Exploring the Interactions between Plant Proanthocyanidins and Thiabendazole: Insights from Isothermal Titration Calorimetry.

Anthelmintic resistance in gastrointestinal nematodes produces substantial challenges to agriculture, and new strategies for nematode control in livestock animals are called for. Natural compounds, including tannins, with proven anthelmintic activity could be a functional option as structurally diverse complementary compounds to be used alongside commercial anthelmintics. However, the dual use of two anthelmintic components requires an understanding of the pharmacological effects of the combination, while information concerning the interactions between plant-based polyphenols and commercial anthelmintics is scarce. We studied the direct interactions of proanthocyanidins (PAs, syn. condensed tannins) and a commercial anthelmintic thiabendazole, as a model substance of benzimidazoles, by isothermal titration calorimetry (ITC). Our results show evidence of a direct interaction of an exothermic nature with observed enthalpy changes ranging from 0 to -30 kJ/mol. The strength of the interaction between PAs and thiabendazole is mediated by structural characteristics of the PAs with the strongest positive correlation originating from the presence of galloyl groups and the increased degree of polymerization.

Alkaline oxidization can increase the in vitro antiparasitic activity of proanthocyanidin-rich plant extracts against Ascarissuum.

Proanthocyanidins (PAs) are a class of plant specialized metabolites with well-documented bioactivities such as antiparasitic effects. However, little is known about how the modification of PAs influences their bioactivity. The objective of this study was to investigate a wide range of PA-containing plant samples to determine if extracts containing PAs modified by oxidation had altered antiparasitic activities, compared to the original extracts that had not been modified in alkaline conditions. We extracted and analyzed samples from 61 proanthocyanidin-rich plants. The extracts were then oxidized under alkaline conditions. We used these non-oxidized and oxidized proanthocyanidin-rich extracts to conduct a detailed analysis of direct antiparasitic effects against the intestinal parasite Ascaris suum in vitro. These tests showed that the proanthocyanidin-rich extracts had antiparasitic activity. Modification of these extracts significantly increased the antiparasitic activity for the majority the extracts, suggesting that the oxidation procedure enhanced the bioactivity of the samples. Some samples that showed no antiparasitic activity before oxidation showed very high activity after the oxidation. High levels of other polyphenols in the extracts, such as flavonoids, was found to be associated with increased antiparasitic activity following oxidation. Thus, our in vitro screening opens up the opportunity for future research to better understand the mechanism of action how alkaline treatment of PA-rich plant extracts increases their biological activity and potential as novel anthelmintics.

Affinity of Tannins to Cellulose: A Chromatographic Tool for Revealing Structure-Activity Patterns.

Food, feed and beverage processing brings tannins into contact with macromolecules, such as proteins and polysaccharides, leading to different chemical and physical interactions. The interactions of tannins with proteins are well known but less is known about the affinity of tannins to polysaccharides. We used bacterial cellulose from nata de coco as a model compound to investigate how tannins and cellulose interact by adsorption measurements using UPLC-DAD. We also explored how the structure of tannins influences these interactions. The model tannins included nine individual structurally different hydrolysable tannins (HTs) and eight well-defined proanthocyanidin (PA) fractions with different monomeric units, mean degree of polymerization and both A- and B-type linkages. Tannins were found to have both strong and weak interactions with bacterial cellulose, depending on the exact structure of the tannin. For HTs, the main structural features affecting the interactions were the structural flexibility of the HT molecule and the number of free galloyl groups. For PAs, prodelphinidins were found to have a higher affinity to cellulose than procyanidins. Similarly to HTs, the presence of free galloyl groups in galloylated PAs and the flexibility of the PA molecule led to a stronger interaction. Adsorption measurements by UPLC-DAD proved to be a sensitive and rapid tool to evaluate the affinity of tannins to cellulose.

Tannins Can Have Direct Interactions with Anthelmintics: Investigations by Isothermal Titration Calorimetry.

