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One of the main obstacles in biocatalysis is the substrate inhibition (SI) of enzymes that play important roles in biosynthesis and metabolic regulation in organisms. The promiscuous glycosyltransferase UGT72AY1 from Nicotiana benthamiana is strongly substrate-inhibited by hydroxycoumarins (inhibitory constant Ki < 20 µM), but only weakly inhibited when monolignols are glucosylated (Ki > 1000 µM). Apocarotenoid effectors reduce the inherent UDP-glucose glucohydrolase activity of the enzyme and attenuate the SI by scopoletin derivatives, which could also be achieved by mutations. Here, we studied the kinetic profiles of different phenols and used the substrate analog vanillin, which has shown atypical Michaelis–Menten kinetics in previous studies, to examine the effects of different ligands and mutations on the SI of NbUGT72AY1. Coumarins had no effect on enzymatic activity, whereas apocarotenoids and fatty acids strongly affected SI kinetics by increasing the inhibition constant Ki. Only the F87I mutant and a chimeric version of the enzyme showed weak SI with the substrate vanillin, but all mutants exhibited mild SI when sinapaldehyde was used as an acceptor. In contrast, stearic acid reduced the transferase activity of the mutants to varying degrees. The results not only confirm the multi-substrate functionality of NbUGT72AY1, but also reveal that the enzymatic activity of this protein can be fine-tuned by external metabolites such as apocarotenoids and fatty acids that affect SI. Since these signals are generated during plant cell destruction, NbUGT72AY1 likely plays an important role in plant defense by participating in the production of lignin in the cell wall and providing direct protection through the formation of toxic phytoalexins.

Eleven phenylpropanoids were isolated from the whole grass of Liparis nervosa, an orchidaceous medicinal plant. Their structures were elucidated as (+)-Syringaresinol (1), (-)-Syringaresinol-4-O-β-D-glucopyranoside (2), Sinapaldehyde (3), Coniferyl aldehyde (4), Syringin (5), Sinapaldehye glucoside (6), 2,3-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone (7), C-Veratroylglycol (8), 7S, 7′S, 8R, 8′R-icariol A2 (9), Erigeside 2 (10), and Methylsyringin (11) by comparing the spectroscopic data and physicochemical constants from the isolated compounds with the data reported in the literature. Compounds 1 and 9 were found to have potent in vitro antioxidant activities in the DPPH and ABTS assays, and their IC50 values were lower than those of vitamin C. More importantly, compound 9 had a strong α-glucosidase inhibitory activity with an IC50 value of 43.76 ± 2.03 µM, which was much lower than that of acarbose (IC50 = 273.12 ± 11.84 µM), indicating that compound 9 has the potential for the development of hypoglycemic drugs. In conclusion, the present study suggests that phenylpropanoids may be the additional representative type of active constituents in L. nervosa, which provides a new line of evidence to understand this medicinal plant.

CmCAD2 and CmCAD3 function more positively than CmCAD1 in oriental melon for lignin synthesis which is important to ensure internal water status and thus for drought tolerance. Well-lignification may be the guarantee of efficient axial water transport and barrier of lateral water flow in oriental melon tolerating drought stress, however remains to be verified. As an important enzyme in monolignol synthesis pathway, five cinnamyl alcohol dehydrogenase (CAD) genes were generally induced in melon seedlings by drought. Here we further revealed the roles of CmCAD1, 2, and 3 in lignin synthesis and for drought tolerance. Results found that overexpressing CmCAD2 or 3 strongly recovered CAD activities, lignin synthesis and composition in Arabidopsis cadc cadd, whose lignin synthesis is disrupted, while CmCAD1 functioned modestly. In melon seedlings, silenced CmCAD2 and 3 individually or collectively decreased CAD activities and lignin depositions drastically, resulting in dwarfed phenotypes. Reduced lignin, mainly composed by guaiacyl units catalyzed by CmCAD3, is mainly due to the limited lignification in tracheary elements and development of Casparion strip. While CmCAD1 and 2 exhibited catalysis to p-coumaraldehyde and sinapaldehyde, respectively. Compared with CmCAD1, drought treatments revealed higher sensitivity of CmCAD2 and/or 3 silenced melon seedlings, accompanying with lower relative water contents, water potentials and relatively higher total soluble sugar contents. Slightly up-regulated expressions of aquaporin genes together with limited lignification might imply higher lateral water loss in stems of silenced lines. In Arabidopsis, CmCAD2 and 3 transgenic lines enhanced cadc cadd drought tolerance through recovering lignin synthesis and root development, accompanying with decreased electrolyte leakage ratios and increased RWCs, thus improved survival rates. Briefly, lignin synthesized by CmCAD2 and 3 functions importantly for drought tolerance in melon.

