Objectives Characterize lignin-derived compounds from the hurd of 10 fiber/grain Cannabis sativa L. cultivars and investigate the antimicrobial activity of select compounds on the wasteful ruminant bacterium, Acetoanaerobium sticklandii SR.Methods Hurd was processed using an aqueous ethanol solvent system and analyzed via GC-MS to identify and characterize lignin derivatives. Antimicrobial activity of the hurd and three monomers was assessed using solid (hurd) and liquid medium (monomers) bioassays against SR.Results 27 compounds were identified, most as monomers but a few were dimers, ethylated derivatives, and organic acids. There were approximately equal G- and S-units with substantially fewer H-units. Several derivatives were present across cultivars, but their abundance appeared varied. Generally, syringaldehyde, vanillin, and syringic acid were most prominent, whereas vanillic acid and p-coumaric acid were less so, and syringaresinol was largely variable. Antimicrobial activity of ground hurd, as well as vanillin, vanillic acid, and p-coumaric acid were tested against SR. Only vanillin exhibited antimicrobial activity (1 mg mL-1) under the testing conditions.Conclusions The current work primarily contributes to the characterization and identification of lignin-derivatives in hemp hurd across fiber/grain cultivars with secondary contributions identifying hemp hurd as a potential source of plant-derived antimicrobials for amino-acid fermenting bacteria.

Objectives Characterize lignin-derived compounds from the hurd of 10 fiber/grain Cannabis sativa L. cultivars and investigate the antimicrobial activity of select compounds on the wasteful ruminant bacterium, Acetoanaerobium sticklandii SR. Methods Hurd was processed using an aqueous ethanol solvent system and analyzed via GC-MS to identify and characterize lignin derivatives. Antimicrobial activity of the hurd and three monomers was assessed using solid (hurd) and liquid medium (monomers) bioassays against SR. Results 27 compounds were identified, most as monomers but a few were dimers, ethylated derivatives, and organic acids. There were approximately equal G- and S-units with substantially fewer H-units. Several derivatives were present across cultivars, but their abundance appeared varied. Generally, syringaldehyde, vanillin, and syringic acid were most prominent, whereas vanillic acid and p-coumaric acid were less so, and syringaresinol was largely variable. Antimicrobial activity of ground hurd, as well as vanillin, vanillic acid, and p-coumaric acid were tested against SR. Only vanillin exhibited antimicrobial activity (1 mg mL −1 ) under the testing conditions. Conclusions The current work primarily contributes to the characterization and identification of lignin-derivatives in hemp hurd across fiber/grain cultivars with secondary contributions identifying hemp hurd as a potential source of plant-derived antimicrobials for amino-acid fermenting bacteria.

Elicitation is an important strategy for producing plant secondary metabolites, which are responsible for a plant's highly dynamic chemical defense against environmental stressors such as UV light, predators, and pathogens. Elicitation strategies have recently employed nanoparticles as carriers to effectively deliver the elicitation agent through the cell membrane. This study examines the elicitation of secondary metabolites in Lobelia cardinalis hairy root cultures (HRCs) in response to the plant hormone jasmonic acid (JA) and a JA-loaded nanoparticle carrier (mesoporous silica nanoparticles (MSNPs)). Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) with high resolution mass spectrometry identified 12 m/z features only upregulated in the treatments with JA (free JA, JA-loaded MSNPs, and physical mixtures of JA and MSNPs) relative to a control experiment (without JA) or exposure to MSNPs alone. Putative identifications for these compounds elicited by JA included sesquiterpenoids (rishitin), monolignols (elemicin), and coumarins (7-hydroxycoumarin derivatives), respectively, and roughly correlated with the levels of JA that was measured in the respective HRCs. Signature m/z analytes associated with exposure to MNSPs were identified across treatment of bare MNSPs, JA-loaded MSNPs, and physical mixtures of JA and MSNPs and are consistent with a general stress response. The study demonstrates the ability to differentiate the elicitation effects of a biotic elicitor and its delivery system in untargeted elicitation studies, with a goal of designing nanocarrier systems for effective production of secondary metabolites.

