.e., CH2Cl2/methanol (4:1 v/v) in the presence of 0.2 M LiClO4), whereas it was not obtained in a totally aqueous medium (0.2 M NaOH). Scanning electron microscopic analysis showed that the polymer film had a porous lamellar structure of ~10 μm thickness. Most of the carboxyl groups remained, and IR spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) analyses revealed that o-quinones existed in the polymer film. After electropolymerization, caffeic acid, sinapic acid, 3,6-bis(4-hydroxy-3-methoxyphenyl)tetrahydrofuro[3,4-c]furan-1,4-dione (1), and 4,4’-dihydroxy-3,3’-dimethoxy-β,β’-bicinnamic acid (2) were detected in the organic reaction medium; this suggested that demethylation, methoxyl group addition, and radical coupling reactions occurred at the electrode surface.

A large amount of monolignol glucosides (MLGs: p-glucocoumaryl alcohol, coniferin, syringin) are found in lignifying soft xylem near cambium and they disappear with the progress of lignification. Recently, it became a matter of debate whether those MLGs are real intermediates in lignin biosynthesis or only a storage form of monolignols outside of the main biosynthetic pathway. The latter is partly based on a misinterpretation of 14C-tracer experiments and partly on the simple generalization of the results of gene manipulation experiments concerning the flexible and complex lignification. In the present paper, it could be confirmed by the most reliable 13C-tracer method that MLGs are real intermediates in the pathway from l-phenylalanine to macromolecular lignin-polysaccharides complexes in the cell walls. This pathway via MLGs is essential for transport and programmed delivery of specific monolignols in a stable form from intracellular space to specific lignifying sites within the cell wall. The pool size of MLGs is large in most gymnosperm trees and some angiosperm species that emerged in an early stage of phylogeny, while the pool size is small in most angiosperms. This difference in pool size is reasonably understandable from the viewpoint of plant evolution, in the course of which the role of MLGs changed to meet variation in type of major cells, reaction wood formation, and postmortem lignification.

Sulfuric acid lignin (SAL), one of the major biorefinery industry residues from the acid saccharification process on lignocellulosic materials for bio-fuel generation, is becoming a major problem for the sustainable development of the bio-refinery industry. Meanwhile, the recalcitrant and highly complex structure of SAL makes it insoluble in water and other organic solvents, resulting in a low reactivity. This study investigated the effects of hydrothermal reactions on SAL to enhance its solubility and reactivity. A hydrothermal reaction was first conducted on SAL from Cryptomeria japonica to obtain a completely water-soluble lignin. Fourier transform infrared spectroscopy, gel permeation chromatography, gas chromatography, and nuclear magnetic resonance (NMR) analyses revealed that the hydrothermal reaction of the SAL caused depolymerisation, demethylation, and the introduction of phenolic hydroxyls. Furthermore, another experiment was conducted on synthesizing a lignin polymer model compound (DHP) with 13C-labelled on the aromatic ring, followed by hydrothermal reaction and acetylation. Detailed structural information of DHP under hydrothermal reaction was obtained by solid-state cross polarization magic angle spinning NMR analysis. Reactivity of the hydrothermally treated SAL was enhanced by the hydrothermal reaction through the reversed increasing of the phenolic hydroxyl/aliphatic hydroxyl ratio in the condensed lignin units.

The structure and biosynthesis of lignin are not yet fully understood, especially the step following the initial dimerization of monolignol. Liquid chromatograph mass spectrometer (LC–MS) was used to analyze the consumption rates of monolignol dimers formed by β-O-4, β-5, and β-β couplings between coniferyl alcohols in efforts to understand the activity of monolignol dimers in enzymatic dehydrogenative polymerization. We investigated the reaction kinetics in single-component and mixed-component reaction systems containing one and two species of the dimers, respectively. A difference was observed between the consumption rates of the three dimers we tested, and the consumption rate of one dimer in the single-component reaction was different from that in a mixed-component reaction. In qualitative LC–MS analyses, coniferyl alcohol oligomers were detected in the reaction products. Some monolignol tetramers were formed by 5-5 and 5-O-4 coupling between the dimers. The results of this work suggested that monolignol dimers with β-5 and β-β linkages could function as radical mediators in enzyme-catalyzed polymerization.