4-Chloro-2,6-pyridinedicarboxylic acid (CPDA) functionalized mesoporous silica SBA-15 nanocomposites (SBA/5CPDA) were synthesized and characterized for the Knoevenagel condensation of syringaldehyde and acetaldehyde to synthesize sinapaldehyde in the liquid phase. The physicochemical characterization of the SBA/CPDA nanocomposites showed the decrease in the surface properties (surface area, pore size, pore volume) compared to SBA-15 confirming the complete functionalization of CPDA inside the mesopores of SBA-15 materials. The catalytic activity results displayed high activity (~90%) and selectivity (90–95%) of the desired product (sinapaldehyde) under environmental friendly conditions. All the reaction parameters were optimized to accomplish the maximum conversion of the selective products. Finally, the mechanism of the formation of the products was proposed and discussed.

Alternative technologies have been extensively investigated towards a more sustainable ageing of red wine, but few studies were made on wine spirits. Moreover, the effect of micro-oxygenation on wine spirits’ ageing is still unknown. This work examined the effect of micro-oxygenation and wood staves applied in 1000 L stainless steel tanks versus 250 L wooden barrels on the phenolic composition and chromatic characteristics acquired by the wine spirit over the first six months of ageing. Two different kinds of wood, Limousin oak and chestnut, were used. Significantly higher total phenolic content and individual contents of low molecular weight phenolic compounds (syringic acid, ellagic acid, vanillin, syringaldehyde, coniferaldehyde, sinapaldehyde and umbelliferone) were obtained with micro-oxygenation and staves than with barrels, for both kinds of wood. Accordingly, greater colour evolution (lower lightness, higher saturation and higher intensities of red, yellow and brown hues) was achieved in the former. These results were ascribed to a faster extraction and its balance with several phenomena involving the wood-derived compounds in the specific environment created by the direct supply of oxygen. A synergistic effect between the alternative technology and the chestnut wood was also observed.

The metabolism of phenolic compounds is a key factor in the development of wound-induced enzymatic browning of fresh-cut lettuce. In the present study, the lettuce midribs discriminant metabolites, selected in a previous untargeted metabolomics study, were thoroughly identified. Our results showed that their basal contents correlated with browning developed after 5 days of storage. 5- trans-Chlorogenic acid and 5- cis-chlorogenic acid were positively correlated with browning, while sinapaldehyde and its 4-β-d-glucoside and 4-(6'-malonyl)-β-d-glucoside conjugates were negatively correlated. Using targeted metabolomics, the metabolites were analyzed in lettuce heads with different degrees of development and different browning susceptibility and these biomarkers were confirmed. Despite the large variability in the browning process of lettuce, the chlorogenic acids/sinapaldehyde derivatives ratio showed a linear correlation ( r2 = 0.79) with the fresh-cut lettuce browning developed in 24 Romaine lettuce cultivars, validating the relevance of these biomarkers. These results show that the analysis of the basal content of these metabolites could be used in lettuce breeding programs to select cultivars that are more appropriate for the fresh-cut industry.

European regulations do not allow the addition of flavouring agents, including vanillin, and permit the caramel to be added quantum satis (as a means to adapt colour) to the wine spirit. Therefore, monitoring the authenticity and the caramel content of this beverage in order to prevent unfair competition between producers and to protect the consumer is imperative. This work presents the use of multivariate analysis and control charts, based on the ratios between furanic aldehydes and between phenolic aldehydes, as a simple, rapid, and reliable tool to discriminate aged wine spirits to which vanillin and/or high amount of caramel was added. A representative set of samples, consisting of 288 authentic aged wine spirits collected from trials and 11 commercial brandies, was used to classify 34 commercial aged wine spirits. Despite the high variability inherent to these authentic aged drinks induced by the kind of wood and toasting level, the Factor Analysis performed allowed to firstly separated the authentic samples from the commercial ones, and discriminate the unknown samples of commercial aged wine spirits as authentic or suspect to have been added of vanillin and/or high amount of caramel. The upper control limit (UCL) of the individuals control charts obtained from trials’ data - 0.621 for HMF/furfural ratio; 1.165 for vanillin/syringaldehyde ratio; 1.379 for vanillin/coniferaldehyde ratio; 1.438 for vanillin/sinapaldehyde ratio; 0.416 for vanillin/syringaldehyde + coniferaldehyde + sinapaldehyde ratio – proved to be of paramount importance in refining the analysis and identifying the samples with added vanillin (non-authentic) and/or containing high amount of caramel.