Plant tannins are known for their anthelmintic and antiparasitic activities and have been increasingly studied to battle the ever-growing problem of anthelmintic resistance. While tannins have been shown to exhibit these activities on their own, one approach would be to use them as complementary nutrients alongside commercial anthelmintics. So far, research on the interactions between tannins and anthelmintics is limited, and few studies have reported both synergistic and antagonistic effects depending on the type of tannin and the method used. These interactions could either strengthen or weaken the efficacy of commercial anthelmintics, especially if tannin-rich diets are combined with anthelmintics used as oral drenches. To study these interactions, a series of hydrolysable tannins (HTs) was selected, and their direct interactions with thiabendazole (TBZ) were evaluated by isothermal titration calorimetry (ITC), which allowed the detection of the exothermic interaction but also the roles and significances of different structural features of HTs in these interactions. Our results show that HTs can have a direct interaction with the benzimidazole anthelmintic TBZ and that the interaction is strengthened by increasing the number of free galloyl groups and the overall molecular flexibility of HTs.

Identification of Oxindoleacetic Acid Conjugates in Quinoa (Chenopodium quinoa Willd.) Seeds by High-Resolution UHPLC-MS/MS.

Quinoa (Chenopodium quinoa Willd.) has a high nutritional value and it contains a high number and high amounts of specialized metabolites. These metabolites include, for example, phenolic acids, flavonoids, terpenoids and steroids. In addition, it is known to contain N-containing metabolites, such as betalains. Here, we report the presence and identification of 14 new oxindoleacetate conjugates in quinoa by high-resolution ultrahigh-performance liquid chromatography quadrupole-time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) and ultrahigh-resolution UHPLC-QOrbitrap-MS/MS. The oxindoleacetate conjugates were extracted from dried and ground quinoa seeds using either methanol/water or acetone/water (4:1, v/v) and were further concentrated into aqueous phase and analyzed by UHPLC with reverse-phase chromatography using acetonitrile and 0.1% aqueous formic acid as eluents. High-resolution hybrid LC-MS/MS techniques, including full scan MS with in-source collision, induced dissociation, and data dependent-MS2(TopN) with stepped normalized collision energies using N2 as collision gas and data-independent acquisition (MSE) using ramped collision energies and argon as collision gas enabled their analysis directly from the crude quinoa seed extract. The oxindoleacetate conjugates were found to be present in both conventional and organic farmed seeds. According to our best knowledge, this is the first time hydroxy-oxindoles have been reported in quinoa.

Chemotaxonomic Markers for the Leaf Buds of Common Finnish Trees and Shrubs: A Rapid UHPLC MS Fingerprinting Tool for Species Identification.

In this study, a chemotaxonomic tool was created on the basis of ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) for the identification of 13 common Finnish deciduous trees and shrubs from their leaf bud metabolites. The bud extracts were screened with UHPLC-ESI-QqQ-MS and UHPLC-ESI-Q-Orbitrap-MS to discover suitable markers for each species. Two approaches were tested in the marker selection: (1) unique species-specific markers to obtain selective fingerprints per species and (2) major markers to maximise the sensitivity of the fingerprints. The markers were used to create two selected ion-recording-based fingerprinting tools with UHPLC-ESI-QqQ-MS. The methods were evaluated for their selectivity, repeatability, and robustness in plant species identification by analysing leaf buds from several replicates of each species. The created chemotaxonomic tools were shown to provide unique chromatographic profiles for the studied species in less than 6 min. A variety of plant metabolites, such as flavonoids, triterpenoids, and hydroxycinnamic acid derivatives, were found to serve as good chemotaxonomic markers for the studied species. In 10 out of 13 cases, species-specific markers were superior in creating selective and repeatable fingerprints.

Interactions between Hydrolysable Tannins and Lipid Vesicles from Escherichia coli with Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) was used to study the interactions between hydrolysable tannins (HTs) and lipid vesicles prepared from a phospholipid extract of Escherichia coli (E. coli). A group of 24 structurally different HTs was selected, and structural differences affecting their affinities to interact with lipid vesicles in aqueous buffered media were identified. In general, the interactions between HTs and lipid vesicles were exothermic in nature, and ITC as a technique functioned well in the screening of HTs for their affinity for lipids. Most notably, the galloyl moiety, the structural flexibility of the entire tannin structure, the hydrophobicity of the tannin, and higher molecular weight were observed to be important for the stronger interactions with the lipids. The strongest interactions with lipids were observed for rugosins D and G. It was also observed that some HTs with moderate hydrophobicities, such as geraniin, chebulagic acid, and chebulinic acid, did not have any detectable interactions with the lipid vesicles, suggesting that a hydrophobic structure alone does not guarantee an affinity for lipids.