Lignin-carbohydrates, one of the major cell wall components, are believed to be the structures that form chemical linkage between lignin and cell wall polysaccharides. Due to the molecular complexity of lignin-containing substances, their isolation and the assignment of their biological activities have so far remained a difficult task. Here, we extracted two lignin-containing carbohydrates, lignin-rich enzyme lignin (LREL) and pure enzyme lignin (PEL), from barley husk and demonstrated that they act as immune stimulators of dendritic cells (DCs), which are particularly important in linking innate and adaptive immunity. Thioacidolysis, acid hydrolysis, and mild alkali hydrolysis of both LREL and PEL revealed that their immunostimulatory activities depended on the lignin structure and/or content, neutral sugar content (especially the characteristic distribution of galactose and mannose), and presence of an ester bond. Furthermore, we showed that the immunostimulatory potency of the lignin-carbohydrate depended on its molecular weight and degree of polymerization. We also demonstrated that the LREL-induced activation of DCs was mediated via TLR4. Thus, LREL-induced increases in the expression levels of several cell surface marker proteins, production of inflammatory cytokines IL-12p40 and TNF-α, and activation and nuclear translocation of transcription factors, as was observed in the WT DCs, were completely abrogated in DCs derived from the TLR4(-/-) mice but not in DCs derived from the TLR2(-/-), TLR7(-/-), and TLR9(-/-) mice. We further demonstrated that LRELs isolated from other plant tissues also activated DCs. These immunostimulatory activities of lignin-carbohydrates, extracted from edible plant tissues, could have potential relevance in anti-infectious immunity and vaccine adjuvants.

In this study, the antioxidant activity of hydrothermal extracts of different parts of watermelons (flesh, white rind, and green rind) was evaluated. Each part of the watermelons was extracted at temperatures ranging from 100 °C to 300 °C for 10, 30, or 60 min. The highest total phenol content was observed for the green rind extracted at 300 °C for 30 min with 7626.52 μg gallic acid equivalents (GAE)/g, whereas the phenol content of the untreated green rind extract was 715.15 μg GAE/g. The antioxidant activity of the watermelon parts increased with increase in treatment temperature and time. In GC–MS analysis, catechol, 4-methylcatechol, pyrogallol, and 1,2,4-benzenetriol were detected after hydrothermal extraction at 300 °C for 30 min. These results indicate that hydrothermal extraction is an efficient process for increasing the antioxidant activity of watermelon extracts.

This study aimed to evaluate the relationship between low-density lipoprotein cholesterol (LDL-C) levels and cardiovascular disease (CVD) in high-risk patients with hypercholesterolemia without a history of CVD. Patients who were receiving or started treatment with pravastatin, were followed-up for 2 years. Patients were divided into quartiles according to on-treatment LDL-C. The maximum contrast method based on the Cox proportional hazards model was used to evaluate the relationship between achieved LDL-C and the incidence of CVD. Incidence of CVD was also compared according to whether a number of risk factor targets were achieved. A total 6,229 patients were enrolled, with 4,916 having reported LDL-C values. During the 2 years, 69 cases of CVD (6.7/1000 patient years), including 36 coronary artery disease (CAD) (3.5/1000 patient years) and 28 strokes (2.7/1000 patient years), occurred. The comparison of on-treatment LDL-C level quartiles suggested that the incidence of all CVD decreased linearly as the LDL-C levels decreased. Incidence of CAD showed a curvilinear relationship to LDL-C levels, suggesting some attenuation of risk below LDL-C of 119 mg/dL. The incidence of all CVD and CAD tended to be decreased as the number of achieved risk factor targets increased. In conclusion, through our observational study, it was shown that a linear relationship between the incidence of CVD and LDL-C was observed in high-risk hypercholesterolemic patients. The low incidence of CVD in the present study may be associated with multifactorial management of conventional risk factors including high LDL-C levels. However, prospective, randomized studies are needed to confirm these findings.

