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Antimicrobial resistance is a global threat. The use of biologically active natural products alone or in combination with the clinically proven antimicrobial agents might be a useful strategy to fight the resistance. The scientific hypotheses of this study were twofold: (1) the natural humic substances rich in dicarboxyl, phenolic, heteroaryl, and other fragments might possess inhibitory activity against β-lactamases, and (2) this inhibitory activity might be linked to the molecular composition of the humic ensemble. To test these hypotheses, we used humic substances (HS) from different sources (coal, peat, and soil) and of different fractional compositions (humic acids, hymatomelanic acids, and narrow fractions from solid-phase extraction) for inhibiting serine β-lactamase TEM-1. Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) was used to characterize the molecular composition of all humic materials used in this study. The kinetic assay with chromogenic substrate CENTA was used for assessment of inhibitory activity. The inhibition data have shown that among all humic materials tested, a distinct activity was observed within apolar fractions of hymatomelanic acid isolated from lignite. The decrease in the hydrolysis rate in the presence of most active fractions was 42% (with sulbactam-87%). Of particular importance is that these very fractions caused a synergistic effect (2-fold) for the combinations with sulbactam. Linking the observed inhibition effects to molecular composition revealed the preferential contribution of low-oxidized aromatic and acyclic components such as flavonoid-, lignin, and terpenoid-like molecules. The binding of single low-molecular-weight components to the cryptic allosteric site along with supramolecular interactions of humic aggregates with the protein surface could be considered as a major contributor to the observed inhibition. We believe that fine fractionation of hydrophobic humic materials along with molecular modeling studies on the interaction between humic molecules and β-lactamases might contribute to the development of novel β-lactamase inhibitors of humic nature.

While liquid biopreparations for water and soil bioremediation are convenient and economically attractive, microbial survival under standard environmental conditions is poor. Microbial immobilization is a common and efficient method to preserve high viable cell titers. We tested the effect of three new materials on the survival of various hydrocarbon oxidizers during long-term storage. The suspended (planktonic) cultures stored under the same conditions served as a control when assessing the viability of immobilized cultures after long-term storage. The cells of Acinetobacter seifertii, Pseudomonas extremaustralis, P. aeruginosa, Rhodococcus erythropolis, and Dietzia maris grown with microcapsules (MCs) of chitosan-modified polyurea (represented by crumpled spheres, 40–200 µm in diameter) attached abundantly to the MC surface. After several months of storage with MCs, CFU titers were two to five times higher than in the control. Cultivation of P. aeruginosa and R. erythropolis with polylactide (PLA) MCs (represented by slightly crumpled thin-walled spheres 25–100 µm in size) resulted in the formation of thick biofilms on MC surface. After 30-day storage with these MCs, CFU titers of R. erytropolis were up to one thousand times higher than in the control. In the presence of PLA MCs, the respiration rates of both cultures were five to eight times higher than in the control without MCs. The reasons of the better survival of immobilized cells are being discussed. After storage for up to 12 months, CFU titers of bacteria (R. erythropolis, A. seifertii, P. aeruginosa, P. extremaustralis) and yeasts (Yarrovia lipolytica) immobilized in the gel based on silanol derivatives of humic substances were ten to one hundred times higher than in the control. After 4-month storage, hydrocarbon degradation by the stabilized cultures began earlier and was faster and more complete than in the control. The tested materials may be used to extend the storage time for the preparations of biotechnologically important bacteria and for application for petroleum bioremediation.

Peat humic acids are well known for their wide range of biological effects which can be attributed to the complex chemical structure of naturally occurring humic substances. One of the promising tools is an ontology-based quantitative analysis of the relationship between physical and chemical parameters describing a chemical structure of peat humic acids and their biological activity. This article demonstrates the feasibility of such an approach to estimate the antioxidant and cell protective properties of the peat humic acids. The structural parameters of the peat humic acids were studied by electronic, fluorescence, infrared, 13C-NMR spectroscopy, titrimetric analysis, elemental C,H,N, and O- analysis, and gel chromatography. Antioxidant and antiradical activities were assessed by physicochemical methods of analysis: electronic paramagnetic resonance, cathodic voltammetry, ABTS•+ scavenging, assay of DPPH radical-scavenging activity, assay of superoxide radical-scavenging activity, iron chelating activity, and scavenging of hydroxyl radicals. Cytoprotective activity was evaluated by the neutral red-based cytotoxicity test in 3T3-L1 cell culture in a wide range of concentrations. Assessment of intracellular ROS production was carried out using a 2,7-dichlorodihydrofluoresceindiacetate (DCFDA) fluorescent probe. Intracellular ROS production was induced using two common prooxidants (tert-butyl hydroperoxide, Fe2+ ions). We suggested an ontology-based model for the antioxidant and cytoprotective activity of humic acids based on experimental data and numerical models. This model establishes the way to further research on the biological effects of humic acids and provides a useful tool for numerical simulation of these effects. Remarkable antioxidant and cell protective activity of humic acids makes them a promising natural source of new pharmaceutical substances that feature a wide range of biological effects.