Aging cachaça in wooden barrels is essential to improve its quality. The level of maturation of distillates can be determined based on the contents of aging-marker phenolic compounds extracted from the lignin of the wooden barrel. This study aimed to characterize the aging process of cachaça by analyzing the mechanism of lignin degradation during its maturation in new barrels made from two oak species, European (Quercus petraea) and American (Quercus alba), for up to 60 months. Evaluation was based on the analyses of cinnamic aldehydes (sinapaldehyde and coniferaldehyde), benzoic aldehydes (syringaldehyde and vanillin), and benzoic acids (syringic and vanillic acids) using high-performance liquid chromatography. Oak species had a significant effect on all the studied phenolic compounds. Higher contents of all the identified phenolic compounds were found in cachaça aged in barrels made from American oak. The total contents of benzoic acids (vanillic and syringic acids) can be considered for predicting the level of maturation of cachaça aged in barrels made from both oak species. Based on the composition of maturation-related congeners, it is likely that for cachaça each year of aging in new oak barrels corresponds to approximately 5 years of aging for spirits in general commercialized worldwide., Food analysis, Food technology, Sugarcane spirit, Aging, Oak species, Lignin, Phenolic compounds

Myrcia splendens is popularly known as “guamirim-de-folha-miúda”, and its occurrence ranges from Mexico to southern Brazil. The aim of this work was to identify and quantify phenolic compounds in the crude hydroalcoholic (EBH), ethyl acetate (EBAE) and dichloromethane (EBDM) extracts using the HPLC-ESI- MS/MS. In total, 15 compounds, including protocatecuic acid, syringic acid, p-coumaric acid, salicylic acid, isoquercetin, ellagic acid, ferulic acid, umbelliferone, coniferaldehyde, sinapaldehyde, carnosol, gallic acid, syringaldehyde, umbelliferone, coniferaldehyde, myricetin and kaempferol were identified. Ellagic acid was the major compound in all extracts. Resumo Myrcia splendens é conhecida popularmente por “guamirim-de-folha-miúda”, e sua ocorrência vai desde o México até o sul do Brasil. Poucos estudos sobre sua composição química existem na literatura, sendo assim, este trabalho teve por objetivo identificar e quantificar substâncias fenólicas nos extratos brutos hidroalcoólico (EBH), acetato de etila (EBAE) e diclorometano (EBDM) através da técnica de HPLC-ESI-MS/MS. Ao total, 15 substâncias foram identificadas, incluindo ácido protocatecuico, ácido siríngico, ácido p-cumárico, ácido salicílico, isoquercetina, ácido elágico, ácido ferúlico, umbeliferona, coniferaldeído, sinapaldeído, carnosol, ácido gálico, siringaldeído, coniferaldeído, miricetina e kaempferol. O ácido elágico foi a substância majoritária em todos os extratos.

Alternative technologies for a more sustainable wine spirits&rsquo, ageing have been studied but a lack of knowledge on the effect of oxygenation level remains. This work examined the behaviour of low molecular weight compounds, iron and copper of a wine spirit aged in 50 L demijohns with chestnut wood staves combined with three levels of micro-oxygenation or nitrogen. Compounds and mineral elements were quantified by HPLC and FAAS, respectively, in samples collected at 8, 21, 60, 180, 270 and 365 days of ageing. Results showed that most of the compounds underwent significant changes in their content over time and behave differently depending on the wine spirit&rsquo, s oxygenation level: higher contents of gallic acid, syringic acid and vanillin were associated with lower micro-oxygenation level while higher contents of ellagic acid, syringaldehyde, coniferaldehyde and sinapaldehyde resulted from higher one, lowest contents of these compounds were found in the nitrogen modality. Weak correlation between copper and the studied compounds was evidenced whereas closer relationship between iron, vanillin, gallic, syringic and ellagic acids at end of ageing was observed. This study provides innovative information on the role of oxygen in wine spirit&rsquo, s ageing, and on chestnut wood effect on wine spirit&rsquo, s mineral composition.

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.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and p-coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum (Sorghum bicolor), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis (Arabidopsis thaliana) CAD5 and Populus tremuloides sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde > p-coumaraldehyde > sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in bmr6 mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in bmr6 plants could produce a phenotype that is more amenable to biomass processing.