Lignin is an essential component of higher plants, which is built by the enzymatic dehydrogenative polymerization of monolignols. First, monolignol is enzymatically oxidized to produce the phenoxy radical, which can form resonance hybrids. Two radical resonant hybrids are coupled with each other to yield dilignol with various linkage types, of which the main structures are β- O -4′ ( I ), β-5′ ( II ) and β-β′ ( III ). However, the reaction mechanism behind the addition lignol radicals to dilignol is not yet fully understood. Here, we show an unexpected reaction with structure II during enzymatic dehydrogenative polymerization, which involves cleavage of a covalent linkage and creation of a new radical coupling site. This implied that the β-5 dilignol diversifies the growing pattern of lignin. This discovery elucidates a novel mechanism in lignin polymerization.

To utilize the by-product lignin (SAL) that is generated by bioethanol production systems that use lignocellulosic materials with a sulfuric acid catalyst, the antioxidant activity, reducing power, and acetylcholinesterase inhibitory activity of SAL were investigated. Hydrothermal treatment effectively increased the total phenolic content (TPC) and had positive effects on the antioxidant activity and reducing power. By using model compounds, the reaction mechanism of the increase in TPC by hydrothermal treatment was determined. The antioxidant activity, reducing power, and acetylcholinesterase inhibitory activity of hydrothermalized SAL were insensitive to the methoxy group content, molecular weight, and differences in nucleus species (such as guaiacyl or syringyl). This suggests that the species of lignocellusic materials have little relationship to the potential utilization of SAL as a bioactive agent.

To clarify the role of coniferin in planta, semi-quantitative cellular distribution of coniferin in quick-frozen Ginkgo biloba L. (ginkgo) was visualized by cryo time-of-flight secondary ion mass spectrometry and scanning electron microscopy (cryo-TOF-SIMS/SEM) analysis. The amount and rough distribution of coniferin were confirmed through quantitative chromatography measurement using serial tangential sections of the freeze-fixed ginkgo stem. The lignification stage of the sample was estimated using microscopic observations. Coniferin distribution visualized at the transverse and radial surfaces of freeze-fixed ginkgo stem suggested that coniferin is stored in the vacuoles and showed good agreement with the assimilation timing of coniferin to lignin in differentiating xylem. Consequently, it is suggested that coniferin is stored in the tracheid cells of differentiating xylem and is a lignin precursor.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an analytical method that has developed just within the last few decades. It is a kind of imaging mass spectrometry and supplies mass information with a submicron lateral resolution. In this chapter, the availability of TOF-SIMS measurements in the research field of woody plants is introduced. The main polymer components of the plant cell wall, cellulose, hemicellulose, and lignin are detected as fragment ions. The comprehension of the fragmentation behavior is essential to interpret the complicated mass spectra of biological samples, and the case of lignin is specifically described. Inorganics and low-molecular-weight extractives are detectable as they are. The latest topics of cryo-TOF-SIMS dealing with frozen-hydrated samples are also presented. Heterogeneous distribution of many chemical components in plants at dried or frozen-hydrated conditions might be useful information to improve our discussion another notch.

The experiments with model compounds revealed that time-of-flight secondary ion mass spectrometry (ToF-SIMS) is able to split the common interunit linkages of lignin, except the 5-5 linkage. In a previous study, ToF-SIMS produced characteristic secondary ions with m/z 137 and 151 (C6-C1 fragments) from the phenylcoumaran-type lignin model compound, the benzofuran ring of which has a β-5 linkage and an α-O-4 linkage. However, it is still unclear whether the fragments are from ring A with the free phenolic OH as a result of the sole cleavage of the α-O-4 link by opening the furan ring or from ring B as a result of the β-5 cleavage. In this study, the phenylcoumaran-type lignin model compound and its deuterium- and/or 13C-labeled analogues were synthesized and analyzed by ToF-SIMS. It could be clarified that the β-5 linkage is not cleaved by ToF-SIMS.