Ellagitannin-Lipid Interaction by HR-MAS NMR Spectroscopy.

Ellagitannins have antimicrobial activity, which might be related to their interactions with membrane lipids. We studied the interactions of 12 different ellagitannins and pentagalloylglucose with a lipid extract of Escherichia coli by high-resolution magic angle spinning NMR spectroscopy. The nuclear Overhauser effect was utilized to measure the cross relaxation rates between ellagitannin and lipid protons. The shifting of lipid signals in 1H NMR spectra of ellagitannin-lipid mixture due to ring current effect was also observed. The ellagitannins that showed interaction with lipids had clear structural similarities. All ellagitannins that had interactions with lipids had glucopyranose cores. In addition to the central polyol, the most important structural feature affecting the interaction seemed to be the structural flexibility of the ellagitannin. Even dimeric and trimeric ellagitannins could penetrate to the lipid bilayers if their structures were flexible with free galloyl and hexahydroxydiphenoyl groups.

Characterization of Natural and Alkaline-Oxidized Proanthocyanidins in Plant Extracts by Ultrahigh-Resolution UHPLC-MS/MS.

In this study, we analyzed the proanthocyanidin (PA) composition of 55 plant extracts before and after alkaline oxidation by ultrahigh-resolution UHPLC-MS/MS. We characterized the natural PA structures in detail and studied the sophisticated changes in the modified PA structures and the typical patterns and models of reactions within different PA classes due to the oxidation. The natural PAs were A- and B-type PCs, PDs and PC/PD mixtures. In addition, we detected galloylated PAs. B-type PCs in different plant extracts were rather stable and showed no or minor modification due to the alkaline oxidation. For some samples, we detected the intramolecular reactions of PCs producing A-type ether linkages. A-type PCs were also rather stable with no or minor modification, but in some plants, the formation of additional ether linkages was detected. PAs containing PD units were more reactive. After alkaline oxidation, these PAs or their oxidation products were no longer detected by MS even though a different type and/or delayed PA hump was still detected by UV at 280 nm. Galloylated PAs were rather stable under alkaline oxidation if they were PC-based, but we detected the intramolecular conversion from B-type to A-type. Galloylated PDs were more reactive and reacted similarly to nongalloylated PDs.

Field-Grown and In Vitro Propagated Round-Leaved Sundew (Drosera rotundifolia L.) Show Differences in Metabolic Profiles and Biological Activities.

Drosera rotundifolia L. is a carnivorous plant used in traditional medicine for its therapeutic properties. Because of its small size, its collection in nature is laborious and different cultivation methods have been studied to ensure availability. However, only a few studies exist where the lab-grown sundew tissue and field-grown sundew would have been compared in their functionality or metabolic profiles. In this study, the antioxidant and antiviral activities of lab-grown and field-grown sundew extracts and their metabolic profiles are examined. The effect of drying methods on the chromatographic profile of the extracts is also shown. Antioxidant activity was significantly higher (5-6 times) in field-grown sundew but antiviral activity against enterovirus strains coxsackievirus A9 and B3 was similar in higher extract concentrations (cell viability ca. 90%). Metabolic profiles showed that the majority of the identified compounds were the same but field-grown sundew contained higher numbers and amounts of secondary metabolites. Freeze-drying, herbal dryer, and oven or room temperature drying of the extract significantly decreased the metabolite content from -72% up to -100%. Freezing was the best option to preserve the metabolic composition of the sundew extract. In conclusion, when accurately handled, the lab-grown sundew possesses promising antiviral properties, but the secondary metabolite content needs to be higher for it to be considered as a good alternative for the field-grown sundew.

