Understanding the bioactivity of plant tannins: developments in analysis methods and structure-activity studies

Tannins, typically segregated into two major groups, the hydrolyzable tannins (HTs) and the proanthocyanidins (PAs), are plant polyphenolic secondary metabolites found throughout the plant kingdom. On one hand, tannins may cause harmful nutritional effects on herbivores, for example insects, and hence they work as plants’ defense against plant-eating animals. On the other hand, they may affect positively some herbivores, such as mammals, for example by their antioxidant, antimicrobial, anti-inflammatory or anticarcinogenic activities. This thesis focuses on understanding the bioactivity of plant tannins, their anthelmintic properties and the tools used for the qualitative and quantitative analysis of this endless source of structural diversity. The first part of the experimental work focused on the development of ultra-high performance liquid chromatography−tandem mass spectrometry (UHPLC-MS/MS) based methods for the rapid fingerprint analysis of bioactive polyphenols, especially tannins. In the second part of the experimental work the in vitro activity of isolated and purified HTs and their hydrolysis product, gallic acid, was tested against egg hatching and larval motility of two larval developmental stages, L1 and L2, of a common ruminant gastrointestinal parasite, Haemonchus contortus. The results indicated clear relationships between the HT structure and the anthelmintic activity. The activity of the studied compounds depended on many structural features, including size, functional groups present in the structure, and the structural rigidness. To further understand tannin bioactivity on a molecular level, the interaction between bovine serum albumin (BSA), and seven HTs and epigallocatechin gallate was examined. The objective was to define the effect of pH on the formation on tannin–protein complexes and to evaluate the stability of the formed complexes by gel electrophoresis and MALDI-TOF-MS. The results indicated that more basic pH values had a stabilizing effect on the tannin–protein complexes and that the tannin oxidative activity was directly linked with their tendency to form covalently stabilized complexes with BSA at increased pH.Siirretty Doriast

Influence of the Hydrolyzable Tannin Structure on the Characteristics of Insoluble Hydrolyzable Tannin-Protein Complexes

Precipitation of bovine serum albumin (BSA) by 21 hydrolyzable tannins (HTs) and the characteristics of the insoluble complexes were studied stoichiometrically by ultra-performance liquid chromatography. With regard to HT monomers, the protein precipitation and the characteristic of the formed precipitates were unique for each studied HT and depended upon the functional groups present in the structures. The monomeric units comprising the oligomers formed the functional units important for the protein precipitation capacity, and small structural differences among the monomer units were less important than the overall oligomer size and flexibility. In addition, the greater tendency of certain HTs to form insoluble complexes when mixed with BSA was partially linked to the higher self-association and consequent stronger cooperative binding of these HTs with BSA

Legacy of agrochemicals in the circular food economy: Glyphosate-based herbicides introduced via manure fertilizer affect the yield and biochemistry of perennial crop plants during the following year

Conventional agricultural practices favoring the use of glyphosate-based herbicides (GBHs) increase the risk of GBH residues ending up in animal feed, feces, and, eventually, manure. The use of poultry manure as organic fertilizer in the circular food economy increases the unintentional introduction of GBH residues into horticultural and agricultural systems, with reportedly negative effects on the growth and reproduction of crop plants. To understand the potential lasting effects of exposure to GBH residues via organic manure fertilizers, we studied strawberry (Fragaria x vescana) plant performance, yield quantity, biochemistry, folivory, phytochemistry, and soil elemental composition the year after exposure to GBH. Although plants exposed to GBH residues via manure fertilizer were, on average, 23% smaller in the year of exposure, they were able to compensate for their growth during the following growing season. Interestingly, GBH residue exposure in the previous growing season led to a trend in altered plant size preferences of folivores during the following growing season. Furthermore, the plants that had been exposed to GBH residues in the previous growing season produced 20% heavier fruits with an altered composition of phenolic compounds compared to non-exposed plants. Our results indicate that GBHs introduced via manure fertilizer following circular economy practices in one year can have effects on perennial crop plants in the following year, although GBH residues in soil have largely vanished.Legacy of agrochemicals in the circular food economy: Glyphosate-based herbicides introduced via manure fertilizer affect the yield and biochemistry of perennial crop plants during the following yearpublishedVersio

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. View Full-TextKeywords: high-resolution mass spectrometry; orbitrap; oxidation; tannins; UHPLC-DAD-MS/MS</div

Isolation of chemically well-defined semipreparative liquid chromatography fractions from complex mixtures of proanthocyanidin oligomers and polymers

In this study, a semipreparative liquid chromatography method was developed for the isolation of chemically well-defined proanthocyanidin (PA) oligomer and polymer fractions. The aim was to achieve better separation than traditionally achieved for the PAs with other chromatographic methods. The method was tested with eleven PA rich Sephadex LH-20 fractions, which originated from eleven different plant species. The resulting semipreparative fractions were analyzed by both triple quadrupole and high-resolution mass spectrometry assisted by ultrahigh-performance liquid chromatography (UPLC) separation. The results showed remarkable differences in the procyanidin to prodelphinidin ratio, mean degree of polymerization, and specific oligomeric and polymeric content. However, some of these features indicated consistent patterns between species as the function of UPLC retention time. The developed method enables the production of tens of well-defined fractions of PA oligomers and polymers from the unresolved chromatographic PA hump. Accordingly, this allows researchers to explore the most bioactive parts of the complex PA humps of any plant species, which have not been possible earlier.</div

Variability in Foliar Ellagitannins of Hippophae rhamnoides L. and Identification of a New Ellagitannin, Hippophaenin C

Berries of common sea-buckthorn (Hippophae rhamnoides L.) are well-known and used for their bioactive components, and while there is a considerable amount of research on the leaves as well, their ellagitannins (ETs) have not been a prominent focus of research. We identified and quantified ten major hydrophilic polyphenols, all ETs, in H. rhamnoides leaves and compared their abundance between 58 plant individuals. Of these compounds, hippophaenin C was characterized as a new ellagitannin by various spectrometric methods. The total concentrations of ETs ranged from 42.5 mg g(-1) dry weight (DW) to 109.1 mg g(-1) DW between individual plants. Among the ETs, hippophaenin C, stachyurin, and casuarinin were on average the most abundant compounds. Sexes did not differ significantly, while cultivars showed variation in some ETs. These results suggest that H. rhamnoides leaves could be a potential and rich source of several ETs

Inhibition of Pneumolysin Cytotoxicity by Hydrolysable Tannins

Streptococcus pneumoniae causes invasive infections such as otitis media, pneumonia and meningitis. It produces the pneumolysin (Ply) toxin, which forms a pore onto the host cell membrane and has multiple functions in the pathogenesis of S. pneumoniae. The Ply C-terminal domain 4 mediates binding to membrane cholesterol and induces the formation of pores composed of up to 40 Ply monomers. Ply has a key role in the establishment of nasal colonization, pneumococcal transmission from host to host and pathogenicity. Altogether, 27 hydrolysable tannins were tested for Ply inhibition in a hemolysis assay and a tannin-protein precipitation assay. Pentagalloylglucose (PGG) and gemin A showed nanomolar inhibitory activity. Ply oligomerization on the erythrocyte surface was inhibited with PGG. PGG also inhibited Ply cytotoxicity to A549 human lung epithelial cells. Molecular modelling of Ply interaction with PGG suggests that it binds to the pocket formed by domains 2, 3 and 4. In this study, we reveal the structural features of hydrolysable tannins that are required for interaction with Ply. Monomeric hydrolysable tannins containing three to four flexible galloyl groups have the highest inhibitory power to Ply cytotoxicity and are followed by oligomers. Of the oligomers, macrocyclic and C-glycosidic structures were weaker in their inhibition than the glucopyranose-based oligomers. Accordingly, PGG-type monomers and oligomers might have therapeutic value in the targeting of S. pneumoniae infections

Legacy of agrochemicals in the circular food economy: Glyphosate-based herbicides introduced via manure fertilizer affect the yield and biochemistry of perennial crop plants during the following year

Conventional agricultural practices favoring the use of glyphosate-based herbicides (GBHs) increase the risk of GBH residues ending up in animal feed, feces, and, eventually, manure. The use of poultry manure as organic fertilizer in the circular food economy increases the unintentional introduction of GBH residues into horticultural and agricultural systems, with reportedly negative effects on the growth and reproduction of crop plants. To understand the potential lasting effects of exposure to GBH residues via organic manure fertilizers, we studied strawberry (Fragaria x vescana) plant performance, yield quantity, biochemistry, folivory, phytochemistry, and soil elemental composition the year after exposure to GBH. Although plants exposed to GBH residues via manure fertilizer were, on average, 23% smaller in the year of exposure, they were able to compensate for their growth during the following growing season. Interestingly, GBH residue exposure in the previous growing season led to a trend in altered plant size preferences of folivores during the following growing season. Furthermore, the plants that had been exposed to GBH residues in the previous growing season produced 20% heavier fruits with an altered composition of phenolic compounds compared to non-exposed plants. Our results indicate that GBHs introduced via manure fertilizer following circular economy practices in one year can have effects on perennial crop plants in the following year, although GBH residues in soil have largely vanished.</div

Comparative analysis of the anthelmintic efficacy of European heather extracts on Teladorsagia circumcincta and Trichostrongylus colubriformis egg hatching and larval motility

Background Gastrointestinal nematode (GIN) control is traditionally achieved with the use of anthelmintic drugs, however due to regulations in organic farming and the rise in anthelmintic resistance, alternatives are sought after. A promising alternative is the use of bioactive plant feeding due to the presence of plant secondary metabolites (PSMs) such as proanthocyanidins (PAs). This study focussed on the perennial shrub heather (Ericaceae family), a plant rich in PAs, highly abundant across Europe and with previously demonstrated anthelmintic potential. Methods In vitro assays were used to investigate heather’s anthelmintic efficacy against egg hatching and larval motility. Heather samples were collected from five European countries across two seasons, and extracts were tested against two GIN species: Teladorsagia circumcincta and Trichostrongylus colubriformis. Polyphenol group-specific ultraperformance liquid chromatography-tandem mass spectrometry analysis was performed to identify relevant polyphenol subgroups present, including the PA concentration and size and ratio of the subunits. Partial least squares analysis was performed to associate efficacy with variation in PSM composition. Results Heather extracts reduced egg hatching of both GIN species in a dose-dependent manner by up to 100%, while three extracts at the highest concentration (10 mg/ml) reduced larval motility to levels that were not significantly different from dead larvae controls. PAs, particularly the procyanidin type, and flavonol derivatives were associated with anthelmintic activity, and the particular subgroup of polyphenols associated with the efficacy was dependent on the GIN species and life stage. Conclusions Our results provide in vitro evidence that heather, a widely available plant often managed as a weed in grazing systems, has anthelmintic properties attributed to various groups of PSMs and could contribute to sustainable GIN control in ruminant production systems across Europe

Large variability of proanthocyanidin content and composition in sainfoin (onobrychis viciifolia)

Proanthocyanidins (PAs) in sainfoin (Onobrychis viciifolia Scop.) are of interest to ameliorate the sustainability of livestock production. However, sainfoin forage yield and PA concentrations, as well as their composition, require optimization. Individual plants of 27 sainfoin accessions from four continents were analyzed with LC-ESI-QqQ-MS/MS for PA concentrations and simple phenolic compounds. Large variability existed in PA concentrations (23.0–47.5 mg g–1 leaf dry matter (DM)), share of prodelphinidins (79–96%), and mean degree of polymerization (11–14) among, but also within, accessions. PAs were mainly located in leaves (26.8 mg g–1 DM), whereas stems had less PAs (7.8 mg g–1 DM). Overall, high-yielding plants had lower PA leaf concentrations (R2 = 0.16, P < 0.001) and fewer leaves (R2 = 0.66, P < 0.001). However, the results show that these two trade-offs between yield and bioactive PAs can be overcome