Lignans: Advances in Biosynthesis, Bioavailability, and Pharmacological Activity.

Lignans, a group of naturally occurring compounds abundant in various plant-based foods, are becoming increasingly popular due to their potential health benefits. The literature suggests that these bioactive substances can reduce the risk of certain types of cancer, such as postmenopausal colon and breast cancer. Moreover, the significance of lignans for improving cardiovascular health has been recognized, as studies have revealed a potential correlation between the intake of lignans and a decreased risk of cardiovascular disease. These complex molecules possess diverse bioactive capabilities, rendering them potential alternatives for preventing chronic diseases. Further research is needed to examine the mechanisms responsible for their beneficial outcomes. Recent research has emphasized the pharmacological properties of lignans as effective substances for human health. Incorporating foods rich in lignans into the diet may be a practical approach to enhancing protection against life-threatening ailments, such as cardiovascular diseases and malignancies.

Phylogeny and functional characterization of the cinnamyl alcohol dehydrogenase gene family in Phryma leptostachya.

Phryma leptostachya has attracted increasing attention because it is rich in furofuran lignans with a wide range of biological activities. Biosynthesis of furofuran lignans begins with the dimerization of coniferyl alcohol, one of the monolignol. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step of monolignol biosynthesis, reducing cinnamyl aldehydes to cinnamyl alcohol. As it is in the terminal position of monolignol biosynthesis, its type and activity can cause significant changes in the total amount and composition of lignans. Herein, combined with bioinformatics analysis and in vitro enzyme assays, we clarified that CAD in P. leptostachya belonged to a multigene family, and identified nearly the entire CAD gene family. Our in-depth characterization about the functions and structures of two major CAD isoforms, PlCAD2 and PlCAD3, showed that PlCAD2 exhibited the highest catalytic activity, and coniferyl aldehyde was its preferred substrate, followed by PlCAD3, and sinapyl aldehyde was its preferred substrate. Considering the accumulation patterns of furofuran lignans and expression patterns of PlCADs, we speculated that PlCAD2 was the predominant CAD isoform responsible for furofuran lignans biosynthesis in P. leptostachya. Moreover, these CADs found here can also provide effective biological parts for lignans and lignins biosynthesis.

Identification, Molecular Cloning, and Functional Characterization of a Coniferyl Alcohol Acyltransferase Involved in the Biosynthesis of Dibenzocyclooctadiene Lignans in Schisandra chinensis.

Schisandra chinensis owes its therapeutic efficacy to the dibenzocyclooctadiene lignans, which are limited to the Schisandraceae family and whose biosynthetic pathway has not been elucidated. Coniferyl alcohol is the synthetic precursor of various types of lignans and can be acetylated to form coniferyl acetate by coniferyl alcohol acyltransferase (CFAT), which belongs to the BAHD acyltransferase family. This catalytic reaction is important because it is the first committed step of the hypothetical biosynthetic pathway in which coniferyl alcohol gives rise to dibenzocyclooctadiene lignans. However, the gene encoding CFAT in S. chinensis has not been identified. In this study, firstly we identified 37 ScBAHD genes from the transcriptome datasets of S. chinensis. According to bioinformatics, phylogenetic, and expression profile analyses, 1 BAHD gene, named ScBAHD1, was cloned from S. chinensis. The heterologous expression in Escherichia coli and in vitro activity assays revealed that the recombinant enzyme of ScBAHD1 exhibits acetyltransferase activity with coniferyl alcohol and some other alcohol substrates by using acetyl-CoA as the acetyl donor, which indicates ScBAHD1 functions as ScCFAT. Subcellular localization analysis showed that ScCFAT is mainly located in the cytoplasm. In addition, we generated a three-dimensional (3D) structure of ScCFAT by homology modeling and explored the conformational interaction between protein and ligands by molecular docking simulations. Overall, this study identified the first enzyme with catalytic activity from the Schisandraceae family and laid foundations for future investigations to complete the biosynthetic pathway of dibenzocyclooctadiene lignans.

Dual Mechanisms of Coniferyl Alcohol in Phenylpropanoid Pathway Regulation.