Direct comparison of high-resolution mass spectrometry (HRMS) data acquired with different instrumentation or parameters remains difficult as the derived lists of molecular species via HRMS, even for the same sample, appear distinct. This inconsistency is the result of inherent inaccuracies caused by instrumental limitations and sample conditions. We propose a method that classifies HRMS data based on the differences in the number of elements between each pair of molecular formulae within the formulae list to preserve the essence of the given sample. The novel metric, Formulae Difference Chains Expected Length (FDCEL), allowed for comparing and classifying samples measured by different instruments. FDCEL metric was implemented for both spectrum quality control and for examination of samples of various nature. We also demonstrate a web application and a prototype for a uniform database for HRMS data serving as a benchmark for future biogeochemical applications.

Interlaboratory comparison on the determination of the molecular composition of humic substances (HS) was undertaken in the framework of IUPAC project 2016-015-2-600. The analysis was conducted using high resolution mass spectrometry, nominally, Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization. Six samples of HS from freshwater, soil, and leonardite were used for this study, including one sample of humic acids (HA) from coal (leonardite), two samples of soil HA (the sod-podzolic soil and chernozem), two samples of soil fulvic acids (FA) (the sod-podzolic soil and chernozem), and one sample of freshwater humic acids (the Suwannee River). The samples were analyzed on five different FTICR MS instruments using the routine conditions applied in each participating laboratory. The results were collected as mass lists, which were further assigned formulae for the determination of molecular composition. The similarity of the obtained data was evaluated using appropriate statistical metrics. The results have shown that direct comparison of discrete stoichiometries assigned to the mass lists obtained by the different laboratories yielded poor results with low values of the Jaccard similarity score – not exceeding 0.56 (not more than 56 % of the similar peaks). The least similarity was observed for the aromatics-rich HA samples from leonardite (coal) and the chernozem soil, which might be connected to difficulties in their ionization. The reliable similarity among the data obtained in this intercomparison study was achieved only by transforming a singular point (stoichiometry) in van Krevelen diagram into a sizeable pixel (a number of closely located stoichiometries), which can be calculated from the population density distribution. The conclusion was made that, so far, these are descriptors of occupation density distribution, which provide the metrics compliant with the data quality requirements, such as the reproducibility of the data measurements on different instruments.

A new approach is described to increasing the numbers of viable cells in long-term stored populations (cultures) of the hydrocarbon-oxidizing bacterium Acinetobacter junii, which involves application of humic substances (HSs). HSs are produced due to condensation and oxidation of phenolic compounds, including alkylresorcinols, factors of intercellular microbial communication with stress-potentiating and antioxidant activity. HS addition to the stationary-phase bacterial culture was shown to result in 10- to 15-fold increase in cell viability for bacterial preparations stored for 1 to 4 months under provocative conditions (free air supply, growth medium, and temperatures of 20–25°C). Analysis of death curves for experimental and control A. junii cultures revealed two phases, with the initial stage of rapid death (0.167 log CFU/mL day), usual for autolysis of the regular stationary cells, and the stage of slower death (log 0.015 CFU/mL day), typical of persister cells. This was previously shown to be the feature determining this type of phenotypic heterogeneity in the populations of opportunistic bacteria. The concentration dependence of HSs effect on persisters formation was determined. While treatment of A. junii stationary cultures with hydrogen peroxide (0.03–0.3%) as a stress factor also resulted in enhanced persisters formation (fourfold), unlike the HS-treated variants they survived for not more than 1.5 to 2 months. Plating of the control and experimental variants (with HSs or H2O2) after long-term storage (2–4 months) revealed their phase variation spectrum to change due to replacement of the dominant colony morphology phenotype by the minor ones (40% and more), which is one of the features of the dormant cystlike bacterial forms. The ratios of the variants in the control and experiments (with HSs) were different. Comparison of the chemical composition of HS preparations indicated that the most oxidized hydrophilic HSs affected formation of persister cells, while less oxidized humates with higher antioxidant activity were responsible for the viability prolongation. Thus, the effect of HSs on bacterial populations includes induction of persister cell formation (their increased number), prolongation of the colony-forming capacity in the cells surviving under provocative storage conditions, and alteration of the phase variant spectrum of the stored populations. Formation of stress-induced persister cells developing into mature dormant forms supporting survival of the population (species) was established for saprotrophic bacteria (in the case of A. junii). In practical terms, HS-induced formation of persister cells may be recommended for development of diverse bacterial preparations.