Bioactive Properties of the Aqueous Extracts of Endophytic Fungi Associated with Scots Pine (Pinus sylvestris) Roots.

Despite the continuing interest in various plant and natural products, only a small portion of the biologically active compounds from nature has been discovered and exploited. In this study, antioxidant and antibacterial properties of aqueous fractions of three endophytic fungi isolated from the roots of 8-year-old Scots pines (Pinus sylvestris) growing on a drained peatland were investigated. The endophytic fungi species were Acephala applanata, Phialocephala fortinii, and Humicolopsis cephalosporioides/Coniochaeta mutabilis. The bioactivities were examined using hydrogen peroxide scavenging and oxygen radical absorbance capacity tests as well as sensitive Escherichia coli-based biosensors, which produce a luminescent signal in the presence of substances with oxidative or genotoxic properties. In addition, cell models for Parkinson's disease, age-related macular degeneration, and osteoarthritis were used to evaluate the potential for pharmaceutical applications. The aqueous extracts of fungi and 19 out of 42 fractions were found to be active in one or more of the tests used. However, no activity was found in the age-related macular degeneration and osteoarthritis cell model tests. Additionally, bioactivity data was connected with metabolites putatively annotated, and out of 330 metabolites, 177 were interesting in view of the bioactivities investigated. A majority of these were peptides and all three fungal species shared a highly similar metabolome. We propose that Scots pine endophytic fungi are a rich source of interesting metabolites, and synergistic effects may cause the bioactivities, as they were found to vary after the fractionation process.

Partition Coefficients (logP) of Hydrolysable Tannins.

The partition coefficients (logP) between n-octanol and water of 47 purified and characterized hydrolysable tannins were measured with the shake flask method utilizing UPLC and HPLC with UV detection. Results show that galloyl glucoses and gallotannins are clearly more hydrophobic than ellagitannins but the differences in hydrophobicity within ellagitannins are more varied than within galloyl glucoses or gallotannins. Most notable structural features that were found to influence the hydrophobicity of ellagitannins were the number of free galloyl groups, acyclic versus cyclic polyol, substitution of the anomeric position of glucose and 4C1 versus 1C4 conformation of the glucopyranose core.

Antimicrobial Activities of Ellagitannins against Clostridiales perfringens, Escherichia coli, Lactobacillus plantarum and Staphylococcus aureus.

In this study, we tested the growth inhibition effect of 22 individual ellagitannins and of pentagalloylglucose on four bacterial species, i.e., Clostridiales perfringens, Escherichia coli, Lactobacillus plantarum and Staphylococcus aureus. All tested compounds showed antimicrobial effects against S. aureus, and almost all against E. coli and C. perfringens. For L. plantarum, no or very weak growth inhibition was detected. The level of inhibition was the greatest for S. aureus and the weakest for C. perfringens. For S. aureus, the molecular size or flexibility of ellagitannins did not show a clear relationship with their antimicrobial activity, even though rugosins E and D and pentagalloylglucose with four or five free galloyl groups had a stronger growth inhibition effect than the other ellagitannins with glucopyranose cores but with less free galloyl groups. Additionally, our results with S. aureus showed that the oligomeric linkage of ellagitannin might have an effect on its antimicrobial activity. For E. coli, the molecular size, but not the molecular flexibility, of ellagitannins seemed to be an important factor. For C. perfringens, both the molecular size and the flexibility of ellagitannin were important factors. In previous studies, corilagin was used as a model for ellagitannins, but our results showed that other ellagitannins are much more efficacious; therefore, the antimicrobial effects of ellagitannins could be more significant than previously thought.

Changes in Feed Proanthocyanidin Profiles during Silage Production and Digestion by Lamb.