Lignin is a complex phenolic polymer that imparts cell wall strength, facilitates water transport and functions as a physical barrier to pathogens in all vascular plants. Lignin biosynthesis is a carbon-consuming, non-reversible process, which requires tight regulation. Here, we report that a major monomer unit of the lignin polymer can function as a signal molecule to trigger proteolysis of the enzyme L-phenylalanine ammonia-lyase, the entry point into the lignin biosynthetic pathway, and feedback regulate the expression levels of lignin biosynthetic genes. These findings highlight the highly complex regulation of lignin biosynthesis and shed light on the biological importance of monolignols as signaling molecules.

Density functional theory study on the coupling and reactions of diferuloylputrescine as a lignin monomer.

Diferuloylputrescine has been found in a variety of plant species, and recent work has provided evidence of its covalent bonding into lignin. Results from nuclear magnetic resonance spectroscopy revealed the presence of bonding patterns consistent with homo-coupling of diferuloylputrescine and the possibility of cross-coupling with lignin. In the present work, density functional theory calculations have been applied to assess the energetics associated with radical coupling, rearomatization, and dehydrogenation for possible homo-coupled dimers of diferuloylputrescine and cross-coupled dimers of diferuloylputrescine and coniferyl alcohol. The values obtained for these reaction energetics are consistent with those reported for monolignols and other novel lignin monomers. As such, this study shows that there would be no thermodynamic impediment to the incorporation of diferuloylputrescine into the lignin polymer and its addition to the growing list of non-canonical lignin monomers.

Production of phenylpropanoids and flavonolignans from glycerol by metabolically engineered Escherichia coli.

Phenylpropanoids are a group of plant natural products with medicinal importance derived from aromatic amino acids. Here, we report the production of two representative phenylpropanoids-coniferyl alcohol (CA) and dihydroquercetin (DHQ)-from glycerol by engineered Escherichia coli. First, an E. coli strain capable of producing 187.7 mg/L of CA from glycerol was constructed by the introduction of hpaBC from E. coli and OMT1, 4CL4, and CCR1 from Arabidopsis thaliana to the p-coumaric acid producer. Next, an E. coli strain capable of producing 239.4 mg/L of DHQ from glycerol was constructed by the introduction of F3H, TT7, and CPR from A. thaliana to the naringenin producer, followed by engineering the signal peptide of a cytochrome P450 TT7. Furthermore, to demonstrate the production of flavonolignans, a group of heterodimeric phenylpropanoids, from glycerol, ascorbate peroxidase 1 from Silybum marianum was employed and engineered to produce 0.04 µg/L of silybin and 1.29 µg/L of isosilybin from glycerol by stepwise culture. Finally, a single strain harboring all the 16 necessary genes was constructed, resulting in 0.12 µg/L of isosilybin production directly from glycerol. The strategies described here will be useful for the production of pharmaceutically important yet complex natural products.

Assessment of attractancy and safeness of (E)-coniferyl alcohol for management of female adults of Oriental fruit fly, Bactrocera dorsalis (Hendel).

BACKGROUND: Bactrocera dorsalis is a devastating pest on fruits and vegetables because the adult female is the key factor that determines the population density of offspring and the degree of host damage. Unfortunately, there is still a lack of effective female attractants for behavioral control. Males of B. dorsalis fed on methyl eugenol (ME) were shown to be more sexually attracted to females and, therefore, were more successful in mating over ME-deprived males. RESULTS: In the current study, we demonstrated that (E)-coniferyl alcohol (E-CF), one of the ME metabolites in males, was highly attractive to sexually-mature females in laboratory bioassays. During the dusk courtship period, mature females showed the highest response to E-CF. However, there were no significant differences in olfactory responses to E-CF between virgin and mated mature females. Moreover, no obvious signs and symptoms of toxicity or death were observed in mice during a 14-day acute oral toxicity test. Toxicologically, no significant changes were observed in body weight, water intake, food consumption and absolute and relative organ weights between control and treated groups of healthy-looking mice, implying that E-CF could be regarded as non-toxic. Furthermore, cytotoxicity assessment revealed that E-CF was non-toxic against human fetal lung fibroblast 1 (HFL1), human breast cancer (MDA-MB-231), mouse embryonic hepatocytes (BNL-CL.2) and Spodoptera frugiperda ovary (SF-9) cell lines. CONCLUSIONS: E-CF proved to be an effective, promising and eco-friendly lure to B. dorsalis females. Therefore, this study may facilitate the development of novel control strategies against B. dorsalis in the field.