Large scale isolation—in gram quantities—of dissolved organic matter (DOM) from natural waters is necessary for detailed investigation of its role in chemical and microbial processes driving carbon cycling under conditions of global climate change. The best candidate for a use in these large-scale experiments is a bulk sorbent Bondesil PPL, which has the same modification as the widely used Bond Elut PPL sorbent. There have been no studies so far reported on interchangeability of these sorbents with regard to DOM isolation. This work was devoted to comparative studies on sorption efficiency and molecular selectivity of these two sorbents—Bond Elut PPL and Bondesil PPL with regard to DOM components. Fulvic acids (FA) from peat water leachate were used as a model DOM. Laboratory solid phase extraction (SPE) setup was used for monitoring sorption recovery and extraction yield. It included three parallel experiments on pre-packed Bond Elut PPL cartridges (500 mg/3 mL) and three self-packed Bondesil PPL cartridges (500 mg/3 mL). Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS) and 13C/1H nuclear magnetic resonance (NMR) spectroscopy were used for determination of molecular and structural group compositions of the FA isolates obtained with a use of two different sorbents. The results of this study allowed a conclusion on interchangeability of the two sorbents used in this study for the purposes of DOM isolation from natural waters. This conclusion was backed up by similarity of sorption behavior of the peat FA components on both sorbents and by high similarity of molecular compositions and carbon distribution among the main structural groups.

Humic substances (HS) are natural supramolecular systems of high- and low-molecular-weight compounds with distinct immunomodulatory and protective properties. The key beneficial biological activity of HS is their antioxidant activity. However, systematic studies of the antioxidant activity of HS against biologically relevant peroxyl radicals are still scarce. The main objective of this work was to estimate the antioxidant capacity (AOC) of a broad set of HS widely differing in structure using an oxygen radical absorption capacity (ORAC) assay. For this purpose, 25 samples of soil, peat, coal, and aquatic HS and humic-like substances were characterized using elemental analysis and quantitative 13C solution-state NMR. The Folin–Ciocalteu method was used to quantify total phenol (TP) content in HS. The determined AOC values varied in the range of 0.31–2.56 μmol Trolox eqv. mg−1, which is close to the values for ascorbic acid and vitamin E. Forward stepwise regression was used to reveal the four main factors contributing to the AOC value of HS: atomic C/N ratio, content of O-substituted methine and methoxyl groups, and TP. The results obtained clearly demonstrate the dependence of the AOC of HS on both phenolic and non-phenolic moieties in their structure, including carbohydrate fragments.

Humification is a ubiquitous natural process of biomass degradation that creates multicomponent systems of nonliving organic matter, including dissolved organic matter (DOM) and humic substances (HS) in water environments, soils, and organic rocks. Despite significant differences in molecular composition, the optical properties of DOM and HS are remarkably similar, and the reason for this remains largely unknown. Here, we employed fluorescence spectroscopy with (sub)picosecond resolution to elucidate the role of electronic interactions within DOM and HS. We revealed an ultrafast decay component with a characteristic decay lifetime of 0.5-1.5 ps and spectral diffusion originating from excitation energy transfer (EET) in the system. The rate of EET was positively correlated to the fraction of aromatic species and tightness of aromatic species packing. Diminishing the number of EET donor-acceptor pairs by reduction with NaBH4 (decrease of the acceptor number), decrease of pH (decrease of the electron-donating ability), or decrease of the average particle size by filtration (less donor-acceptor pairs within a particle) resulted in a lower impact of the ultrafast component on fluorescence decay. Our results uncover the role of electronic coupling among fluorophores in the formation of DOM and HS optical properties and provide a framework for studying photophysical processes in heterogeneous systems of natural fluorophores.

This article considers the influence that suspensions of nanoparticles (sized from 10 to 60 nm) of iron oxo-compounds in different oxidation states have on biological objects. The suspension is formed by the interaction of metallic iron with aqueous solutions of humic substances. Based on the example of green microalgae Scenedesmus quadricauda (Turp.) Breb., it is shown that suspensions that contain iron oxo-compound nanoparticles stabilized with humic substances at an iron concentration from 0.14 to 2036 µM do not have a toxic effect on microalgae. The availability of iron contained in the suspensions was evaluated in the experiment on sprouts of wheat Triticum aestivum L., which had been grown under iron-deficient conditions. The root uptake of the ionic form of iron contained in the suspension was confirmed. It is shown that the studied suspensions of iron nanoparticles stabilized by humic substances accumulate on the surface of plant roots. These suspensions are supposed to be a source of iron with prolonged action for plants.

Addition of humic compounds (9 preparations) to the cultures of hydrocarbon-oxidizing bacteria was shown to result in up to 15-fold increase in the number of viable cells in the cultures stored in the growth media with access to air at 20‒25°C. The most oxidized humates exhibited the highest activity in induction of persister formation, which subsequently matured to dormant forms, while unoxidized humates with antioxidant activity supported viability persistence.