Initially, we investigated the distribution of chemicals in dried wood samples using a standard TOF-SIMS and obtained two-dimensional images of chemical distribution in various wood samples. However, during drying as part of the sample preparation, some artifacts could occur because water-soluble chemicals could be moved, drained, or changed at that time. To understand the function of chemicals in living trees, it is important to reveal the distribution of chemicals in planta. For this purpose, we developed the new cryo-TOF-SIMS/SEM system to analyze chemical distribution in frozen-hydrated samples. This new system consists of a glove box, a cryo-SEM, and a cryo-TOF-SIMS, which are interconnected by a cryo-vacuum shuttle. This system was then applied to freeze-fixed Cryptomeria japonica xylem. Potassium, ferruginol, a major wood chemical of C. japonica, and water were simultaneously detected in frozen-hydrated wood tissues. For accurate detection, the sublimation of ice from sample surfaces must be prevented. After the TOF-SIMS analysis of the surface, the sample was cut to make the next sample surface in the glove box and cryo-TOF-SIMS analysis was performed again. Continuing with this procedure, we obtained a three-dimensional analysis data of TOF-SIMS images. The maximum height was 1000 µm. These results suggest that this new system is useful for revealing the chemical distribution of wood tissue in planta. Copyright © 2012 John Wiley & Sons, Ltd.

The distribution of ethyl acetate extracts and 4,8-dihydroxy-5-methoxy-2-naphthaldehyde (compound I), which is a major constituent of the extracts obtained from the blackened heartwood of Diospyros kaki, was analyzed via gas chromatography-mass spectrometry (GC-MS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). According to GC-MS, the extracts and compound I are high in concentration at the pith and at the edges of the blackened heartwood. ToF-SIMS analysis revealed a peak at a mass-to-charge ratio of (m/z) 218, which is characteristic of the ionic form of compound I. The ToF-SIMS imaging of compound I in the blackened heartwood based on m/z 218 shows that compound I is located in parenchyma cells and their neighboring axial elements.

This study showed that hydrogenated palm oil was an effective conditioning agent for repair of damaged hair because it inhibits water-induced swelling of damaged hair. To understand the functional mechanism of hydrogenated palm oil, we conducted TOF-SIMS analysis of the penetration of this oil into damaged hair. TOF-SIMS analysis revealed that tripalmitin, a constituent of hydrogenated palm oil, penetrated into the cuticle and the outer cortex of damaged hair. This is considered as the mechanism responsible for inhibition of hair swelling by hydrogenated palm oil.

Lignin, one of the main structural polymer of plant cell walls, varies in amount and monomeric composition among tissue and cell types, as well as among plant species. However, few analytical methods are available that can conveniently and accurately determine the morphological distribution of lignin units at the cellular level. In this report, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly map guaiacyl (G) and syringyl (S) lignin units in several successive growth rings of the maple xylem. TOF-SIMS imaging and a semiquantitative approach revealed clear difference in the annual distribution of lignins between the fiber and vessel. While the vessel walls were constantly G-rich with varied S/G ratios through a growth ring, the fibers showed fairly regular annual distribution of lignins in which the earlywood was S-rich with an almost constant S/G ratio and the latewood was G-rich resulting from a decrease of the S unit. The reliability of TOF-SIMS results was demonstrated by its high correlation with the results of thioacidolysis on radial distribution of the S/G ratio in several contiguous tree rings and also in the latewood and earlywood of each ring. These results indicate that TOF-SIMS allows direct visualization of lignin composition in plant tissues.

In this study, polyesters were prepared from sulfuric acid lignin (SAL) that was generated as a by-product during the production of bioethanol from lignocellulosic materials using ɛ-caprolactone (CL). Carboxylic groups were introduced into SAL by a hydrothermal reaction under alkali conditions. The acid form (-COOH) of the obtained polymer (HSAL) was dissolved into ɛ-caprolactone and the mixture was heated at 150°C for 12 h with ZnCl2 as a catalyst. The structure of the obtained polymer (PCL-HSAL) was proposed based on results of differential scanning calorimetric analysis, swelling tests, and dynamic mechanical thermal analysis. The hydroxyl group in HSAL participated in the ring-opening polymerization of CL. PCL-HSAL with a high HSAL content formed a three-dimensional network that did not dissolve in organic solvents and did not melt at high temperature. PCL-HSAL showed a high swelling property for a suitable ratio of CL to HSAL.