Antifungal Activity against Botryosphaeriaceae Fungi of the Hydro-Methanolic Extract of Silybum marianum Capitula Conjugated with Stevioside.

Silybum marianum (L.) Gaertn, viz. milk thistle, has been the focus of research efforts in the past few years, albeit almost exclusively restricted to the medicinal properties of its fruits (achenes). Given that other milk thistle plant organs and tissues have been scarcely investigated for the presence of bioactive compounds, in this study, we present a phytochemical analysis of the extracts of S. marianum capitula during the flowering phenological stage (stage 67). Gas chromatography-mass spectroscopy results evidenced the presence of high contents of coniferyl alcohol (47.4%), and secondarily of ferulic acid ester, opening a new valorization strategy of this plant based on the former high-added-value component. Moreover, the application of the hydro-methanolic extracts as an antifungal agent has been also explored. Specifically, their activity against three fungal species responsible for the so-called Botryosphaeria dieback of grapevine (Neofusicoccum parvum, Dothiorella viticola and Diplodia seriata) has been assayed both in vitro and in vivo. From the mycelial growth inhibition assays, the best results (EC90 values of 303, 366, and 355 µg·mL-1 for N. parvum, D. viticola, and D. seriata, respectively) were not obtained for the hydroalcoholic extract alone, but after its conjugation with stevioside, which resulted in a strong synergistic behavior. Greenhouse experiments confirmed the efficacy of the conjugated complexes, pointing to the potential of the combination of milk thistle extracts with stevioside as a promising plant protection product in organic Viticulture.

Vanillic acid and methoxyhydroquinone production from guaiacyl units and related aromatic compounds using Aspergillus niger cell factories.

BACKGROUND: The aromatic compounds vanillin and vanillic acid are important fragrances used in the food, beverage, cosmetic and pharmaceutical industries. Currently, most aromatic compounds used in products are chemically synthesized, while only a small percentage is extracted from natural sources. The metabolism of vanillin and vanillic acid has been studied for decades in microorganisms and many studies have been conducted that showed that both can be produced from ferulic acid using bacteria. In contrast, the degradation of vanillin and vanillic acid by fungi is poorly studied and no genes involved in this metabolic pathway have been identified. In this study, we aimed to clarify this metabolic pathway in Aspergillus niger and identify the genes involved. RESULTS: Using whole-genome transcriptome data, four genes involved in vanillin and vanillic acid metabolism were identified. These include vanillin dehydrogenase (vdhA), vanillic acid hydroxylase (vhyA), and two genes encoding novel enzymes, which function as methoxyhydroquinone 1,2-dioxygenase (mhdA) and 4-oxo-monomethyl adipate esterase (omeA). Deletion of these genes in A. niger confirmed their role in aromatic metabolism and the enzymatic activities of these enzymes were verified. In addition, we demonstrated that mhdA and vhyA deletion mutants can be used as fungal cell factories for the accumulation of vanillic acid and methoxyhydroquinone from guaiacyl lignin units and related aromatic compounds. CONCLUSIONS: This study provides new insights into the fungal aromatic metabolic pathways involved in the degradation of guaiacyl units and related aromatic compounds. The identification of the involved genes unlocks new potential for engineering aromatic compound-producing fungal cell factories.

Metabolic Changes of Cholangiocarcinoma Cells in Response to Coniferyl Alcohol Treatment.

Cholangiocarcinoma (CCA) is a major cause of mortality in Northeast Thailand with about 14,000 deaths each year. There is an urgent necessity for novel drug discovery to increase effective treatment possibilities. A recent study reported that lignin derived from Scoparia dulcis can cause CCA cell inhibition. However, there is no evidence on the inhibitory effect of coniferyl alcohol (CA), which is recognized as a major monolignol-monomer forming a very complex structure of lignin. Therefore, we aimed to investigate the effect of CA on CCA cell apoptosis. We demonstrated that a half-inhibitory concentration of CA on KKU-100 cells at 48 h and 72 h was 361.87 ± 30.58 and 268.27 ± 18.61 µg/mL, respectively, and on KKU-213 cells 184.37 ± 11.15 and 151.03 ± 24.99 µg/mL, respectively. Furthermore, CA induced CCA cell apoptosis as demonstrated by annexin V/PI staining in correspondence with an increase in the BAX/Bcl-2 ratio. A metabonomic study indicated that CA significantly decreased the intracellular concentrations of glutathione and succinate in KKU-213 cells and increased dihydrogen acetone phosphate levels in KKU-100 cells treated with 200 µg/mL of CA compared to the control group. In conclusion, CA induced cellular metabolic changes which are involved in the antioxidant defense mechanism, glycerophospholipid metabolism and the tricarboxylic acid cycle. CA may serve as a potent anticancer agent for CCA treatment by inducing CCA cellular apoptosis.