Humic acids (HAs) make up to 30–50% of the soil organic matter, which is the main reservoir of organic carbon in the biosphere. The common isolation protocol for HAs implies alkaline extraction from soils followed by acidification of the extract with HCl to pH 2. International Humic Substances Society (IHSS) recommends isolation of HAs in oxygen-free atmosphere (e.g., under nitrogen or inert gas purging) to prevent oxidative transformations of HAs during the extraction process. In the Russian school of soil science, extraction is usually conducted without the use of nitrogen. In the present work, we compared the physicochemical properties of HAs isolated from A1 horizons of soddy-podzolic soil (Retisol) and chernozem (Chernozem) by 0.1 M NaOH in the presence and absence of oxygen. The soils used in this study represented zonal types of southern taiga and steppe, respectively, and differed markedly with respect to humus formation conditions. The yield of humic substances (Corg content in the extracts), their elemental composition, functional groups content, molecular-weight distributions (gel filtration on Sephadex G-75), paramagnetic properties, and absorption spectra in the visible, UV, and IR regions were studied. For both soils, no statistically significant differences were found in the quantitative yield, molecular weight distribution, absorption spectra in the visible, UV and IR regions between HAs isolated by alkaline extraction in the presence and absence of oxygen. At the same time, for the HAs extracted from the Retisol soil in the presence of oxygen, higher O : C ratios, higher contents of quinone and carboxyl groups, and significantly higher content of free radicals were observed. This was revealed with the use of elemental analysis, potentiometric titration, 1H and 13C NMR spectroscopy, and electron paramagnetic resonance spectroscopy. For the Chernozem HAs, these differences were not observed. The obtained results suggest that partial oxidation of the soil organic matter components takes place during alkaline extraction from the Retisol in the presence of oxygen. In the Chernozem, humification process is apparently accompanied by significant oxidative transformation of organic residues, so the presence of molecular oxygen does not cause further oxidation of HAs under alkaline conditions. Our results indicate that, for the isolation of HAs from the mineral horizons of Chernozems, the use of oxygen-free atmosphere is optional. In the case of Retisols, the use of oxygen-free atmosphere is desirable, especially if it is intended to study the reactions of HA oxidation upon, for example, enzymatic catalysis.

The dynamics of interaction between metallic iron and aqueous solutions of three preparations of humic coal substances from different manufacturers is analyzed. Humic substances are characterized via elemental analysis, NMR spectroscopy, size-exclusion chromatography, electronic absorption spectroscopy, and chemical analysis. Distinctions are observed between the investigated humic substances in the content of aromatic moieties, the degree of their substitution, the content of carboxylic groups, and the degree of oxidation. It is shown that regardless of the background of humic substances, the interaction between their solutions and iron is based on the same set of consecutive reactions, which can be divided into three basic stages of interaction using data on the changes in easily controllable parameters (redox potential, pH, and solution absorbance). These stages of interaction are qualitatively described in Pourbaix diagram coordinates. Features of the structural and group composition of humic substances result in different times of the corresponding stages of interaction. Based on an analysis of the main components, a way of describing the complicated sequence of reactions between humic substances and iron in the coordinates of a two-factor plane is proposed to allow the clear attribution of experimentally measured parameters of the reaction medium to a certain stage of interaction between metallic iron and aqueous solutions of humic substances, regardless of the specific properties of the latter.

Nature-like technologies can be considered as a logical development of green chemistry principles implemented to design novel materials and processes aimed at mimicking and reproducing natural life-sustaining mechanisms on molecular level. Humic substances which penetrate throughout the entire environment and represent from 50 to 90% of organic matter in soil and water ecosystems, play multiple life-sustaining functions on Earth. To name a few, HS regulate transport and availability of biogenic elements to plants, immobilize and mitigate toxicity of hazardous elements in the contaminated ecosystems, protect plants from non-specific abiotic stresses, play key role for fertility of soils determining water-retention and structure. Here we represent a novel platform for nature-inspired synthesis of soft and hybrid (nano)materials aimed at their use for soil and water clean up, carbon sequestration, soil fertility restoration. It is based on a smart use of natural hyperbranched polyelectrolytes – humic substances, which possess multiple functional groups including carboxyl, hydroxyl, amide, and others. Multiple functional groups of HS make them amenable both for classical chemical modification as well as for producing interpolyelectrolyte complexes. In this work, we present both approaches for manufacturing silicon-containing humic derivatives and supramolecular complexes with acquired new property – self-adhesion to both inorganic and bio-surfaces. The synthesis is conducted using humic materials from different sources and functional organosilanes. Self-assembly of the supramolecular silicon-humic systems occurs with formation of humic-silsesquioxane networks capable to adhere to mineral surfaces. This process is similar to immobilization of organic coatings to mineral surfaces. We have shown how this process can be realized in the ground waters for the purposes of the environmental clean up. We have also proposed to use the silicon-humic complexes for improving humus content of soils and for reconstructing soil restoration processes both in the lab and in the field. Another field of our research is synthesis of iron-containing humics-stabilized nanoparticles (NPs), which can be used as a source for plants nutrition instead of synthetic iron chelates. The idea is based on the natural phenomenon that in soils, water-stable sols of iron-containing NPs are formed due to complexing with HS, which can bind large amounts of poorly ordered iron (hydr)oxides providing for stabilization of colloidal iron in the form of NPs. It has been numerously shown that the presence of HS improves iron acquisition by plants in soils, but there was no systematic study so far with respect to a relationship between size and crystallinity of humics-stabilized iron-containing NPs and their availability to plants. We have conducted such a study and could establish conditions when humics-stabilized NPs could be taken up by plants with similar efficiency as FeEDTA. The presented data demonstrate good prospects for a use of green humics-based materials in nature-like technologies. We also hope that these studies will give rise to new branch of chemistry and technology which can be called ecoadaptive chemistry and technology.