4,8-Dihydroxy-5-methoxy-2-naphthaldehyde (Compound I) was isolated from blackened heartwood of Diospyros kaki and was methylated with diazomethane. Antioxidant and cytotoxic activities of Compound I and two methylated derivatives [4-hydroxy-5,8-dimethoxy-2-(2-oxopropyl)-naphthalene (D-1) and 2-glycidyl-4-hydroxy-5,8-dimethoxy naphthalene (D-2)] were evaluated. Compound I showed higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and reducing power than D-1 and D-2. However, D-1 and D-2 exhibited slightly stronger 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)+ (ABTS+) radical scavenging activity than Compound I. Compound I also exhibited stronger cytotoxic activity than D-1 and D-2 against the growth of HT-29 colon cancer cells. The results supported the hypothesis that methylation of naphthalene derivatives slightly increased ABTS+ radical scavenging activity, but significantly decreased DPPH radical scavenging activity, reducing power, and cytotoxic activity.

In this study, saccharification of the inner bark of Eucalyptus was carried out by enzymatic hydrolysis to produce bioethanol from non-food biomass. To enhance the accessibility of the enzyme to the polysaccharides such as cellulose and holocellulose in the cell wall of the bark, the bark was subjected to hydrothermal pre-treatment with carbon dioxide. This pre-treatment considerably influenced enzymatic hydrolysis. The main component (over 90%) of the generated monosaccharide was glucose, and the yield of glucose on the basis of alpha-cellulose reaches about 80%. This result suggests that the secondary wall, whose main component is cellulose, was effectively hydrolyzed by the enzyme. Microscopic analysis revealed that after pre-treatment, the phloem parenchyma cell had a considerably swollen primary wall and the phloem fibre showed many nano-clefts within its secondary wall. These structural changes appeared to promote enzymatic hydrolysis, because of high accessibility of enzymes to cellulose in the secondary wall.

The antioxidant activities of two persimmon (Diospyros kaki T.) branches were evaluated. After preparing acetone, 70% acetone, and methanol extracts from Fuyu and Nishimurawase persimmon branches, DPPH radical scavenging activity (RSA), ABTS RSA, reducing power (RP), and total phenol contents (TPC) were determined. The acetone extracts of Fuyu and Nishimurawase showed higher antioxidant properties than the other extracts. The highest TPC (22.48 mg/g), DPPH RSA ($IC_{50}

The lignin syringyl to guaiacyl (S/G) ratio, the composition of monomeric units, is an important parameter that is widely used to obtain information on the complex polymer structure of lignin present in plant cell walls. We assessed the reliability of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for determining the S/G ratio using monomer, dimer and polymer model compounds of lignin. In the case of the monomer and dimer, the S/G ratios were much higher than the actual molar S/G ratios. In contrast, for the aryl ether 8-O-4'-linked polymers, the S/G ratios estimated were similar to the actual S/G molar ratios, suggesting that the ionization efficiencies of the characteristic ions of S and G units detected from the synthetic lignin polymer compounds were similar. The results suggest that TOF-SIMS is useful for rapid and convenient determination of the lignin S/G ratio in plants.

Human hair damage is caused by chemical treatments and various daily factors. A number of cosmetic ingredients have been developed to repair hair damage. It is important to know the localization of cosmetic ingredients distributed on human hair to understand the interaction mechanism of these ingredients with the hair. In this work, the distribution of cosmetic ingredients on damaged hair was investigated using time-of-flight secondary ion mass spectrometry (TOF-SIMS). We examined the penetration of L-theanine and the adsorption of cationic chitosan. The TOF-SIMS imaging analysis revealed that L-theanine penetrated deep and was distributed uniformly inside the damaged hair. Further, cationic chitosan was uniformly adsorbed on the hair surface. The analysis results showed that TOF-SIMS is one of the most convenient methods to estimate the distribution of cosmetic ingredients on the hair.