Fraxinus mandshurica 4-coumarate-CoA ligase 2 enhances drought and osmotic stress tolerance of tobacco by increasing coniferyl alcohol content.

4-Coumarate-CoA ligase (4CL) is an important branch point in the phenylpropane pathway and plays important roles in plant growth and development. In this study, the 4CL2 gene from Fraxinus mandshurica (designated Fm4CL2) was identified and isolated. Sequence analysis revealed that Fm4CL2 is a type I 4CL gene involved in lignin biosynthesis. Analysis of cell wall components revealed that Fm4CL2-overexpressing (OE-Fm4CL2) tobacco showed increased lignin content (by 58.9%) and decreased hemicellulose content (by 41.2%). Detection of small-molecule metabolites in the lignin pathway revealed that coumaric acid content decreased by 48% and coniferyl alcohol content increased by 250% compared with the control values. Compared with wild type, OE-Fm4CL2 tobacco showed increased xylem cell layer number (by 120%) and cell wall thickness (by 54.5%). Under osmotic stress, transgenic tobacco showed higher growth than wild-type tobacco. The germination rate of transgenic tobacco was higher than that of wild type. Reactive oxygen species accumulation and malondialdehyde content were significantly lower in transgenic tobacco than in wild type. Under drought, the expression of stress-related genes was higher in 35S-Fm4CL2-infected Fraxinus mandshurica plants than in control plants. These results indicate that Fm4CL2 overexpression can enhance drought and osmotic stress tolerance of plants.

Subcritical Methanol Extraction of the Stone of Japanese Apricot Prunus mume Sieb. et Zucc.

The pits of Japanese apricot, Prunus mume Sieb. et Zucc., which are composed of stones, husks, kernels, and seeds, are unused by-products of the processing industry in Japan. The processing of Japanese apricot fruits generates huge amounts of waste pits, which are disposed of in landfills or, to a lesser extent, burned to form charcoal. Mume stones mainly consist of cellulose, hemicellulose, and lignin. Herein, we attempted to solubilize the wood-like carapace (stone) encasing the pit by subcritical fluid extraction with the aim of extracting useful chemicals. The characteristics of the main phenolic constituents were elucidated by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analyses. The degrees of solubility for various treatments (190 °C; 3 h) were determined as follows: subcritical water (54.9%), subcritical 50% methanol (65.5%), subcritical 90% methanol (37.6%), subcritical methanol (23.6%), and subcritical isopropyl alcohol (14.4%). Syringaldehyde, sinapyl alcohol, coniferyl alcohol methyl ether, sinapyl alcohol methyl ether, 5-(hydroxymethyl)-2-furfural, and furfural were present in the subcritical 90% methanol extract. Coniferyl and sinapyl alcohols (monolignols) are source materials for the biosynthesis of lignin, and syringaldehyde occur in trace amounts in wood. Our current findings provide a solubilization method that allows the main phenolic constituents of the pits to be extracted under mild conditions. This technique for obtaining subcritical extracts shows great potential for further applications.

Pulsed-ozonolysis assisted oxidative treatment of forestry biomass for lignin fractionation.