The objective of this study was to synthesize iron (hydr)oxide nanoparticles (IONPs) stabilized by humic substances, and to estimate the feasibility of their use for foliar application on iron deficient plants. The IONPs were synthesized by rapid hydrolysis of iron(III) nitrate in a solution of potassium humate. The iron speciation and nanoparticle morphologies were characterized using X-ray diffraction, transmission electron microscopy, and M&ouml, ssbauer spectroscopy. The obtained sample of IONPs was applied at concentrations of 1- and 10-mM Fe, and 0.2% urea was used as an adjuvant. Wheat plants (Triticum aestivum L. cv. L15) were used for the iron uptake test. For both of the concentrations tested, spraying the nanoparticles resulted in a 70&ndash, 75% higher iron content in wheat leaves compared to ferric ammonium salt of ethylenediaminetetraacetic acid (Fe-EDTA). The synergistic effect of humic substances acting as a surfactant seemed to promote an increase in the iron uptake of the ferrihydrite nanoparticles compared to the aqueous Fe-EDTA solution used in this study. We concluded that humic-stabilized IONPs are much better suited to foliar application as compared to soil amendment when applied as a source of iron for plants. This is because humic substances act as a capping agent for nanoparticles and the surfactants enhance iron penetration into the leaf.

The introduction of various concentrations of chemically modified humic compounds (HC) with different redox characteristics into the media with free and immobilized anaerobic consortia accumulating landfill gases was studied as approach to their functioning management. For this purpose, quinone (hydroquinone, naphthoquinone or methylhydroquinone) derivatives of HC were synthesized, which made it possible to vary the redox and antioxidant properties of HC as terminal electron acceptors in methanogenic systems. The highest acceptor properties were obtained with potassium humate modified by naphthoquinone. To control possible negative effect of HC on the cells of natural methanogenic consortia, different bioluminescent analytical methods were used. The addition of HC derivatives, enriched with quinonones, to nutrient media at concentrations above 1 g/L decreased the energetic status of cells and the efficiency of the methanogenesis. For the first time, the significant decrease in accumulation of biogas was reached as effect of synthetic HC derivatives, whereas both notable change of biogas composition towards increase in the CO2 content and decrease in CH4 were revealed. Thus, modification with quinones makes it possible to obtain low-potential HC derivatives with strongly pronounced acceptor properties, promising for inhibition of biogas synthesis by methanogenic communities.

Many natural substances exhibit anti-inflammatory activity and considerable potential in prophylaxis and treatment of allergies. Knowing exact molecular targets, which is required for developing these as medicinal products, is often challenging for multicomponent compositions. In the present study we examined novel polyphenolic substance, a water-soluble fraction of wood lignin (laboratory code BP-Cx-1). In our previous study, a number of polyphenolic components of BP-Cx-1 (flavonoids, sapogenins, phenanthrenes etc.) were identified as the major carriers of biological activity of BP-Cx drug family, and several molecular targets involved in cancer and/or inflammation signaling pathways were proposed based on the results of the in vitro and in silico screening studies. In the present study, half maximal inhibitory concentration (IC50) of BP-Cx-1 was established with a radioligand method and a range of IC50 values between 22.8 and 40.3 μg/ml were obtained for adenosine receptors A1, A2A and prostaglandin receptors EP2, IP (PGI2). IC50 for serotonin 5-HT1 and for glucocorticoid GR receptors were 3.0 μg/ml and 12.6 μg/ml, respectively, both being within the range of BP-Cx-1 concentrations achievable in in vivo models. Further, distribution of [3H] labelled BP-Cx-1 in NIH3T3 murine fibroblasts and MCF7/R carcinoma cells was studied with autoradiography. [3H]-BP-Cx-1 (visualized as silver grains produced by tritium beta particles) was mainly localized along the cell membrane, in the perinuclear region and in the nucleus, suggesting ability of BP-Cx-1 to enter cells and bind to membrane or cytosol receptors. In our experiment, we observed the effect of BP-Cx-1 on maturation of dendritic cells (DCs): downregulation of expression of the lipid-presentation molecule CD1a, co-stimulatory molecules CD80, CD83 and CD 40, decreased production of pro-inflammatory cytokines IL-4 and TNF-α and increased production of anti-inflammatory cytokine IL-10. It is hypothesized that [3H]-BP-Cx-1 detectable in the nucleus is part of the activated GR complex, known to be involved in regulation of transcription of genes responsible for the anti-inflammatory response. Based on IC50, cell distribution data and results of the experiment with DCs it is suggested that the in vivo effects of BP-Cx-1 are mediated via GR and 5-HT1 receptors thus promoting development of tolerogenic effector function in dendritic cells.