BACKGROUND:Thinopyrum intermedium (Host) Barkworth & D.R. Dewey, or intermediate wheat grass (IWG), is being developed as the first widely-available perennial grain candidate. However, because the crop is still in development, grain yields are lower than those of traditional cereals. Utilization of its non-grain biomass (e.g. for biofuel production and as a source of fine chemicals) would increase the economic value of its cultivation. The present study provides a structural characterization of the lignin and cell wall carbohydrates in IWG biomass and qualitative profiling of biomass extractives and compares them to those of annual wheat (Triticum aestivum) biomass grown in the same location and growing season. RESULTS:The monosaccharide composition and ester-linked phenolic acid contents of vegetative biomass material from annual wheat and IWG were similar. IWG vegetative biomass is rich in feruloylated arabinoxylans (AX) with a very low substitution rate, whereas the AX from IWG bran have a slightly higher substitution rate. The structure of IWG lignin was investigated using both the quantitative derivatization followed by reductive cleavage method and 2D-NMR analysis, revealing an H:G:S lignin that incorporates tricin and is acylated with coumaric acid and smaller amounts of ferulates. IWG and wheat extractives contained fatty acids, various free phenolic compounds (tricin, monolignols and phenolic acids), phenolic conjugates and phytosterols. CONCLUSION:The present study provides firm support for the further exploration of T. intermedium biomass as a carbohydrate feedstock (e.g, abundant in lightly substituted AX and cellulose polymers) for biofuel production and source of high-value fine chemicals, such as tricin. © 2024 Society of Chemical Industry.

The gas phase delivery of lignin into the hot zone of cw-CO2 laser powered homogeneous pyrolysis (LPHP) reactor under “wall-less” conditions led to break down of lignin macromolecules into neutral oligomers and paramagnetic fragments which deposited onto the reactor cell walls. Formation of PAHs was observed in parallel to the defragmentation of lignin which accelerated with increased laser power. Remarkably, no phenolic compounds were detected among lignin fragments (intermediate radicals and oligomers). It is concluded that the PAH and soot like particulate formation occurs in the hot zone of the LPHP reactor and resembles combustion-related processes. The key role of the resonantly stabilized radicals in formation of low molecular weight PAHs is outlined. An alternative channel for formation of cyclopentadienyl radicals adsorbed/trapped (CPDa) on lignin macromolecules as precursors for PAHs formation is hypothesized.

Structural elucidation of lignin degradation products is a requirement for successfully developing lignin valorization technology. Most of mass spectrometry–based techniques have utilized negative ion mode mass spectrometry for structural elucidation of lignin-derived compounds. Unfortunately, simple deprotonation can lead to in-source fragmentation and may not be suitable for condensed lignin structures without acidic moieties. Herein, we present a lithium cationization methodology for mass spectrometry sequencing of advanced lignin oligomers having β-β′ and β-O-4′ bonding motifs. To do so, two advanced lignin oligomers were first synthesized through a step-by-step synthetic route, and then subjected to two different ESI mass spectrometry techniques in positive ion mode using lithium cations for ionization. An orbitrap mass spectrometer was used to obtain exact mass information, and higher-energy collisional dissociation (HCD) was used to sequence the lignin model oligomers. Based on the sequence-specific fragment ions, sequence rules were proposed. Multi-stage (MSn) collision-induced dissociation (CID) using an ion trap mass spectrometer provided data to investigate the origin of each fragment ion and to further confirm proposed fragmentation pathways. In addition to β-O-4′ bond cleavage, the presented lithium cationization approach led to cleavage of β-β′ bonds on the model oligomers in both ion trap and orbitrap mass spectrometry experiments. Additionally, MSn experiments were used to investigate possible lithium cationization sites on the model oligomers. Lithium cationization in positive ion mode mass spectrometry proved to be a robust tool for characterization and sequencing of advanced lignin oligomers with different bonding motifs.

Successful strategies for the attachment of oligopeptides to mesoporous silica with pores large enough to load biomolecules should utilize the high surface area of pores to provide an accessible, protective environment. A two-step oligopeptide functionalization strategy is examined here using diazirine-based heterobifunctional linkers. Mesoporous silica nanoparticles (MSNPs) with average pore diameter of ~8 nm and surface area of ~730 m2/g were synthesized and amine-functionalized. Tetrapeptides Gly-Gly-Gly-Gly (GGGG) and Arg-Ser-Ser-Val (RSSV), and a peptide comprised of four copies of RSSV (4RSSV), were covalently attached via their N-terminus to the amine groups on the particle surface by a heterobifunctional linker, sulfo-succinimidyl 6-(4,4′-azipentanamido)hexanoate (sulfo-NHS-LC-diazirine, or SNLD). SNLD consists of an amine-reactive NHS ester group and UV-activable diazirine group, providing precise control over the sequence of attachment steps. Attachment efficiency of RSSV was measured using fluorescein isothiocyanate (FITC)-tagged RSSV (RSSV-FITC). TGA analysis shows similar efficiency (0.29, 0.31 and 0.26 mol peptide/mol amine, respectively) for 4G, RSSV and 4RSSV, suggesting a generalizable method of peptide conjugation. The technique developed here for the conjugation of peptides to MSNPs provides for their attachment in pores and can be translated to selective peptide-based separation and concentration of therapeutics from aqueous process and waste streams.