A detailed chemical investigation of the extractives of the blackened heartwood of Diospyros kaki was carried out to understand their chemical characteristics and to obtain chemotaxonomic information. Three novel naphthalene derivatives were isolated, i.e., 4-hydroxy-5,6-dimethoxy-2-naphthaldehyde (1), 5,6,8-trimethoxy-3-methyl-1-naphthol (2), and 4,8-dihydroxy-5-methoxy-2-naphthaldehyde (3), in addition to two previously reported 2-naphthaldehydes: 4-hydroxy-5,8-dimethoxy-2-naphthaldehyde (4) and 4-hydroxy-5-methoxy-2-naphthaldehyde (5) Their structures were identified by 1D and 2D nuclear magnetic resonance spectroscopy, as well as by high-resolution mass spectrometry.

To make use of technical lignins as a nonionic polymeric surfactant, we have already reported the modification of acetic acid lignin (AL) to amphiphilic derivatives by polyoxyethylation using two types of polyethylene glycol (PEG) with diglycidyl (PEGDE) and monoglycidyl (EPEG) groups.[ 1 ] Kraft lignin (KL) was converted to amphiphiles in a similar manner. The resultant KL derivatives also indicated high surface activity. Polyethylene glycol with long alkyl chain was introduced to AL, KL, and lignosulfonate (LS) to prepare surfactants of high performance, using dodecyloxy-polyethylene glycol glycidyl ether (DAEO). The resultant DAEO-derivatives showed lower critical micelle concentration by 2–4 orders of magnitude than the corresponding PEGDE- and EPEG-derivatives. The DAEO-derivatives from LS showed better dispersibility for gypsum paste, one of cement components, than LS.

Electropolymerization of coniferyl alcohol was carried out in an aqueous system (0.2 M NaOH) and in an organic solvent system [CH2Cl2/methanol (4:1 v/v) in the presence of 0.2 M LiClO4] to produce a dehydrogenation polymer (DHP) - artificial lignin. In both systems, the polymerization of coniferyl alcohol was visually confi rmed. In the aqueous system, no dimer was detected in the reaction medium after electropolymerization, suggesting that endwise polymerization occurred on the electrode surface. Thioacidolysis degradation revealed that the obtained polymers had numerous 8-O-4′ linkages. The electropolymerization products obtained in the organic solvent system also had numerous 8-O-4′ linkages; in particular, the polymers obtained in the initial polymerization stage. This was probably because of the limited area available for reaction and the orientation of coniferyl alcohol on the electrode surface controlled the polymerization.

Acetic acid lignin (AL), one of the organosolv lignins, was modified by polyoxyethylation using commercially available polyethylene glycol diglycidylethers (PEGDE) having various chain lengths in order to generate novel nonionic polymeric surfactants. AL could be converted to the amphiphile by modifying with PEGDE (PEGDE-AL) having more than 9 of the ethylene oxide (EO) repeating units. Although the surface activities of PEG and AL were very limited, PEGDE-AL did strongly depress surface tension of water, and showed clear critical micelle concentrations (CMC). The CMC value of PEGDE-AL could be comparable to a commercial anionic lignin surfactant, lignosulfonate. The surface activity of AL amphiphile was further improved by modification with monoepoxides, ethoxy-(2-hydroxy)-propoxy-polyethylene glycol glycidylether (EPEGGE). The surface tension of water was depressed by the addition of the EPEGGE-AL to the same level as Triton® X-100, which is a commercial PEG-based nonionic surfactant, although ...

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to investigate the distribution of elements, Na, Mg, Al, K, and Ca, and lignin, in the contiguous growth rings including the sapwood/heartwood boundary in Hinoki cypress (Chamaecyparis obtusa). Lignin was distributed almost uniformly from sapwood to heartwood. The concentrations of most of the elements showed a drastic increase or decrease in the transition zone between sapwood and heartwood. The ToF-SIMS mapping analysis showed that most of the elements predominantly localized in the ray parenchyma cells in the inner transition zone and heartwood, while the elements showed no localization and distributed almost uniformly in the outer transition zone near sapwood. The result suggests that the ray parenchyma cells play a role in behaviors of elements during the transition from sapwood to heartwood.