Natural products, such as humic substances (HS) and shilajit, are known to possess antiviral activity. Humic-like components are often called as carriers of biological activity of shilajit. The goal of this study was to evaluate anti-HIV activity of well characterized HS isolated from coal, peat, and peloids, and compare it to that of water-soluble organic matter (OM) isolated from different samples of Shilajit. The set of humic materials included 16 samples of different fractional composition: humic acid (HA), hymatomelanic acid (HMA), fulvic acid (FA). The set of shilajit OM included 19 samples of different geographic origin and level of alteration. The HIV-1 p24 antigen assay and cell viability test were used for assessment of antiviral activity. The HIV-1 Bru strain was used to infect CEM-SS cells. The obtained EC50 values varied from 0.37 to 1.4 mg·L-1 for the humic materials, and from 14 to 142 mg·L-1 for the shilajit OM. Hence, all humic materials used in this study outcompeted largely the shilajit materials with respect to anti-HIV activity: For the humic materials, the structure-activity relationships revealed strong correlation between the EC50 values and the content of aromatic carbon indicating the most important role of aromatic structures. For shilajit OM, the reverse relationship was obtained indicating the different mechanism of shilajit activity. The FTICRMS molecular assignments were used for ChEMBL data mining in search of the active humic molecules. As potential carriers of antiviral activity were identified aromatic structures with alkyl substituents, terpenoids, N-containing analogs of typical flavonoids, and aza-podophyllotoxins. The conclusion was made that the typical humic materials and Shilajit differ greatly in molecular composition, and the humic materials have substantial preferences as a natural source of antiviral agents as compared to shilajit., Highlights • Comparison of anti-HIV activity measured for the two large sets of humic materials and Shilajit water extracts demonstrated much higher activity of the humic materials. • Structure-activity relationships revealed leading role of aromatic structures in anti-HIV activity of humic substances, and the opposite trend was observed for shilajit. • 13C NMR and FTICR MS studies revealed structural similarity of shilajit extract to fulvic acid. • Different mode of antiviral action is suggested for aromatics-rich humic materials (humic acids, hymatomelanic acids) and N-rich shilajit. • This work was partially supported by the Russian Science Foundation: project 20-63-47070 in the part of structural characterization of the humic materials used in this study, and project 19-75-00092 in the part of FTICR MS data-mining and chemometrics.

More than 50% of engineered nanomaterials released into the environment contain silver nanoparticles (Ag-NPs). The mobility, bioavailability, and toxicity of engineered Ag-NPs are known to depend on their properties and the environmental conditions. However, almost nothing is known about the fate of naturally occurring Ag-NPs, which are formed during the reduction of Ag+ by natural organic matter, primarily humic substances (HSs). The aim of this work was to study the interaction of soils and plants with simulated natural Ag-NPs, i.e., Ag-NPs stabilized with HSs (Ag-HS-NPs). To reach this goal, Ag-HS-NPs were synthesized, and their sorption-desorption behavior on two contrasting soils (a mineral soil and one rich in organic matter) was evaluated, including alterations in the mineral composition of the soil solution. In parallel, the influence of Ag-HS-NPs on wheat seedling growth was estimated. Introduction of Ag-HS-NPs into the soils resulted in a 1.3- to 2-fold or greater increase in the concentration of many elements in the soil solution (Al, Cr, Cu, Fe, etc.), and this effect was more pronounced for the organic soil than for the mineral soil. To explain this effect, we hypothesized that this phenomenon was due to the partial dissolution of Ag-HS-NPs leading to the production of Ag+ that could be further reduced by soil organic matter, which was correspondingly oxidized. Therefore, the partial breaking of soil aggregates because of the decomposition of soil organic matter in the presence of Ag-HS-NPs could be expected. Plants treated with Ag-HS-NPs demonstrated a lower rate of water uptake, which decreased by over 81%. The shoot and root biomass decreased by 15–17% and by 13–15%, respectively. This study clearly demonstrates an underestimated hazard of Ag-NPs formed in nature in terms of their ability to adversely affect the environment.

Nanodiamonds produced by the detonation method are used as lubricants, polishing compositions, polymer composites, etc. To reveal how nanodiamonds differ in terms of surface properties and interact with natural organic matter, we used tritium-labelled humic substances to quantitively describe their adsorption onto the nanodiamond surface. It was shown that the adsorption of humic substances onto nanodiamonds resulted in fractionation of humic substances that was strongly dependent on the zeta potential of nanodiamonds in water but did not significantly affect the uptake of nanodiamonds by wheat seedlings. The uptake of nanodiamond particles by plants was determined by the functional composition of the particle surface.

The goal of this study was to establish reactivity of lignin-derived synthetic polyphenolic material under irradiation by ultraviolet (254 nm) and visible (460 and 525 nm) light in order to deeper examine relationships between the optical properties of this complex mixture and its individual constituents. In all photoirradiation experiments, blue shift of the fluorescence spectrum was observed. We aimed at understanding whether these changes could be explained on the basis of the chromophore interactions hypothesis, which implies destruction of electron-acceptor pairs via free radical transformations to be responsible for the alteration of optical properties. For this, changes in molecular composition were explored by Fourier transform ion cyclotron resonance mass spectrometry. Irradiation with UV resulted in a pronounced oxidation of polyphenols, which was manifested in the van Krevelen diagram by the formation of components with higher O/C ratio. At the same time, irradiation by visible light had led to the appearance of more condensed molecules depleted of oxygen. Consideration of changes in relative contribution of 500 most abundant components in polyphenol materials revealed higher transformation yields under UV light as compared to the visible light. Further studies using deuteromethylation followed by Fourier transform ion cyclotron resonance mass spectrometry enabled to enumerate the number of carboxylic groups in individual components of the parent polyphenol material. It was shown that at all wavelengths irradiation mainly impacted carboxylic-rich unsaturated and aromatic compounds, which can be considered as strong electron-acceptors. We suggest that their transformation is responsible for the blue shift of fluorescence spectrum, thus emphasizing the role of chromophore interaction mechanism of the optical properties formation.