Lignin derived from lignocellulosic biomass is the largest source of renewable bioaromatics present on earth and requires environmentally sustainable separation strategies to selectively obtain high-value degradation products. Applications of supramolecular interactions have the potential to isolate lignin compounds from biomass degradation fractions by the formation of variable inclusion complexes with cyclodextrins (CDs). CDs are commonly used as selective adsorbents for many applications and can capture guest molecules in their internal hydrophobic cavity. The strength of supramolecular interactions between CDs and lignin model compounds that represent potential lignocellulosic biomass degradation products can be characterized by assessing the thermodynamics of binding stability. Consequently, the inclusion interactions of β-CD and lignin model compounds G-(β-O-4')-G, G-(β-O-4')-truncG (guaiacylglycerol-β-guaiacyl ether), and G-(β-β')-G (pinoresinol) were investigated empirically by electrospray ionization mass spectrometry and isothermal titration calorimetry, complemented by molecular dynamics (MD) simulations. Empirical results indicate that there are substantial differences in binding stability dependent on the linkage type. The lignin model β-β' dimer showed more potential bound states including 1:1, 2:1, and 1:2 (guest:host) complexation and, based on binding stability determinations, was consistently the most energetically favorable guest. Empirical results are supported by MD simulations that reveal that the capture of G-(β-β')-G by β-CD is promising with a 66% probability of being bound for G-(β-O-4')-truncG compared to 88% for G-(β-β')-G (unbiased distance trajectory and explicit counting of bound states). These outcomes indicate CDs as a promising material to assist in separations of lignin oligomers from heterogeneous mixtures for the development of environmentally sustainable isolations of lignin compounds from biomass fractions.

Lignin and lignans are natural products found in plant cell walls. Lignin research has historically focused on lignin degradation techniques in the hope of converting lignin into useful aromatic carbon feedstocks. In contrast, investigations of lignans existing as natural product dimers, have been focused on thier interesting biological activities. Many lignan compounds are chemically identical to dimers derived from lignin, and both lignin and lignan dimers can possess multiple chiral centers leading to observations of diastereomer pairs where one diastereomer exhibits the bulk of the activity. For example, the G-(β-O-4′)-G dimer was reported to have a pro-angiogenic activity with one diastereomer of the pair showing enhanced pro-angiogenic activity. Traditional analytical techniques such as nuclear magnetic resonance (NMR) can differentiate the diastereomer pairs of β-O-4 compounds; however, isolation of a pure sample is often required for analysis. This work was aimed at exploring the potential use of tandem mass spectrometry to differentiate diastereomer pairs in the β-O-4 dimer series. Each diastereomer pair in the nine-dimer series was separated by HPLC and interrogated by tandem mass spectrometry. To understand the chromatographic behavior of the diastereomer pair in the β-O-4 dimer series, three commercially available reverse phase HPLC columns were evaluated. A temperature programming experiment using water/acetonitrile isocratic elution showed that the chromatographic retention mechanism of these diastereomers was hydrophobically driven with analytes having more methoxy groups exhibiting larger ΔH0 and higher octanol-water partition coefficient values. Tandem mass spectrometry performed on each of the diastereomers produced fragment ions having different ion abundances. A mechanistic study based on the ion abundance of “sequence-specific ions” and “-48 ions” was used to assign a configuration to each of the pairs of diastereomers in the nine-dimer series.

A study of the interaction between cell membranes and small molecules derived from lignin, a protective phenolic biopolymer found in vascular plants, is crucial for identifying their potential as pharmacological and toxicological agents. In this work, the interactions of model cell membranes [supported 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers] are compared for three βO4 dimers of coniferyl alcohol (G lignin monomer): guaiacylglycerol guaiacol ester with a hydroxypropenyl (HOC3H4-) tail (G-βO4'-G), a truncated GG dimer without HOC3H4- (G-βO4'-truncG), and a benzylated GG dimer (benzG-βO4'-G). The uptake of the lignin dimers (per mass of lipid) and the energy dissipation (a measure of bilayer disorder) are higher for benzG-βO4'-G and G-βO4'-truncG than those for G-βO4'-G in the gel-phase DPPC bilayer, as measured using quartz crystal microbalance with dissipation (QCM-D). A similar uptake of G-βO4'-truncG is observed for a fluid-phase bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine, suggesting that the effect of the bilayer phase on dimer uptake is minimal. The effects of increasing lignin dimer concentration are examined through an analysis of density profiles, potential of mean force curves, lipid order parameters, and bilayer area compressibilities (disorder) in the lipid bilayers obtained from molecular dynamics simulations. Dimer distributions and potentials of mean force indicate that the penetration into bilayers is higher for benzG-βO4'-G and G-βO4'-truncG than that for G-βO4'-G, consistent with the QCM-D results. Increased lipid tail disorder due to dimer penetration leads to a thinning and softening of the bilayers. Minor differences in the structure of lignin derivatives (such as truncating the hydroxypropenyl tail) have significant impacts on their ability to penetrate lipid bilayers.