Aromatic aldehydes and acids derived from lignin were studied by capillary zone electrophoresis in barrel matured wine distillates used in brandy production. Different conditions of analysis were applied to set up the best separation and reproducibility. The optimal separation was performed using 25 mM borate buffer containing 8% v/v ethanol as modifier. Total amount of guaiacol-type compounds (coniferaldehyde, vanillin, and vanillic acid) and syringol-type compounds (sinapaldehyde, syringaldehyde, and syringic acid) increased with increasing ageing time for distillates aged from 1 to 25 years. Sum of concentrations of syringic and vanillic acids can be also used for characterizing aged distillates according to their ageing level. The developed analysis method is simple, precise and cost efficient. It provides good separation efficiency, and it is ideally suited for the analysis of aged wine distillates for determination of their overall quality, approximate ageing time and signs of counterfeiting.

Green-colored fiber (GCF) is the unique raw material for naturally colored cotton textile but we know little about the pigmentation process in GCF. Here we compared transcriptomes and metabolomes of 12, 18 and 24 days post-anthesis (DPA) fibers from a green fiber cotton accession and its white-colored fiber (WCF) near-isogenic line. We found a total of 2047 non-redundant metabolites in GCF and WCF that were enriched in 80 pathways, including those of biosynthesis of phenylpropanoid, cutin, suberin, and wax. Most metabolites, particularly sinapaldehyde, of the phenylpropanoid pathway had a higher level in GCF than in WCF, consistent with the significant up-regulation of the genes responsible for biosynthesis of those metabolites. Weighted gene co-expression network analysis (WGCNA) of genes differentially expressed between GCF and WCF was used to uncover gene-modules co-expressed or associated with the accumulation of green pigments. Of the 16 gene-modules co-expressed with fiber color or time points, the blue module associated with G24 (i.e., GCF at 24 DPA) was of particular importance because a large proportion of its genes were significantly up-regulated at 24 DPA when fiber color was visually distinguishable between GCF and WCF. A total of 56 hub genes, including the two homoeologous Gh4CL4 that could act in green pigment biosynthesis, were identified among the genes of the blue module that are mainly involved in lipid metabolism, phenylpropanoid biosynthesis, RNA transcription, signaling, and transport. Our results provide novel insights into the mechanisms underlying pigmentation of green fibers and clues for developing cottons with stable green colored fibers.

Most of the known 4-coumarate:coenzyme A ligase (4CL) isoforms lack CoA-ligation activity for sinapic acid. Therefore, there is some doubt as to whether sinapic acid contributes to sinapyl alcohol biosynthesis. In this study, we characterized the enzyme activity of a protein mixture extracted from the developing xylem of Robinia pseudoacacia. The crude protein mixture contained at least two 4CLs with sinapic acid 4-CoA ligation activity. The crude enzyme preparation displayed negligible sinapaldehyde dehydrogenase activity, but showed ferulic acid 5-hydroxylation activity and 5-hydroxyferulic acid O-methyltransferase activity; these activities were retained in the presence of competitive substrates (coniferaldehyde and 5-hydroxyconiferaldehyde, respectively). 5-Hydroxyferulic acid and sinapic acid accumulated in the developing xylem of R. pseudoacacia, suggesting, in part at least, sinapic acid is a sinapyl alcohol precursor in this species.

We evaluated the anti-inflammatory effects of SNAH in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages by performing nitric oxide (NO) assays, cytokine enzyme-linked immunosorbent assays, Western blotting, and real-time reverse transcription-polymerase chain reaction analysis. SNAH inhibited the production of NO (nitric oxide), reactive oxygen species (ROS), tumor necrosis factor (TNF)-&alpha, and interleukin (IL)-6. Additionally, 100 &mu, M SNAH significantly inhibited total NO and ROS inhibitory activity by 93% (p &lt, 0.001) and 34% (p &lt, 0.05), respectively. Protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) stimulated by LPS were also decreased by SNAH. Moreover, SNAH significantly (p &lt, 0.001) downregulated the TNF-&alpha, IL-6, and iNOS mRNA expression upon LPS stimulation. In addition, 3&ndash, 100 &micro, M SNAH was not cytotoxic. Docking simulations and enzyme inhibitory assays with COX-2 revealed binding scores of &minus, 6.4 kcal/mol (IC50 = 47.8 &mu, M) with SNAH compared to &minus, 11.1 kcal/mol (IC50 = 0.45 &mu, M) with celecoxib, a known selective COX-2 inhibitor. Our results demonstrate that SNAH exerts anti-inflammatory effects via suppression of ROS and NO by COX-2 inhibition. Thus, SNAH may be useful as a pharmacological agent for treating inflammation-related diseases.