Complex plant-derived polyphenols are promising for biomedical application. Their high complexity prevents the use of conventional pharmacopoeia techniques to perform quality control. The goal of this study was to apply ultra-high-resolution mass spectrometry to evaluate the batch-to-batch consistency of the molecular composition of a polyphenolic ligand using appropriate statistical metrics. METHODS Polyphenols were obtained by hydrolyzed-lignin oxidation. Manufacturing was performed under a range of reaction conditions: heating cycles, oxygen flows, purification. Direct-injection Fourier transform ion cyclotron resonance mass spectrometry (DI FTICR-MS) was applied to analyze reaction products. For pairwise comparison Jaccard and Tanimoto similarities calculations were proposed. In addition, principal component analysis (PCA) was applied for sample grouping based on the molecular class contributions. RESULTS FTICR-MS analysis revealed moderate Jaccard similarity of products synthesized under the same conditions, which shared about 50% of the formulae calculated in each sample. The intensity-based Tanimoto index indicated high similarity of major components distribution of samples synthesized under standard conditions, while products obtained with variations in synthetic conditions were significantly different. PCA of molecular class contributions showed similar grouping with a high cumulative score. CONCLUSIONS FTICR-MS provides robust metrics for the examination of batch-to-batch consistency of synthetic polyphenol materials. This approach can be proposed for the analysis of reference samples and for development of complementary methods for quality control of medicinal agents based on various biologically active matrices.

The goal of this study was to establish a relationship between the optical properties of soil dissolved organic matter (DOM) and acidic functions carried out by its individual constituents. We obtained 12 fractions of DOM samples using sequential solid phase extraction on nonionic sorbent at steadily lowered pH values: 7, 5, 3, 2, which correspond to low bounds of p

The Ob-Irtysh River system is the seventh-longest one in the world. Unlike the other Great Siberian rivers, it is only slightly impacted by the continuous permafrost in its low flow. Instead, it drains the Great Vasyugan mire, which is the world largest swamp, and receives huge load of the Irtysh waters which drain the populated lowlands of the East Siberian Plain. The central challenge of this paper is to understand the processes responsible for molecular transformations of natural organic matter (NOM) in the Ob-Irtysh river system along the South-North transect. For solving this task, the NOM was isolated from the water samples collected along the 3,000 km transect using solid-phase extraction. The NOM samples were further analyzed using high resolution mass spectrometry and optical spectroscopy. The obtained results have shown a distinct trend both in molecular composition and diversity of the NOM along the South-North transect: the largest diversity was observed in the Southern “swamp-wetland” stations. The samples were dominated with humic and lignin-like components, and enriched with aminosugars. After the Irtysh confluence, the molecular nature of NOM has changed drastically: it became much more oxidized and enriched with heterocyclic N-containing compounds. These molecular features are very different from the aliphatics-rich permafrost NOM. They witnesses much more conservative nature of the NOM discharged into the Arctic by the Ob-Irtysh river system. In general, drastic reduction in molecular diversity was observed in the northern stations located in the lower Ob flow.

Here, we report on sequential solid-phase extraction of leonardite hymatomelanic acid (CHM) on a non-ionic sorbent at four steadily lowered pH values: 7, 5, 3, and 2, yielding fractions with different acidic properties. Using nuclear magnetic resonance (NMR) spectroscopy and ultrahigh-resolution mass spectrometry, we revealed a gradual shift of dominating scaffolds in the fractions of CHM from reduced saturated to oxidized aromatic compounds. An increase on the average aromaticity of the CHM fractions was accompanied by a red shift in fluorescence spectra. These results were supported by heteronuclear single quantum coherence and heteronuclear multiple bond correlation NMR experiments. We have demonstrated that the CHM fraction isolated at pH 5 was dominated by aliphatic carboxyl carriers, while the pH 3 fraction was dominated by aromatic carboxyl acids. The developed fractionation technique will enable deeper insight on structure-property relationships and the design of the humic-based materials with tailored reactive properties.