Lignocellulosic biomass has been used to produce biomolecules of industrial interest through thermochemical, biological, and chemical transformation. However, few works have been developed over lignin fractionation to obtain monolignols with commercial potentials, such as sinapyl, coniferyl, and p-coumaryl alcohols. This study is focused on developing a thermochemical method to delignify biomass. Additionally, an oxidative treatment with ozone was studied to increase the release of monolignol compounds. The results showed that with 30 sec of ozonation in liquid samples from softwood sawdust a total concentration of 368.50 ± 0.73 mg/kg of monolignols was released after microwave-assisted extraction (256.5 ± 0.51 mg/kg of sinapyl alcohol and 112 ± 0.22 mg/kg of coniferyl alcohol) and 629.20 ± 0.21 mg/kg was released after thermal treatment (453.70 ± 0.15 mg/kg of sinapyl alcohol and 175.5 ± 0.06 mg/kg of coniferyl alcohol). For p-coumaryl alcohol, 16.32 mg/kg was obtained only in hardwood samples. The results of the present study showed that ozonolysis improves monolignols release from forestry residues.

Characterization of a sweet basil acyltransferase involved in eugenol biosynthesis.

Sweet basil (Ocimum basilicum) plants produce its characteristic phenylpropene-rich essential oil in specialized structures known as peltate glandular trichomes (PGTs). Eugenol and chavicol are the major phenylpropenes produced by sweet basil varieties whose synthetic pathways are not fully elucidated. Eugenol is derived from coniferyl acetate by a reaction catalysed by eugenol synthase. An acyltransferase is proposed to convert coniferyl alcohol to coniferyl acetate which is the first committed step towards eugenol synthesis. Here, we perform a comparative next-generation transcriptome sequencing of different tissues of sweet basil, namely PGT, leaf, leaf stripped of PGTs (leaf-PGT), and roots, to identify differentially expressed transcripts specific to PGT. From these data, we identified a PGT-enriched BAHD acyltransferase gene ObCAAT1 and functionally characterized it. In vitro coupled reaction of ObCAAT1 with eugenol synthase in the presence of coniferyl alcohol resulted in eugenol production. Analysis of ObCAAT1-RNAi transgenic lines showed decreased levels of eugenol and accumulation of coniferyl alcohol and its derivatives. Coniferyl alcohol acts as a common substrate for phenylpropene and lignin biosynthesis. No differences were found in total lignin content of PGTs and leaves of transgenic lines, indicating that phenylpropene biosynthesis is not coupled to lignification in sweet basil.

Green production of silybin and isosilybin by merging metabolic engineering approaches and enzymatic catalysis.

Silymarin extracted from milk thistle seeds, is used for treating hepatic diseases. Silybin and isosilybin are its main components, and synthesized from coupling of taxifolin and coniferyl alcohol. Here, the biosynthetic pathways of taxifolin and coniferyl alcohol were reconstructed in Saccharomyces cerevisiae for the first time. To alleviate substantial burden caused by a great deal of genetic manipulation, expression of the enzymes (e.g. ZWF1, TYR1 and ARO8) playing multiple roles in the relevant biosynthetic pathways was selectively optimized. The strain YT1035 overexpressing seven heterologous enzymes and five native enzymes and the strain YC1053 overexpressing seven heterologous enzymes and four native enzymes, respectively produce 336.8 mg/L taxifolin and 201.1 mg/L coniferyl alcohol. Silybin and isosilybin are synthesized from taxifolin and coniferyl alcohol under catalysis of APX1t (the truncated milk thistle peroxidase), with a yield of 62.5%. This study demonstrates an approach for producing silybin and isosilybin from glucose for the first time.

Kinetic study on the heterogeneous degradation of coniferyl alcohol by OH radicals.

Coniferyl alcohol derived from lignin pyrolysis, is a potential tracer for wood burning emissions, but its atmospheric stability toward OH radicals is not well known. In this work, the degradation kinetics of coniferyl alcohol by OH radicals was studied using a flow reactor at different OH concentrations, temperatures, and relative humidity (RH). The results showed that coniferyl alcohol could be degraded effectively by OH radials, and the average second-order rate constant (k2) was (11.6 ±â€¯0.5) × 10-12 cm3 molecule-1 s-1 at the temperature and RH of 25 °C and 40%, respectively. Additionally, increasing temperature facilitated the degradation of coniferyl alcohol and the Arrhenius equation could be expressed as k2 = (1.7 ±â€¯0.3) × 10-9exp [-(1480.2 ±â€¯55.6)/T] at 40% RH. Meanwhile, increasing RH had a negative impact on the degradation of coniferyl alcohol. According to the k2 obtained under different conditions, the atmospheric lifetime of coniferyl alcohol was in the range of 13.5 ±â€¯0.4 h to 22.9 ±â€¯1.4 h. The results suggested that the atmosphere lifetime of coniferyl alcohol was predominantly controlled by OH radicals.