To investigate the effect of pollination on the fruit quality of &lsquo, Dangshan Su&rsquo, pear, &lsquo, was fertilized by the pollen of &lsquo, Wonhwang&rsquo, (Pyrus pyrifolia Nakai.) (DW) and &lsquo, Jingbaili&rsquo, (Pyrus ussuriensis Maxim.) (DJ). The analysis of primary metabolites was achieved through untargeted metabolomics, and the quantitative analysis of intermediate metabolites of lignin synthesis was undertaken using targeted metabolomics. The untargeted metabolomics analysis was performed via gas chromatography-mass spectrometry (GC-MS). The targeted metabolomics analysis was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) under the multiple reaction monitoring (MRM) mode. The results showed that the metabolite content was significantly different between DW and DJ. Compared with that in DJ, the sugar and amino acid content in DW was higher and the fatty acid content was lower at 47 days after pollination (DAPs), and the sugar, amino acid, and fatty acid content in DW was lower at 63 DAPs. The intermediate metabolites of lignin synthesis were analyzed using the orthogonal partial least squares discriminant analysis (OPLS-DA) model, and the differential metabolites at 47 DAPs were p-coumaric acid, ferulic acid, sinapaldehyde, coniferyl alcohol, and sinapyl alcohol. The differential significant metabolite at 63 DAPs was p-coumaric acid. At 47 DAPs and 63 DAPs, the p-coumaric acid level was significantly different, and the p-coumaric acid content was positively correlated with lignin synthesis. The pollination pollen affects the quality of &lsquo, pear fruit through regulation of the sugar, amino acid, and fatty acid content, at the same time, regulating the levels of intermediate metabolites of lignin synthesis, especially the p-coumaric acid content, to affect lignin synthesis ultimately affects the stone cell content and improves the quality of the pears.

Lignin, the planet's most abundant renewable source of aromatic compounds, is difficult to degrade efficiently to welldefined aromatics. We developed a microwave-assisted catalytic Swern oxidation system using an easily prepared catalyst, MoO2 Cl2 (DMSO)2 , and DMSO as the solvent and oxidant. It demonstrated high efficiency in transforming lignin model compounds containing the units and functional groups found in native lignins. The aromatic ring substituents strongly influenced the selectivity of β-ether phenolic dimer cleavage to generate sinapaldehyde and coniferaldehyde, monomers not usually produced by oxidative methods. Time-course studies on two key intermediates provided insight into the reaction pathway. Owing to the broad scope of this oxidation system and the insight gleaned with regard to its mechanism, this strategy could be adapted and applied in a general sense to the production of useful aromatic chemicals from phenolics and lignin.

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content.

Cachaca is the most important distilled spirit in Brazil and the fourth most consumed in the world. Aging is not mandatory for cachaca. When aging is carried out, casks generally do not undergo the final wood toasting. However the level of thermal degradation of wood influences the formation of new chemical compounds, which are released during the aging process and improve the quality of the spirit. The objective of this study was to verify the influence of origin of wood and toasting intensity of oak chips on the profile of aging markers in cachaca. Staves of oak wood from the forests of Allier, Vosges and Nievre (France) were milled and screened at 9 mesh. The chips underwent light, medium and heavy toasting. The toasted chips were added to cachaca (1 g/L) and kept in a closed system at room temperature, without agitation, for 15 days. The aging congeners (gallic acid, 5-hydroxymethylfurfural, furfural, vanillin, vanillic acid, syringaldehyde, sinapaldehyde, syringic acid and coniferaldehyde) were analysed using HPLC. The increase in toasting intensity enhanced the content of the aging markers in cachaca, especially syringic acid, vanillin, syringaldehyde, coniferaldehyde and sinapaldehyde. The generation of aging marker compounds was influenced only by the toasting intensity and not by the origin of the oak wood. Copyright © 2015 The Institute of Brewing & Distilling

Summary Lignin confers recalcitrance to plant biomass used as feedstocks in agro‐processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3‐dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3‐dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild‐type plants, lines expressing QsuB contain higher amounts of protocatechuate, p‐coumarate, p‐coumaraldehyde and p‐coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D‐NMR spectroscopy and pyrolysis‐gas chromatography/mass spectrometry (pyro‐GC/MS) reveal an increase of p‐hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size‐exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain‐of‐function strategy for spatiotemporal reduction of lignin in plant biomass.