The objective of this study was to shed light on structural features which underlay intensity of long wave absorbance of natural organic matter (NOM) using 1H NMR spectroscopy. For this purpose, a set of the NOM samples was assembled from arctic and nonarctic sampling sites (the Kolyma river basin and Moscow region, respectively). It was to ensure a substantial difference in the humification degree of the isolated organic matter-the biogeochemical proxy of the long-wave absorbance of NOM. The assembled NOM set was analyzed using solution-state 1H NMR spectroscopy. The distribution of both backbone and exchangeable protons was determined using acquisition of spectra in three different solvents. The substantially higher contribution of nonfunctionalized aliphatic moieties CHn (e.g., materials derived from linear terpenoids, MDLT) in the arctic NOM samples was revealed as compared to the nonarctic ones. The latter were characterized with the higher content of CHα protons adjacent to electron-withdrawing groups which belong to carboxyl rich alicyclic moieties (CRAMs) or to aromatic constituents of NOM. We have calculated a ratio of CHn to CHα protons as a structural descriptor which showed significant inverse correlation to intensity of long wave absorbance assessed with a use of E4/ E6 ratio and the slope of absorption spectrum. The steric hindrance of aromatic chromophoric groups of the NOM ensemble by bulky nonfunctionalized aliphatic moieties (e.g., MDLT) was set as a hypothesis for explanation of this phenomenon. The bulky aliphatics might increase a distance between the interacting groups resulting in inhibition of electronic (e.g., charge-transfer) interactions in the NOM ensemble. The obtained relationships were further explored using Fourier transform mass spectrometry as complementary technique to 1H NMR spectroscopy. The data obtained on correlation of molecular composition of NOM with 1H NMR data and optical properties were very supportive of our hypothesis that capabilities of NOM ensemble of charge transfer interactions can be dependent on structural arrangement and relative abundance of nonabsorbing aliphatic moieties.

The use of humic substances is under thorough discussion of state-of-the-art agricultural science. They are marketed mostly as concentrated aqueous solutions of potassium or sodium humates, which are produced by alkali extraction of raw organic matter such as lignite, leonardite, or peat. Due to the presence of clay minerals in the raw materials, humate solutions are characterized with a substantial content of silicates. At the same time, rapid quantification techniques for selective determination of humic components and silicates in humate solutions are missing. The aim of this work was to develop an IR spectroscopic technique for rapid quantification of humic substances (HSs) and silicates in concentrated humate solutions with the minimum sample preparation, which could be used for quality control of humic fertilizers. Sodium humate from Sigma-Aldrich and two potassium humate fertilizers available on the market were used for the experiments. For FTIR measurements, thin-layer open-cell (DialPath) transmission and attenuated total reflectance (ATR) accessories were used. The secondary focus of this work was the use of a compact portable IR spectrometer, which can be used in the field. Total carbon analyzer and ICP-AES for determination of silicon and aluminum contents were used. FTIR spectra were registered for both dry samples and aqueous solutions of the humates. The most intense bands in IR spectra of HS were characterized with linear concentration dependences in the range of concentrations of 2–200 g L−1 of HS. The most sensitive band was shown to be 1560 cm−1. It corresponded to carboxyl groups (COO−) of humates (limits of detection [recalculated to carbon] for transmission and ATR modes are 3 and 1 g L−1, respectively). The band at 1015 cm−1 was attributed to silicate. It did not overlap with the bands of organic constituents and could be used for silicate quantification. The proposed technique can identify different trademarks of the fertilizers by the amount of both HS and silicate. Rapid determination of humate and silicate components comprising three samples of humic fertilizers was proposed without isolation of the analytes from solution.

Identification of molecular targets and mechanism of action is always a challenge, in particular - for natural compounds due to inherent chemical complexity. BP-Cx-1 is a water-soluble modification of hydrolyzed lignin used as the platform for a portfolio of innovative pharmacological products aimed for therapy and supportive care of oncological patients. The present study describes a new approach, which combines in vitro screening of potential molecular targets for BP-Cx-1 using Diversity Profile - P9 panel by Eurofins Cerep (France) with a search of possible active components in silico in ChEMBL - manually curated chemical database of bioactive molecules with drug-like properties. The results of diversity assay demonstrate that BP-Cx-1 has multiple biological effects on neurotransmitters receptors, ligand-gated ion channels and transporters. Of particular importance is that the major part of identified molecular targets are involved in modulation of inflammation and immune response and might be related to tumorigenesis. Characterization of molecular composition of BP-Cx-1 with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and subsequent identification of possible active components by searching for molecular matches in silico in ChEMBL indicated polyphenolic components, nominally, flavonoids, sapogenins, phenanthrenes, as the major carriers of biological activity of BP-Cx-1. In vitro and in silico target screening yielded overlapping lists of proteins: adenosine receptors, dopamine receptor DRD4, glucocorticoid receptor, serotonin receptor 5-HT1, prostaglandin receptors, muscarinic cholinergic receptor, GABAA receptor. The pleiotropic molecular activities of polyphenolic components are beneficial in treatment of multifactorial disorders such as diseases associated with chronic inflammation and cancer.

We conducted a comparative evaluation of the protective effect of humic substances (HSs) derived from coal and peat in seedlings of soft wheat Triticum aestivum L. in water deficit conditions caused by a PEG-6000 hyperosmotic solution. The protective effect of HSs was found to increase with an increase in the content of phenolic fragments. This finding may indicate that the antioxidant activity of HS was the main protective activity mechanism of HS in water deficit conditions.