Infrared and Raman spectra of lignin substructures: Coniferyl alcohol, abietin, and coniferyl aldehyde.

Anatomical and chemical information can be linked by Raman imaging. Behind every pixel of the image is a Raman spectrum, which contains all the information as a molecular fingerprint. Yet to understand the spectra, the bands have to be assigned to components and their molecular structures. Although the lignin distribution is easily tracked in plant tissues, the assignment of the spectra is not good enough to allow in-depth analysis of the composition. Assignments of three lignin model compounds were derived from polarization measurements and quantum-chemical computations. Raman spectra of coniferyl alcohol crystals showed orientation dependence, which helped in band assignment. Abietin showed a Raman spectrum that was very similar to the spectrum of coniferyl alcohol, whereas its IR spectrum was very different due to bands of the sugar moiety. The Raman spectrum of coniferyl aldehyde is affected by the crystal order of molecules. All three compounds show much stronger band intensities than unconjugated single aromatic rings, indicating that the bulk of the lignin structure has significantly reduced contribution to Raman band intensities. Therefore, it is possible to highlight certain structures of lignin with Raman spectroscopy, because low amounts of a compound do not necessarily mean weak features in the spectrum.

Radical coupling reactions of piceatannol and monolignols: A density functional theory study.

Recent experimental work has revealed that the hydroxystilbene piceatannol can function as a monomeric unit in the lignification of palm fruit endocarp tissues. Results indicated that piceatannol homo-couples and cross-couples with monolignols through radical reactions and is integrally incorporated into the lignin polymer. The current work reports on the thermodynamics of the proposed reactions using density functional theory calculations. The results indicated that, in general, the energetics of both homo-coupling and cross-coupling are not dissimilar from those of the monolignol coupling, demonstrating the compatibility of piceatannol with the lignification process. Moreover, the DFT methods appear to predict the correct courses of post-coupling rearomatization reactions.

First Total Synthesis of (ß-5)-(ß-O-4) Dihydroxytrimer and Dihydrotrimer of Coniferyl Alcohol (G): Advanced Lignin Model Compounds.

To investigate lignin degradation, scientists commonly use model compounds. Unfortunately, these models are most of the time simple ß-O-4 dimers and do not sufficiently mimic the wide complexity of lignin structure (i.e., aliphatic side chains and robust C-C bonds). Herein, we present a methodology to access advanced lignin models through the first synthesis of two trimers of monolignol G-possessing side-chains and both robust ß-5 bond and labile ß-O-4 bond-via a chemo-enzymatic pathway. Key steps were (1) the C-C coupling via laccase-mediated oxidation, (2) the C-O coupling via a simple SN2 between a phenolate and a bromoketoester, and (3) a modified Upjohn dihydroxylation or a palladium-catalyzed hydrogenation. (ß-5)-(ß-O-4) dihydroxytrimer and dihydrotrimer of coniferyl alcohol (G) were obtained in good global yield, 9 and 20%, respectively, over nine steps starting from ferulic acid.

Improving bioconversion of eugenol to coniferyl alcohol by in situ eliminating harmful H2O2.

Coniferyl alcohol is a valuable chemical. However, the current approaches to obtain coniferyl alcohol are either unefficient or expensive. Penicillium simplicissimum vanillyl alcohol oxidase (PsVAO) can be used to produce coniferyl alcohol. However, PsVAO intrinsically produces harmful byproduct H2O2. Utilizing catalase to decompose H2O2 is a potential straightforward approach; however, catalase can also exhibit peroxidase activity to facilitate coniferyl alcohol over-oxidation. In this study, catalases exhibiting both high catalase activity and low peroxidase activity were found out, and introduced into the bioconversion systems. Our results showed that eliminating H2O2in situ released H2O2 inhibition of PsVAO, improved coniferyl alcohol production and eliminated coniferyl alcohol over-oxidation. Finally, coniferyl alcohol titer, molar yield, and productivity reached 22.9 g/L, 78.7%, and 0.5 g/(L × h) respectively. An efficient coniferyl alcohol production method was developed by overcoming the intrinsic disadvantages of PsVAO.