Despite the reported cytotoxic activity of the endocarp of Cocos nucifera (Arecaceae), the active principles were never characterized. The total extract (T), methylene chloride (M) and ethyl acetate (E) fractions were investigated for selective cytotoxic activity towards prostate cancer cell line (PC3) compared to normal cells (WI38), T showed remarkable activity (IC50=10.89 ± 2.1 μg/ml), M and E showed comparable moderate activity. All showed significantly higher IC50 values towards WI38 with selectivity index (SI) values of >18.3, >11.8 and 6.6, respectively. The phytochemical investigation of M has led to isolation of coniferaldehyde (1), sinapaldehyde (2), p-hydroxy benzoic acid (3), protocatechuic acid (4), vanillic acid (5), protocatechuic aldehyde (6), balanophonin (7), guaiacylglycerol-β-coniferyl aldehyde ether (8), E-piceatannol (9), kompasinol A (10) and apigenin (11). The cytotoxic and antioxidant potential of the isolates was investigated. Compounds 1, 4, 7–9 and 11 showed selective cytotoxic activity against PC3. Compounds 6, 9 and 10 showed remarkable antioxidant activity. . These results added a medicinal value for the coconut endocarp, instead of being as a wasted resource; it could be renewable inexpensive source of potential leads for selective cytotoxic drugs.

This study investigates the effect of ageing on the qualitative and quantitative composition of plum distillate in contact with oak wood chips. Maturation was performed with lightly toasted French oak (Quercus sessiflora and Quercus robur) chips or oak chips made from fragments of empty barrels that had been used for ageing cognac. The effects of oak chip dose, process temperature, ageing system (static or circulatory) and ultrasound treatment were assessed. Maturation of plum distillate samples with oak chips resulted in higher levels of extractable organics (including tannins) and colour changes, which were correlated with the type and dose of oak chips, and the conditions of maturation. The content of sugars such as glucose, xylose and arabinose also increased, depending on the conditions and type of oak chips. Degradation of lignin resulted in liberation of sinapaldehyde, syringaldehyde, coniferaldehyde and vanillin, with intensities depending on the applied parameters. In terms of volatiles, decreases in the concentration of higher alcohols and aliphatic aldehydes were observed in the majority of maturation experiments, while concentrations of furanic aldehydes increased depending on the type and dose of oak chips, as well as on the conditions of maturation. The quantities of esters such as ethyl acetate decreased in the majority of experimental variants, whereas concentrations of ethyl caproate, ethyl caprylate and ethyl caprate increased gradually. Some phenols and lactones were detected in all matured samples, with the lowest levels found in the samples aged with oak chips made from cognac barrels.

article en open access; International audience; In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar (Populus tremula 3 Populus alba) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1) by a hairpin-RNA-mediated silencing approach, which resulted in only 5% residual CAD1 transcript abundance. These transgenic lines showed no biomass penalty despite a 10% reduction in Klason lignin content and severe shifts in lignin composition. Nuclear magnetic resonance spectroscopy and thioacidolysis revealed a strong increase (up to 20-fold) in sinapaldehyde incorporation into lignin, whereas coniferaldehyde was not increased markedly. Accordingly, ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals. However, no additional cinnamaldehyde coupling products could be detected in the CAD1-deficient poplars. Instead, the transgenic lines accumulated a range of hydroxycinnamate-derived metabolites, of which the most prominent accumulation (over 8,500-fold) was observed for a compound that was identified by purification and nuclear magnetic resonance as syringyl lactic acid hexoside. Our data suggest that, upon down-regulation of CAD1, coniferaldehyde is converted into ferulic acid and derivatives, whereas sinapaldehyde is either oxidatively coupled into S'(8-8) S' and lignin or converted to sinapic acid and derivatives. The most prominent sink of the increased flux to hydroxycinnamates is syringyl lactic acid hexoside. Furthermore, low-extent saccharification assays, under different pretreatment conditions, showed strongly increased glucose (up to +81%) and xylose (up to +153%) release, suggesting that down-regulating CAD1 is a promising strategy for improving lignocellulosic biomass for the sugar platform industry.

A new coruleoellagic acid derivative, 3,3′,4,4′,5′-pentamethylcoruleoellagic acid (1) together with nine known compounds, hexamethylcoruleoellagic acid (2), 3,4,3′-tri-O-methylellagic acid (3), heptaphylline (4), 7-methoxymukonal (5), dentatin (6), sinapaldehyde (7), gallic acid (8), 2,6-dimethoxy-4H-pyran-4-one (9) and β-sitosterol (10) were isolated from the stems of Rhodamnia dumetorum. Their structures were identified by physical and spectroscopic data (IR, 1D and 2D NMR, and MS). Compounds 1, 2 and 7-10 were tested for antibacterial activity against six pathogenic bacterial strains (Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Methicillin resistant S. aureus (MRSA)).