Lignin bio-oils with selective antimicrobial properties against lactic acid bacteria were prepared by depolymerizing corn stover lignin with peracetic acid. Bio-oil treatments significantly increased ethanol yields of contaminated fermentations.

The dopamine transporter (DAT) is targeted in substance use disorders (SUDs), and "non-classical"" DAT inhibitors with low abuse potential are therapeutic candidates. Lobinaline, from Lobelia cardinalis, is an atypical DAT inhibitor lead. Chemical synthesis of lobinaline is challenging; thus, "target-directed evolution" was used for lead optimization. A target protein is expressed in plant cells, and a mutant cell population is selected under conditions where target protein functional inhibition confers a survival advantage. Surviving mutants are "mined" for the targeted activity. Applied to a mutant L. cardinalis cell population expressing the human DAT, we identified 20 mutants overproducing DAT inhibitors. Microanalysis prioritized novel lobinaline derivatives, and we first investigated the more water-soluble lobinaline N-oxide. It inhibited rat synaptosomal [3H]DA uptake with an IC50 similar to lobinaline. Against repeated DA microinjections into the rat striatum, lobinaline produced transient DA clearance reductions. In contrast, lobinaline N-oxide prolongingly increased DA peak amplitudes, particularly in the ventral striatum. Lobinaline N-oxide also produced complex changes in post-peak DA clearance inconsistent with simple DAT inhibition. This unusual DAT interaction may prove therapeutically useful for treating SUDs. This study demonstrates the value of target-directed evolution of plant cells for optimizing lead compounds difficult to synthesize chemically.

Sensitive, high-throughput methods for pharmacokinetic (PK) profiling are essential for potential therapeutics during critical stages of clinical trials. The application of a microfluidic capillary zone electrophoresis mass spectrometry (CZE-MS) method for PK profiling allows for rapid, sensitive and in-depth analysis of multiple samples within a short timeframe. Here, a CZE-MS approach for PK analysis was compared with a traditional UHPLC-MS approach when analyzing serum extracts from rats treated with a potential Alzheimer's disease therapeutic, BNC-1. Resulting PK data generated from both methods displayed statistical similarities. Additionally, the separation efficiency attributed to the use of the CZE-MS method provided substantial metabolic regulation data that was not apparent in the UHPLC-MS method. Additionally, the coupling of the CZE-MS method to the data processing software, MZmine2, was used to monitor changes in metabolism and observe putative BNC-1-derived metabolites. The ability to perform fast analyses without sacrificing sensitivity or metabolic information suggests that this CZE-MS method is ideal for metabolomics-inclusive, high-throughput PK profiling.

Pyrolysis of hydrolytic lignin (HL) in newly designed, gas phase continuous droplet evaporation (CDE) and continuous atomization (CA) reactors was studied. The product distribution was strongly dependent on the heterogeneous character of either delivery of lignin solution into the CDE reactor (in situ formation of solid phase) or sampling conditions using quartz wool in both CDE and CA reactors. The effect of residence time, initial concentration of HL solution, and injection temperature on product distribution in the CDE reactor was investigated and discussed in terms of mass and heat transfer limitation. The experimental data confirm that at low initial mass delivery rates of lignin (micrograms per second) and by increasing initial lignin concentration (up to 40 times), the formation of phenolics is slightly intensified (six times). However, the solid surface or any condensed phase that forms in situ during the reaction in the gas phase may largely govern the pyrolysis processes. The detailed experimental examination of homogeneous pyrolysis of lignin in both gas-phase reactors by implication of diverse analytical techniques (gas chromatography, gel permeation chromatography, laser desorption/ionization, Fourier transform infrared spectroscopy, electron paramagnetic resonance, nuclear magnetic resonance ) revealed break down of HL macromolecules into oligomer fragments after pyrolysis at negligible amounts of phenolics detected. A mechanistic interpretation of primary steps for formation of dominant intermediate products, oligomers and oligomer stable radicals, is represented.