Proanthocyanidins are plant specialized metabolites which are beneficial to animal nutrition and health. This study determined how proanthocyanidin profiles of sainfoin (Onobrychis viciifolia) and birdsfoot trefoil (Lotus corniculatus) change during the forage conservation process and along the digestive tract of lamb. We determined soluble, protein- and fiber-bound proanthocyanidins by spectrophotometric methods and soluble proanthocyanidin profiles by UPLC-MS/MS. During the conservation process, the total proanthocyanidin contents reduced in both forages and the relative proportion of insoluble proanthocyanidins increased, especially in sainfoin. The soluble proanthocyanidins, their mean degree of polymerization and the relative prodelphinidin share declined in both feed species. In the abomasum of lambs fed sainfoin silage, most of the proanthocyanidins were in insoluble form bound to proteins and fibers, but in the small and large intestines, the proportion of soluble proanthocyanidins increased again. For lambs fed birdsfoot trefoil, the trend was not so clear as proanthocyanidins were already mainly soluble in the abomasum. Nevertheless, a large part of soluble proanthocyanidins was recovered in the digestive tract but could not be detected by the UPLC-MS/MS method used. This study suggests that proanthocyanidins have probably been metabolized in the digestive tract by the resident microbiota.

Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC-Orbitrap-MS.

Endophytes are microorganisms living inside plant hosts and are known to be beneficial for the host plant vitality. In this study, we isolated three endophytic fungus species from the roots of Scots pine seedlings growing on Finnish drained peatland setting. The isolated fungi belonged to dark septate endophytes (DSE). The metabolic profiles of the hot water extracts of the fungi were investigated using Ultrahigh Performance Liquid Chromatography with Diode Array Detection and Electron Spray Ionization source Mass Spectrometry with Orbitrap analyzer (UPLC-DAD-ESI-MS-Orbitrap). Out of 318 metabolites, we were able to identify 220, of which a majority was amino acids and peptides. Additionally, opine amino acids, amino acid quinones, Amadori compounds, cholines, nucleobases, nucleosides, nucleotides, siderophores, sugars, sugar alcohols and disaccharides were found, as well as other previously reported metabolites from plants or endophytes. Some differences of the metabolic profiles, regarding the amount and identity of the found metabolites, were observed even though the fungi were isolated from the same host. Many of the discovered metabolites have been described possessing biological activities and properties, which may make a favorable contribution to the host plant nutrient availability or abiotic and biotic stress tolerance.

Correction: Tienaho, J., et al. Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC-Orbitrap-MS. Molecules 2019, 24, 2330.

The authors wish to make the following corrections to this paper published in Molecules[...].

Oxidatively Active Plant Phenolics Detected by UHPLC-DAD-MS after Enzymatic and Alkaline Oxidation.

We developed a combination of methods to estimate the alkaline oxidative conditions of the midgut of insect larvae and to reveal the alkaline and enzymatic oxidative activities for individual phenolic compounds present in the larval host plants. First, we monitored the in vitro isomerization of 5-O-caffeoylquinic acid (5-CQA) into 3-CQA, 4-CQA and 5-CQA at pH 9.0-11.0. Then we calculated the isomer ratios of 3-CQA, 4-CQA and 5-CQA from the frass of eight species of insect herbivores fed on foliage containing 5-CQA. The isomer ratios suggested that the midgut pH of these larvae ranged from 9.4 to around 10.1. Second, we developed an in situ enzymatic oxidation method that enabled oxidation of phenolics in a frozen plant sample at 30 °C by species- and tissue-specific enzymes. Then we measured the alkaline and enzymatic oxidative activities of the individual phenolics in 20 plant species by quantifying the proportion of the compound concentration lost due to the auto-oxidation of a plant extract at pH 10 and due to the enzymatic oxidation of the frozen plant sample at 30 °C. Our results showed that both of the oxidative activity types depended primarily on the type of phenolic compound, but the enzymatic oxidative activity depended also on the plant species and tissue type. This combination of methods offers an approach to characterize a wide array of phenolics that are susceptible to oxidation by the plant enzymes and/or by the alkaline conditions estimated to prevail in the insect midgut. We propose that these kinds of compound-specific results could guide future studies on specific plant-herbivore interactions to focus on the phenolics that are likely to be active rather than inactive plant phenolics.

Preparation of Thin Melanin-Type Films by Surface-Controlled Oxidation.