Equimolar reactions of cinnamaldehyde or its 3,5-dimethoxy-4-hydroxy derivative (sinapaldehyde) with RP(CH2OH)2 (R = Ph or CH2OH) were studied in MeOH or CD3OD at room temperature by NMR spectroscopy. In MeOH, nucleophilic attack of the phosphine at the CC bond, with concomitant loss of CH2O, affords the tertiary phosphine HOCH2P(R)CH(Ar)CH2CHO (3) that rapidly converts mainly into a 1,3-oxaphosphorinane derivative (5) formed as a mixture of four diastereomers. Conformational analysis reveals that the Ar group in these is exclusively in an equatorial position while the OH and R groups can be equatorial-oriented or axial-oriented. In CD3OD, 1,3-oxaphosphorinanes monodeuterated in the C5 position are obtained as a mixture of eight diastereomers where the dominate diastereomers have an axial D-atom. Diastereomeric ratios depend on the nature of the Ar and R groups.

Nine CAD/CAD-like genes in P. tomentosa were classified into four classes based on expression patterns, phylogenetic analysis and biochemical properties with modification for the previous claim of SAD. Cinnamyl alcohol dehydrogenase (CAD) functions in monolignol biosynthesis and plays a critical role in wood development and defense. In this study, we isolated and cloned nine CAD/CAD-like genes in the Populus tomentosa genome. We investigated differential expression using microarray chips and found that PtoCAD1 was highly expressed in bud, root and vascular tissues (xylem and phloem) with the greatest expression in the root. Differential expression in tissues was demonstrated for PtoCAD3, PtoCAD6 and PtoCAD9. Biochemical analysis of purified PtoCADs in vitro indicated PtoCAD1, PtoCAD2 and PtoCAD8 had detectable activity against both coniferaldehyde and sinapaldehyde. PtoCAD1 used both substrates with high efficiency. PtoCAD2 showed no specific requirement for sinapaldehyde in spite of its high identity with so-called PtrSAD (sinapyl alcohol dehydrogenase). In addition, the enzymatic activity of PtoCAD1 and PtoCAD2 was affected by temperature. We classified these nine CAD/CAD-like genes into four classes: class I included PtoCAD1, which was a bone fide CAD with the highest activity; class II included PtoCAD2, -5, -7, -8, which might function in monolignol biosynthesis and defense; class III genes included PtoCAD3, -6, -9, which have a distinct expression pattern; class IV included PtoCAD12, which has a distinct structure. These data suggest divergence of the PtoCADs and its homologs, related to their functions. We propose genes in class II are a subset of CAD genes that evolved before angiosperms appeared. These results suggest CAD/CAD-like genes in classes I and II play a role in monolignol biosynthesis and contribute to our knowledge of lignin biosynthesis in P. tomentosa.

Semi-preparative and preparative high-speed counter-current chromatography (HSCCC) were successfully used for isolation of five glycosides from 50% ethanol extract of the dried barks of Ilex rotunda Thunb. (Aquifoliaceae) by using a two-phase solvent system composed of ethyl acetate-n-butanol-water (1:6:7, v/v/v). From 1.0 g of the extract, syringaresinol 4′,4″-di-O-β-D-glucopyranoside ( I , 20.2 mg, 55.4% purity), syringin ( II , 56.8 mg, 98.1% purity), sinapaldehyde glucoside ( III , 26.2 mg, 68.6% purity), syringaresinol 4′-O-β-D-glucopyranoside ( IV , 20.4 mg, 95.3% purity), and pedunculoside ( V , 45.1 mg, 97.3% purity) were obtained by one run of TBE-1000A HSCCC instrument with 1000 mL of column volume. Glycosides I (9.8 mg, 95.5% purity) and III (14.2 mg, 97.8% purity) were further purified by preparative high performance liquid chromatography (HPLC). Their structures were identified by IR, MS, and 1H and 13C NMR studies. Glycoside I was isolated from this plant for the first time.