Converting lignin to value-added products at high yields provides an avenue for making ethanol biorefineries more profitable while reducing the carbon footprint of products generally derived from petroleum. In this study, corn stover lignin was depolymerized by catalytic transfer hydrogenolysis (CTH) in supercritical ethanol with a Ru/C catalyst. The lignin-derived bio-oil was then sequentially extracted utilizing hexane, petroleum ether, chloroform, and ethyl acetate as solvents in order of less polar to polar, and the subsequent bio-oils were characterized using GPC, GC/MS, and HSQC NMR. Results show that the monomers in the bio-oil fractions contained primarily alkylated phenols, hydrogenated hydroxycinnamic acid derivatives, syringol and guaiacoltype lignins created from reductive cleavages of ether linkages, which were sequentially extracted into groups depending on the solvent polarity. The antimicrobial properties of the bio-oils were screened against Gram-positive (Bacillus subtilis, Lactobacillus amylovorus, and Staphylococcus epidermidis) and Gram-negative (Escherichia soli) bacteria and yeast (Saccharomyces cerevisiae) by examining microbial growth inhibition. Results show that CTH-derived bio-oils inhibited all tested organisms at concentrations less than 3 mg/mL. Total monomer concentration and the presence of specific monomers (i.e., syringyl propane) showed correlations to antimicrobial activity, likely due to cell death or membrane damage. This study provides insights into using sequential extraction to fractionate lignin-derived compounds and correlations between the properties of the extracted compounds and their antimicrobial activity.

• Cook’s kinetic method and the extension of comparisons by the ladder method. • Novel lithium cation basicity estimates of lignin β-O-4 dimers. • Comparison of results on LTQ linear ion trap, and LCQ DECA mass spectrometers. • Impact of the β-O-4 bond on LCB and its implications for mass spectrometry of lignin compounds by lithium adduction.

The potential to impart surfaces with specific lignin-like properties (i.e. resistance to microbes) remains relatively unexplored due to the lack of well-defined lignin-derived small molecules and corresponding surface functionalization strategies. Here, allyl-modified guaiacyl beta-O-4 eugenol (G-eug) lignin-derived dimer is synthesized and attached to mesoporous silica nanoparticles (MSNPs) via click chemistry. The ability of G-eug lignin-dimer functionalized particles to interact with and disrupt synthetic lipid bilayers is compared to that of eugenol, a known natural antimicrobial. Spherical MSNPs (similar to 150 nm diameter with 4.5 nm pores) were synthesized using surfactant templating. Post-synthesis thiol (SH) attachment was performed using (3-mercaptopropyl) trimethoxysilane and quantified by Ellman's test. The resultant SH-MSNPs were conjugated with the Geug dimers or eugenol by a thiol-ene reaction under ultraviolet light in the presence of a photo initiator. From thermogravimetric analysis (TGA), attachment densities of approximately 0.22 mmol eugenol/g particle and 0.13 mmol G-eug dimer/g particle were achieved. The interaction of the functionalized MSNPs with a phospholipid bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (representing model cell membranes) supported on gold surface was measured using Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). Eugenol-grafted MSNPs in PBS (up to 1 mg/mL) associated with the bilayer and increased the mass adsorbed on the QCM-D sensor. In contrast, MSNPs functionalized with G-eug dimer show qualitatively different behavior, with more uptake and evidence of bilayer disruption at and above a particle concentration of 0.5 mg/mL. These results suggest that bio-inspired materials with conjugated lignin-derived small molecules can serve as a platform for novel antimicrobial coatings and therapeutic carriers.

Application of a microfluidic CE* device for CZE-MS allows for fast, rapid, and in-depth analysis of large sample sets. This microfluidic CZE-MS device, the 908 Devices ZipChip, involves minimal sample preparation and is ideal for small cation analytes, such as alkaloids. Here, we evaluated the microfluidic device for the analysis of alkaloids from Lobelia cardinalis hairy root cultures. Extracts from wild-type, transgenic, and selected mutant plant cultures were analyzed and data batch processed using the mass spectral processing software MZmine2 and the statistical software Prism 8. In total 139 features were detected as baseline resolved peaks via the MZmine2 software optimized for the electrophoretic separations. Statistically significant differences in the relative abundance of the primary alkaloid lobinaline (C27H34N2), along with several putative "lobinaline-like" molecules were observed utilizing this approach. Additionally, a method for performing both targeted and untargeted MS/MS experiments using the microfluidic device was developed and evaluated. Coupling data-processing software with CZE-MS data acquisition has enabled comprehensive metabolomic profiles from plant cell cultures to be constructed within a single working day.