The preparation of thin melanin films suitable for applications is challenging. In this work, we present a new alternative approach to thin melanin-type films using oxidative multilayers prepared by the sequential layer-by-layer deposition of cerium(IV) and inorganic polyphosphate. The interfacial reaction between cerium(IV) in the multilayer and 5,6-dihydroxyindole (DHI) in the adjacent aqueous solution leads to the formation of a thin uniform film. The oxidation of DHI by cerium(IV) proceeds via known melanin intermediates. We have characterized the formed DHI-melanin films using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), UV-vis spectroscopy, and spectroelectrochemistry. When a five-bilayer oxidative multilayer is used, the film is uniform with a thickness of ca. 10 nm. Its chemical composition, as determined using XPS, is typical for melanin. It is also redox active, and its oxidation occurs in two steps, which can be assigned to semiquinone and quinone formation within the indole structural motif. Oxidative multilayers can also oxidize dopamine, but the reaction stops at the dopamine quinone stage because of the limited amount of the multilayer-based oxidizing agent. However, dopamine oxidation by Ce(IV) was studied also in solution by UV-vis spectroscopy and mass spectrometry in order to verify the reaction mechanism and the final product. In solution, the oxidation of dopamine by cerium shows that the indole ring formation takes place already at low pH and that the mass spectrum of the final product is practically identical with that of commercial melanin. Therefore, layer-by-layer formed oxidative multilayers can be used to deposit functional melanin-type thin films on arbitrary substrates by a surface-controlled reaction.

Design, discovery, modelling, synthesis, and biological evaluation of novel and small, low toxicity s-triazine derivatives as HIV-1 non-nucleoside reverse transcriptase inhibitors.

A set of top-ranked compounds from a multi-objective in silico screen was experimentally tested for toxicity and the ability to inhibit the activity of HIV-1 reverse transcriptase (RT) in cell-free assay and in cell-based assay using HIV-1 based virus-like particles. Detailed analysis of a commercial sample that indicated specific inhibition of HIV-1 reverse transcription revealed that a minor component that was structurally similar to that of the main compound was responsible for the strongest inhibition. As a result, novel s-triazine derivatives were proposed, modelled, discovered, and synthesised, and their antiviral activity and cellular toxicity were tested. Compounds 18a and 18b were found to be efficient HIV-1 RT inhibitors, with an IC50 of 5.6±1.1µM and 0.16±0.05µM in a cell-based assay using infectious HIV-1, respectively. Compound 18b also had no detectable toxicity for different human cell lines. Their binding mode and interactions with the RT suggest that there was strong and adaptable binding in a tight (NNRTI) hydrophobic pocket. In summary, this iterative study produced structural clues and led to a group of non-toxic, novel compounds to inhibit HIV-RT with up to nanomolar potency.

A study of the structure-activity relationship of oligomeric ellagitannins on ruminal fermentation in vitro.

The aim of this study was to investigate how the degree of oligomerization of ellagitannins (ET) influences their ability to alter ruminal fermentation. Dimeric to heptameric ET were isolated from rosebay willowherb (Epilobium angustifolium) flowers and purified. Ellagitannins were tested in vitro on a mixture of grass silage and buffered rumen fluid. Total gas production was measured in real time using an automated pressure evaluation system. Methane production was monitored at regular interval by gas chromatography for 72h. The effect of ET was evaluated on 2 sources of rumen fluid using a randomized block design. Ammonia nitrogen, volatile fatty acid concentration, and pH were measured at the end of the experiment. Results show that oligomeric ET decreased gas production and total volatile fatty acid concentration proportionally to their degree of oligomerization. Methane production was also decreased by all the tested compounds and dimer was less effective than the larger ET, which showed similar levels of activity. Additionally, willowherb's oligomeric ET decreased ammonia-nitrogen and branched-chain volatile fatty acid concentrations, thus indicating reduced protein degradation by ruminal bacteria. This effect showed a quadratic relationship with the degree of oligomerization and was maximal with the tetramer. In conclusion, this study shows that the degree of oligomerization of ET has more than a simple linear effect on fermentation parameters in vitro. Large oligomers, in fact, have more detrimental effects on volatile fatty acid and gas production than small ones, while being similarly effective at inhibiting methane production.