Beetroot (Beta vulgaris L.) residues, leaves and stems, normally disposed as animal feed or compost, were investigated for the recovery of valuable compounds by pressurized liquid extraction (PLE) at temperature of 40 °C and pressures of 7.5, 10 and 12.5 MPa, and flow rate of 3 mL min-1. This abundant residue, combined with the lack of related studies of beetroot waste by applying the PLE methods to obtain bioactive extracts is the main novelty of this work. Then, the objective was to explore the PLE process to enhance the value of the residues, evaluating process yield, total phenolic content (TPC) and antioxidant activity (DPPH, ABTS and FRAP methods) of the recovered extracts. Chemical composition was analyzed using LC-ESI-MS/MS and GC-MS analysis. Antioxidant potential results suggest the recovered extracts are comparable with synthetic antioxidant (BHT). Ferulic acid, vitexin and sinapaldehyde, were the most abundant phenolic compounds obtained from the extracts. These results indicate that PLE technique can be a good alternative for extracting phenolic compounds of beetroot resides with high biological potential that could be used in formulations for nutraceuticals, functional food or pharmaceutical industry.

Jiubiying, the dried barks of Ilex rotunda Thunb. (Aquifoliaceae), has been used as herbal tea and traditional Chinese medicine for heat-clearing, detoxifying, dehumidification, and odynolysis. Pedunculoside and syringin are two main bioactive components. For the new drug development, we tried to isolate and purify several chemical constituents from Jiubiying by high-speed counter-current chromatography (HSCCC). The two-phase solvent system used was composed of ethyl acetate-n-butanol-water (1:6:7, v/v/v). From 1.0 g of Jiubiying extracts syringaresinol 4',4″-bis-O-β-D- glucopyranoside (I, 20.2 mg), syringin (II, 56.8 mg), sinapaldehyde glucoside (III, 26.2 mg), syringaresinol 4'-O-β-D-glucopyranoside (IV, 20.4 mg), and pedunculoside (V, 45.1 mg) were obtained by one run of TBE-1000A HSCCC machine with 1000 mL of column volume. Their structures were identified by IR, MS, and extensive NMR studies. Syringaresinol 4',4″-bis-O-β-D-glucopyranoside (I) was isolated from this plant for the first time.

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic ( Medicago truncatula ) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1 . NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure.

Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H.

The organized lignocellulosic assemblies of cell walls provide the structural integrity required for the large statures of terrestrial plants. Silencing two CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes in Nicotiana attenuata produced plants (ir-CAD) with thin, red-pigmented stems, low CAD and sinapyl alcohol dehydrogenase activity, low lignin contents, and rubbery, structurally unstable stems when grown in the glasshouse (GH). However, when planted into their native desert habitat, ir-CAD plants produced robust stems that survived wind storms as well as the wild-type plants. Despite efficient silencing of NaCAD transcripts and enzymatic activity, field-grown ir-CAD plants had delayed and restricted spread of red stem pigmentation, a color change reflecting blocked lignification by CAD silencing, and attained wild-type-comparable total lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-CAD plants were protected from wind, herbivore attack, and ultraviolet B exposure and grown in restricted rooting volumes; conversely, it was lost when ir-CAD plants were experimentally exposed to wind, ultraviolet B, and grown in large pots in growth chambers. Transcript and liquid chromatography-electrospray ionization-time-of-flight analysis revealed that these environmental stresses enhanced the accumulation of various phenylpropanoids in stems of field-grown plants; gas chromatography-mass spectrometry and nuclear magnetic resonance analysis revealed that the lignin of field-grown ir-CAD plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems with normal xylem element traits, which collectively enabled field-grown ir-CAD plants to compensate for the structural deficiencies associated with CAD silencing. Environmental stresses play an essential role in regulating lignin biosynthesis in lignin-deficient plants.

A 1:1 reaction of [HO(CH2)3]3P with 4-hydroxy-3-methoxy-cinnamaldehyde (coniferaldehyde) or 3,5-dimethoxy-4-hydroxycinnamaldehyde (sinapaldehyde) in acetone at room temperature affords phosphonium zwitterions of the type R3P+CH(4-O−-Ar)CH2CHO; other phosphines [R = Et, n-Bu, (CH2)2CN, and p-Tol] do not react under the same conditions. In alcohols R’OH(D) [R’ = CD3, Et, (CD3)2CD, s-Bu, HOCH2CH2], the above phosphines (except the cyano-derivative) and those where R = i-Pr, Cy, Me2Ph, MePh2 do react within an equilibrium established between the reactants and the zwitterion-hemiacetal products R3P+CH(4-O−-Ar)CH2CH(OH)(OR′) that are formed as a mixture of two diastereomers. The nature of the phosphine and the alcohol affects the equilibrium and the diastereomeric ratio.

The synthesis of deoxyanthocyanidins from the reaction between cinnamic aldehydes (coniferaldehyde and sinapaldehyde) and phloroglucinol is reported herein. The resulting structures were characterized by visible, MS, and NMR spectroscopy.

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD.