Polymeric Proanthocyanidins Research Articles

Novel flavan-3-ol-glutathione conjugates from the degradation of proanthocyanidins as highly bioactive antioxidants

Synthesis, preparation and antioxidant capacity evaluation of flavan-3-ol-glutathione conjugates.

Accumulation of Catechin and Proanthocyanidins in Black Poplar Stems After Infection by Plectosphaerella populi: Hormonal Regulation, Biosynthesis and Antifungal Activity.

Flavan-3-ols including the monomeric catechin and the polymeric proanthocyanidins (PAs) are abundant phenolic metabolites in poplar (Populus spp.) previously described to protect leaves against pathogen infection. However, it is not known whether stems are also defended in this way. Here we investigated flavan-3-ol accumulation, activity, and the regulation of formation in black poplar (P. nigra) stems after infection by a newly described fungal stem pathogen, Plectosphaerella populi, which forms canker-like lesions in stems. We showed that flavan-3-ol contents increased in P. populi-infected black poplar stems over the course of infection compared to non-infected controls. Transcripts of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes involved in the last steps of flavan-3-ol biosynthesis were also upregulated upon fungal infection indicating de novo biosynthesis. Amending culture medium with catechin and PAs reduced the mycelial growth of P. populi, suggesting that these metabolites act as anti-pathogen defenses in poplar in vivo. Among the hormones, salicylic acid (SA) was higher in P. populi-infected tissues compared to the non-infected controls over the course of infection studied, while jasmonic acid (JA) and JA-isoleucine (JA-Ile) levels were higher than controls only at the early stages of infection. Interestingly, cytokinins (CKs) were also upregulated in P. populi-infected stems. Poplar saplings treated with CK showed decreased levels of flavan-3-ols and SA in stems suggesting a negative association between CK and flavan-3-ol accumulation. Taken together, the sustained upregulation of SA in correlation with catechin and PA accumulation suggests that this is the dominant hormone inducing the formation of antifungal flavan-3-ols during P. populi infection of poplar stems.

Low-Cost Ru/C-Catalyzed Depolymerization of the Polymeric Proanthocyanidin-Rich Fraction from Bark To Produce Oligomeric Proanthocyanidins with Antioxidant Activity.

A new method has been developed for the high-value utilization of larch bark, which is regarded as a low-value byproduct of the logging industry. Polymeric proanthocyanidins (PPCs) were extracted from the Larix gmelinii bark and depolymerized by catalytic hydrogenolysis, using ruthenium/carbon (Ru/C) as the catalyst. The method has been found that although the molecular weight of the depolymerized product was significantly lower, the basic structural units were not destroyed, and the product retained a condensed flavanol polyphenol structure; the depolymerized product contains very little Ru metal and thus complies with food safety standards; the antioxidant properties of both the depolymerized products and PPCs were better than those of the commonly used antioxidant 2,6-di-tert-butyl-4-methylphenol. The relative molecular weight and steric hindrance of the depolymerized products were lower than those of the PPCs, leading to better antioxidant performance. A new technical route for the depolymerization of PPCs from the L. gmelinii bark is provided. The route offers practical and commercial advantages, and the product could have many applications as an antioxidant.

Understanding toward the Biophysical Interaction of Polymeric Proanthocyanidins (Persimmon Condensed Tannins) with Biomembranes: Relevance for Biological Effects.

Persimmon condensed tannins (PT) are highly polymerized (mDP = 26) and highly galloylated (72%) proanthocyanidins. Its pleiotropic effects in oxidation resistance, neuroprotection, hypolipidemia, and cardio-protection both in vitro and in vivo were widely reported. Because large proanthocyanidins are unlikely to be absorbed in the gastrointestinal tract, it is believed that the interaction of PT with biological membranes may play a crucial role in its biological activities. In the present study, the capacities of PT adsorbing to membrane, partitioning into membrane, and its influence on the membrane fluidity were investigated by fluorescence quenching, isothermal titration calorimetry (ITC) and fluorescence anisotropy measurements in a biomembrane-mimetic system composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), sphingomyelin (SPM), and cholesterol (CHOL). Besides, the effects of PT on the morphology and integrity of the cell membrane were studied by scanning electron microscopy (SEM) and fluorescence staining in the 3T3-L1 cell model. The results suggested that PT could affect cell membrane rafts domains, destroy the cell membrane morphology, and regulate cell membrane fluidity, which might contribute to its biological effects.

Characterisation of high molecular weight hop proanthocyanidins using Analytical Ultracentrifugation

We report the novel application of Analytical Ultracentrifugation (AUCF) to characterise the polymeric proanthocyanidin fraction of hops. Extraction of hop samples with 70% acetone (aq) followed by a C-18 Solid Phase Extraction yielded polyphenolic fractions for AUCF analysis. Sedimentation velocity experiments demonstrated the presence of discrete molecular weight bands of proanthocyanidins, as opposed to a continuous distribution of molecular weights. There were 4 such bands for Saaz hop (0.15, 1.1, 2.7 and 4.4S) and 3 bands for Magnum (0.15, 1.6 and 3.0S). The method resulted in a reproducible size (sedimentation coefficient) distribution for replicate runs of the same extract and for extracts prepared from different samples of the same hop variety. Sedimentation equilibrium experiments were then used to fit molecular weight distributions using the new SEDFIT-MSTAR method for the same samples. Thus we report for the first time polymeric proanthocyanidins in hops with molecular weights of up to 100 kDa in Saaz hop (or up to 56 kDa in Magnum). This represents the first application of AUCF to characterise complex fractions of polyphenolics extracted from botanical sources and the methodology developed should find wider application in the study of this diverse and bioactive class of compounds.

Selecting ultrafiltration membranes for fractionation of high added value compounds from grape pomace extracts

The purpose of the current study is to investigate the use of ultrafiltration membrane for the fractionation of phenolic compounds from subcritical water grape pomace extract and the separation of these compounds from other co-extracted components. The extract was assayed in a cross-flow apparatus against eleven membranes with molecular weight ranging from 100 to 2 kDa. Monitoring of the process was executed by determining performance parameters and retention coefficients of proteins, polysaccharides, sugars, phenolic and anthocyanin classes. Results indicated that retention of solutes was affected, not by size exclusion, but primarily by severe fouling phenomena due to polar solutes adsorption on the membrane surface. With the exception of the separation obtained between polymeric and monomeric proanthocyanidins, polysulfone membranes were not able to fractionate phenolic classes. Membranes starting of 20 kDa and over retained high percentages (>60%) of polysaccharides and proteins.

Influence of different drying methods on the quality of Japanese quince fruit

The aim of this study was to determine the effect of different drying methods – convective drying (at 50, 60, 70 °C), vacuum-microwave drying (at 120, 240, 480 W and 480 W with reduction to 120 W), a combination (of convective pre-drying (at 50, 60, 70 °C) and by vacuum-microwave finish drying (at 120 W)) and freeze-drying – on the quality factors of Japanese quince fruit, including phenolic compounds, antioxidant activity, and color. Drying kinetics were determined. The highest content of bioactive compounds was in samples after freeze-drying (total phenolic content – 57 g/kg dw; polymeric proanthocyanidins – 41 g/kg dw). The antioxidant activity measured by oxygen radical absorbance capacity (ORAC) assay was the highest for samples after vacuum-microwave drying at 480 W (0.8 mol Trolox/kg dw). Unfavorable changes in color, formation of hydroxymethylfurfural (HMF) and degradation of l-ascorbic acid as well as phenolic compounds were noted along with the increasing drying temperature and increasing magnetron power. The method that will ensure the proper appearance of dried fruits, storage stability and at the same time guarantee the retention of bioactive compounds seems to be freeze-drying.

VvLAR1 and VvLAR2 Are Bifunctional Enzymes for Proanthocyanidin Biosynthesis in Grapevine.

Proanthocyanidins (PAs) in grapevine (Vitis vinifera) are found mainly in berries, and their content and degree of polymerization are important for the mouth feel of red wine. However, the mechanism of PA polymerization in grapevine remains unclear. Previous studies in the model legume Medicago truncatula showed that 4β-(S-cysteinyl)-epicatechin (Cys-EC) is an epicatechin-type extension unit for nonenzymatic PA polymerization, and that leucoanthocyanidin reductase (LAR) converts Cys-EC into epicatechin starter unit to control PA extension. Grapevine possesses two LAR genes, but their functions are not clear. Here, we show that both Cys-EC and 4β-(S-cysteinyl)-catechin (Cys-C) are present in grapevine. Recombinant VvLAR1 and VvLAR2 convert Cys-C and Cys-EC into (+)-catechin and (-)-epicatechin, respectively, in vitro. The kinetic parameters of VvLARs are similar, with both enzymes being more efficient with Cys-C than with Cys-EC, the 2,3-cis conformation of which results in steric hindrance in the active site. Both VvLARs also produce (+)-catechin from leucocyanidin, and an inactive VvLAR2 allele reported previously is the result of a single amino acid mutation in the N terminus critical for all NADPH-dependent activities of the enzyme. VvLAR1 or VvLAR2 complement the M. truncatula lar:ldox double mutant that also lacks the leucoanthocyanidin dioxygenase (LDOX) required for epicatechin starter unit formation, resulting in increased soluble PA levels, decreased insoluble PA levels, and reduced levels of Cys-C and Cys-EC when compared to the double mutant, and the appearance of catechin, epicatechin, and PA dimers characteristic of the ldox single mutant in young pods. These data advance our knowledge of PA building blocks and LAR function and provide targets for grapevine breeding to alter PA composition.

Optimizing Nucleophilic Depolymerization of Proanthocyanidins in Grape Seeds to Dimeric Proanthocyanidin B1 or B2.

Depolymerization of polymeric proanthocyanidins (PPCs) in grape seeds into oligomeric proanthocyanidins (OPCs), especially the dimers, has important academic significance and practical value. Reaction conditions including nucleophilic reagent/PPC mass ratio, HCl concentration, reaction time, and temperature were systematically optimized by central composite design to maximize the yield of the dimeric product B2 or B1. The yield of B2 reached 3.35 mg mL-1 under the conditions of (-)-epicatechin/PPC mass ratio 2.8, HCl concentration 0.06 mol, reaction time 16 min and temperature 36 °C, and that of B1 reached 3.64 mg mL-1 under the conditions of (+)-catechin/PPC mass ratio 2.8, HCl concentration 0.07 mol, reaction time 17 min, and temperature 34 °C. Overall, this study has provided theoretical guidance and a practical approach to improvethe reaction process and economic value of proanthocyanidins in grape seed proanthocyanidin extract.

Biodegradation Profiles of Proanthocyanidin-Accumulating Alfalfa Plants Coexpressing Lc- bHLH and C1-MYB Transcriptive Flavanoid Regulatory Genes.

The utilization of the nutrient potential of alfalfa ( Medicago sativa L.) cannot be maximized because of its rapidly degradable protein content in the rumen, leading to waste and various digestive disorders. This might be alleviated if protein-binding proanthocyanidins are present in aerial parts of alfalfa forage in adequate amounts. The Lc (bHLH) and C1 (MYB) genes of maize are transcription factors known to be collectively involved in the regulation of anthocyanin biosynthetic pathways. The objective of this study was to investigate the effect of Lc and C1 gene transformations on the proanthocyanidin content, nutrient composition, and degradation characteristics of proteins and carbohydrates by comparing the transgenic alfalfa with its parental nontransgenic (NT) alfalfa and commercial AC-Grazeland cultivar. The DNA extracted from transgenic plants was tested for the presence of respective transgenes by amplification with specific primers of respective transgenes using PCR. Both Lc-single and LcC1-double transgenic alfalfa accumulated both monomeric and polymeric proanthocyanidins with total proanthocyanidins ranging from ca. 460 to 770 μg/g of DM. The C1-transgenic alfalfa did not accumulate proanthocyanidins similar to NT alfalfa. The C1 gene increased the NPN content significantly only in C1-single and Lc1C1-double transgenic alfalfa. The LcC1 combination seemed to have a synergic effect on reducing sugar in alfalfa. In contrast, the Lc gene appears to have a negative effect on starch content. The C1 gene tended to lower the PB3 content irrespective of the presence of the Lc gene. Although the cotransformation of Lc and C1 increased the total N/CHO ratio compared to Lc single gene transformation, the total N/CHO ratio of transgenic alfalfa was not significantly different from NT. In conclusion, Lc-bHLH single and LcC1 double gene transformation resulted in the accumulation of proanthocyanidins and affected the chemical profiles in alfalfa, which altered ruminal degradation patterns and impacted the nutrient availability of alfalfa in ruminant livestock systems.

The use of nanofiltration membranes for the fractionation of polyphenols from grape pomace extracts

Filtration experiments in batch concentration mode (with recycling of the retentate stream) of grape pomace extract were performed in laboratory filtration membrane equipment by using nine commercial nanofiltration (NF) membranes with an approximate molecular weight cut-off (MWCO) of 1000‒150 Da. The filtration experiments of the selected pomace extract were performed by modifying the most important operating variables: transmembrane pressure, tangential velocity, temperature, and the nature and MWCO of the membranes. The evolution of the cumulative permeate volumes and permeate fluxes with processing time was analyzed till a volume reduction factor (VRF) of 10 was reached. The effect of the mentioned operating conditions was discussed. The effectiveness of the filtration treatments was determined by the evaluation of the rejection coefficients for several families of polyphenols. Membranes possessing MWCO between 1000 and 500 Da were able to quantitatively recover polymeric proanthocyanidins in the concentrate stream and separate them from phenols that passed through the membrane into the permeate stream. On the other hand, the 600 to 300 Da membranes could also be used for the fractionation of monomeric phenolic families. The membranes were able to partially remove the anthocyanin fragments of phenolic acid derivatives and flavonols in the concentrate stream and at the same time.

Pea polyphenolics and hydrolysis processing alter microbial community structure and early pathogen colonization in mice

Health benefits associated with pea consumption have been attributed to the fiber and polyphenolic content concentrated within the pea seed coat. However, the amount of pea polyphenols can vary between cultivars, and it has yet to be studied whether pea polyphenols impact the intestinal microbiota. We hypothesized that pea polyphenols promote a healthy microbiome that supports intestinal integrity and pathogen colonization resistance. To investigate the effects of pea polyphenols, pea cultivars rich and poor in proanthocyanidins were supplemented in raw or acid hydrolyzed form to an isocaloric diet in mice. Acid hydrolysis increases the absorption of pea polyphenols by cleaving polymeric proanthocyanidins to their readily absorbable anthocyanidin monomers. After 3 weeks of diet, mice were challenged with Citrobacter rodentium and pathogen colonization and inflammation were assessed. Counter to our hypothesis, pea seed coat fraction supplementation, especially the non-hydrolyzed proanthocyanidin-rich fraction diet adversely increased C. rodentium pathogen load and inflammation. Ileal, cecal and colon microbial communities were notably distinct between pea seed cultivar and hydrolysis processing. The consumption of intact proanthocyanidins decreased microbial diversity indicating that proanthocyanidins have antimicrobial properties. Together our results indicate supplementation of raw pea seed coat rich in proanthocyanidins adversely affect intestinal integrity. However, acid hydrolysis processing restored community structure and colonization resistance, and the anthocyanidin-rich fractions reduced weight gain on a high fat diet. Establishing a clear understanding of the effects of pea fiber and polyphenolic form on health will help to develop research-based pea products and dietary recommendations.

Resistant starch formation through intrahelical V-complexes between polymeric proanthocyanidins and amylose

Reducing starch digestibility can significantly benefit efforts to combat obesity and associated chronic diseases. Polymeric proanthocyanidins (PA) form complexes with starch via unknown mechanisms, resulting in dramatically decreased starch digestibility. We hypothesized that V-type complexes are involved in these interactions. Sorghum derived PA was complexed with amylose, amylopectin, and granular maize starches in regular and deuterated solvents, and structural properties and in vitro digestibility of the complexes investigated. Based on iodine binding, X-ray diffraction patterns, crystallinity, and thermal properties, we demonstrated, for the first time, that type II semi-crystalline V-complexes are formed between amylose and PA. Furthermore, suppression of H-bonding led to amorphous complexes, suggesting extensive H-bonding facilitate and/or stabilize the V-complexes. We speculate that the complexation involves inclusion of B-rings of the PA units into the amylose helical cavity. The V-complex formation significantly increased resistant starch in gelatinized normal starch and pure amylose (by 35–45%), indicating likely physiological benefits.

In vitro Antifungal Activity of a New Bioproduct Obtained from Grape Seed Proanthocyanidins on Botrytis cinerea Mycelium and Spores

Botrytis cinerea is a necrotrophic fungus that affects over 200 plant species. In vineyards, this pathogen is responsible for one of the most important diseases, commonly known as botrytis bunch rot or grey mould. Keeping infection under control with synthetic fungicides leads to an increased biological resistance of pathogen populations. An alternative way to synthetic products is to obtain natural fungicides by using bioactive compounds of plants. This study focuses on the antifungal properties of a new bioproduct obtained from polymeric proanthocyanidins extracted from grape seeds of ‘Fetească neagră’ variety. The bioproduct in solid state presented a total content of polyphenols of 0.625 mg GAE mg-1, a polyphenolic index of 17.40 and an antioxidant activity of 91.27% scavenged DPPH. The bioproduct with polyphenolic structure showed a moderate effect on the radial growth of fungal mycelium, at EC50 values between 11.23 and 12.15 mg mL-1. Effective antifungal activity was showed in the inhibition of spore germination, where the EC50 values varied from 1.14 to 1.47 mg mL-1. These in vitro results sustain the possibility of including the bioproduct in the category of natural fungicides for biological control against Botrytis cinerea fungus.

Proanthocyanidins with a Low Degree of Polymerization are Good Inhibitors of Digestive Enzymes Because of their Ability to form Specific Interactions: A Hypothesis.

Inhibition of target digestive enzymes is an accepted strategy to prevent diseases such as obesity and diabetes. Proanthocyanidins (PACs) are known for their ability to bind, inhibit, and precipitate enzymes, which makes them potential bioDrugs with an impact on the digestive process. PAC degree of polymerization (DP) is one of the structural features responsible for their differential inhibitory potency but the explanation for this phenomenon is still unclear. Pecan nut (Carya illinoinensis L.) kernels and nutshells are rich in oligomeric and polymeric PACs. We have used thiolysis and HPLC analyses to propose four theoretical model structures of PACs representative of four semipurified fractions obtained from pecan kernel and shell, which showed different inhibitory activity against intestinal lipases, amylases, and proteases. The noncovalent interactions between PACs and digestive enzymes were predicted by in silico methods through computational software. These observations are discussed in view of current literature on the biological effects of PACs with different DPs and allowed us to propose the hypothesis that "small oligomeric PACs could be digestive enzyme inhibitors due to their capacity to enter and bind the enzymes' specific cavities better than polymers and oligomers of medium and high molecular weight."

Ultrasound-assisted extraction of proanthocyanidins from vine shoots of Vitis vinifera

ABSTRACTProanthocyanidins are high added value polyphenols which have received considerable attention for their health-protective activities. An alternative vine-shoots exploitation as a cheap source of proanthocyanidins obtained by ultrasound-assisted extraction was studied. Amplitude (50–100%), liquid-to-solid ratio (L/S) (50–100 mL/g) and extraction time (10–30 min) were the variables studied by Box–Behnken design to maximize extraction of oligomeric proanthocyanidins (OPCs) and polymeric proanthocyanidins (PPCs) extracted from vine shoots at 80°C. Under the optimal conditions found and validated the OPCs resulted 89.18 ± 0.08 mg catechin /100 g DM and the PPCs 95.46 ± 0.65 mg catechin /100 g DM. The total proanthocyanidins content found in vine shoots was 1.82 mg/g DM and resulted higher than those reported for barley, whole grain rice and stored vine shoots.

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.

Antioxidant and Fluorescence Properties of Hydrogenolyzised Polymeric Proanthocyanidins Prepared Using SO₄2-/ZrO₂ Solid Superacids Catalyst.

Larix bark oligomeric proanthocyanidins (LOPC) were prepared from larix bark polymeric proanthocyanidins (LPPC) by catalytic hydrogenolysis using SO42−/ZrO2 solid superacid as the catalyst. The catalyst to polymeric proanthocyanidins ratio was 0.2:1 (m/m). The LOPC, obtained after hydrogenolysis at 100 °C for 4 h under 3 MPa hydrogen pressure, retained the structural characteristics of proanthocyanidins. The average degree of polymerization was reduced from 9.50% to 4.76% and the depolymerization yield was 53.85%. LOPC has good antioxidant properties and, at the same concentration, the reducing ability of LOPC was much higher than that of LPPC. The IC50 values of LOPC for scavenging DPPH• and ABTS•+ radicals were 0.046 mg/mL and 0.051 mg/mL, respectively. LOPC is biocompatible and has fluorescent properties that are affected by external factors, such as solvent polarity, pH and the presence of different metal ions. These features indicate that LOPC could be developed as a new biological fluorescent marker. The depolymerization of low-value polymeric proanthocyanidins to provide high-value oligomeric proanthocyanidins and the development of new applications for proanthocyanidins represent significant advances.

Dioic acid glycosides, tannins and methylated ellagic acid glycosides from Morella salicifolia bark

The bark of Morella salicifolia (Hochst.exA. Rich.) Verdc. &Polhill (Myricaceae) is a common herbal drug and especially utilized by the Maasai community of northeastern Tanzania. It is traditionally used to treat swellings on the cheek and face, cough, toothache, stomach problems, headaches, and skin diseases. Unless, there is still a lack of information considering the specialized metabolites that may be responsible for the traditional use of this medicinal plant. An isolation study on a methanolic extract revealed three methylated ellagic acids, castalagin, two monomeric flavan-3-ols, three prodelphinidins and three dioic acid glycosides. Among these metabolites, myriside (3,4-dimethyl ellagic acid 4′-O-β-d-xylopyranoside, 1), epigallocatechin-3-O-gallate-(2β→O→7, 4β→8)-gallocatechin (9), 2,7-dimethyl-8-O-(6′-O-galloyl)-β-d-glucopyranoside-deca-2E,4E-dienedioic acid (10), 2,7-dimethyl-8-O-β-d-glucopyranoside-deca-2E,4E-dienedioic acid (11) as well as 4,9-dimethyl-10-O-β-d-glucopyranoside-dodeca-2E,4E,6E-trienedioic acid (12) were isolated for the first time. To the best of our knowledge, the dioic acid glycosides represent a generally novel structure type. To provide comprehensive information on the condensed tannins, the polymeric proanthocyanidins were enriched and characterized via 13C-NMR. Results confirm chemo-taxonomic relationships within the genera Morella and Myrica in the Myricaceae.

Molecular Controls of Proanthocyanidin Synthesis and Structure: Prospects for Genetic Engineering in Crop Plants.

Proanthocyanidins (PAs) are widespread oligomeric and polymeric flavan-3-ols with significant benefits to human and animal health. As products of the general flavonoid pathway, the biosynthesis of the flavan-3-ols is well-understood and the major enzyme-encoding genes that determine PA structure have been identified. However, the mechanism of PA polymerization remains unknown. The most important transcription factors regulating PA biosynthesis are the MYB factors, potent tools for enhancing PA biosynthesis in plants. In some species, simple overexpression of these transcription factors has led to spectacular successes in upregulating PA synthesis. However, targeted metabolic engineering of the PA structure has not yet been achieved.