Halogenated Analogs to Natural A-Type Proanthocyanidins: Evaluation of Their Antioxidant and Antimicrobial Properties and Possible Application in Food Industries.

A description of new antimicrobial agents suitable for food industries has become necessary, and natural compounds are being considered as promising sources of new active derivatives to be used with the aim of improving food safety. We have previously described desirable antimicrobial and antibiofilm activities against foodborne bacteria by analogs to A-type proanthocyanidins (PACs) with a nitro (NO2) group at carbon 6 of the A-ring. We report herein the synthesis of eight additional analogs with chloro and bromo atoms at the A-ring and the systematic study of their antimicrobial and antioxidant activities in order to evaluate their possible application as biocides or food preservatives, as well as to elucidate new structure-activity relationships. The results from this study show that halogenated analogs to natural A-type proanthocyanidins rise above the nitro derivatives previously reported in their antimicrobial activities. Gram-positive bacteria are the most sensitive to all the analogs and combinations assayed, showing MICs from 10 to 50 µg/mL in most cases, as well as reductions in biofilm formation and the disruption of preformed biofilms of at least 75%. Some structure-activity relationships previously described have also been corroborated. Analogs with just one OH group at the B-ring show better antimicrobial activities than those with two OH groups, and those analogs with two or three OH groups in the whole structure are more active than those with four OH groups. In addition, the analogs with two OH groups at the B-ring and chloro at the A-ring are the most effective when antibiofilm activities are studied, especially at low concentrations.

Synthesis and Isolation of Phenol- and Thiol-Derived Epicatechin Adducts Prepared from Avocado Peel Procyanidins Using Centrifugal Partition Chromatography and the Evaluation of Their Antimicrobial and Antioxidant Activity.

Polyphenols from agro-food waste represent a valuable source of bioactive molecules that can be recovered to be used for their functional properties. Another option is to use them as starting material to generate molecules with new and better properties through semi-synthesis. A proanthocyanidin-rich (PACs) extract from avocado peels was used to prepare several semi-synthetic derivatives of epicatechin by acid cleavage in the presence of phenol and thiol nucleophiles. The adducts formed by this reaction were successfully purified using one-step centrifugal partition chromatography (CPC) and identified by chromatographic and spectroscopic methods. The nine derivatives showed a concentration-dependent free radical scavenging activity in the DPPH assay. All compounds were also tested against a panel of pathogenic bacterial strains formed by Listeria monocytogenes (ATCC 7644 and 19115), Staphylococcus aureus (ATCC 9144), Escherichia coli (ATCC 11775 and 25922), and Salmonella enterica (ATCC 13076). In addition, adducts were tested against two no-pathogenic strains, Limosilactobacillus fermentum UCO-979C and Lacticaseibacillus rhamnosus UCO-25A. Overall, thiol-derived adducts displayed antimicrobial properties and, in some specific cases, inhibited biofilm formation, particularly in Listeria monocytogenes (ATCC 7644). Interestingly, phenolic adducts were inactive against all the strains and could not inhibit its biofilm formation. Moreover, depending on the structure, in specific cases, biofilm formation was strongly promoted. These findings contribute to demonstrating that CPC is a powerful tool to isolate new semi-synthetic molecules using avocado peels as starting material for PACc extraction. These compounds represent new lead molecules with antioxidant and antimicrobial activity.

Preparation, characterization and biological activity of proanthocyanidin-chitosan nanoparticles.

In this study, proanthocyanidin-loaded chitosan nanoparticles (PC-CS-NPs) were produced using ionotropic gelation and characterized using Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and dynamic light scattering (DLS). The synthesized nanoparticles were smaller than 300 nm and had a spherical shape, smooth topography and homogenous morphology as observed through scanning electron microscopy (SEM). In vitro release study showed that proanthocyanidins (PC) had a sustainable release from PC-CS-NPs in different buffer media. PC-CS-NPs had higher or comparable potency in scavenging DPPH and ABTS free radicals as compared to native drugs. Furthermore, PC-CS-NPs also inhibited the growth of four bacteria species, whose degree of inhibition depended on the bacterial strain. The results of SEM confirmed the changes in the microstructure of bacteria. Our findings support the use of chitosan nanoparticles to encapsulate PC and improve its bioactivity in food products.

Synthesis and experimental/computational characterization of sorghum procyanidins-gelatin nanoparticles.

In this research, sorghum procyanidins (PCs) and procyanidin B1 (PB1) were encapsulated in gelatin (Gel) to form nanoparticles as a strategy to maintain their stability and bioactivity and for possible applications as inhibitors of metalloproteinases (MMPs) of the gelatinase type. Encapsulation was carried out by adding either PCs or PB1 to an aqueous solution of A- or B-type Gel (GelA or GelB) at different concentrations and pH. Under this procedure, the nanoparticles PCs-GelA, PCs-GelB, PB1-GelA, and PB1-GelB were synthesized and subsequently characterized by experimental and computational methods. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that all types of nanoparticles had sizes in the range of 22-138 nm and tended to adopt an approximately spherical morphology with a smooth surface, and they were immersed in a Gel matrix. Spectral analysis indicated that the nanoparticles were synthesized by establishing hydrogen bonds and hydrophobic interactions betweenGel and the PCs or PB1. Study of simulated gastrointestinal digestion suggested that PCs were not released from the Gel nanoparticles, and they maintained their morphology (SEM analysis) and antioxidant activity determined by Trolox-equivalent antioxidant capacity (TEAC) assay. Computational characterization carried out through molecular docking studies of PB1 with Gel or (pro-)metalloproteinase-2 [(pro-)MMP-2], as a model representative of the PCs, showed very favorable binding energies (around -5.0 kcal/mol) provided by hydrogen bonds, van der Waals interactions, and desolvation. Additionally, it was found that PB1 could act as a selective inhibitor of (pro-)MMP-2.

Synthesis of A-Type Proanthocyanidins and Their Analogues: A Comprehensive Review.

Proanthocyanidins (PACs) are oligomers or polymers composed of units of flavanols. A-type PACs are a subclass of PACs characterized by the presence of at least a double linkage between two consecutive monomers of flavanol. These A-type PACs are found in some fruits and spices and possess potential health benefits as a result of their interesting biological activities, and consequently, their isolation and synthesis have given rise to great interest in the past. This review summarizes the synthetic efforts made to obtain both naturally occurring A-type PACs and their structurally simplified analogues. Most of the synthetic protocols reported involve the addition of a π-nucleophilic molecule over a molecule with two electrophilic carbons, such as a chalcone, a flavylium salt, or a flavanol derivative, among others. Synthesis of A-type PACs remains an issue at a very early stage of development compared to that of PACs with single linkages between monomers (B-type PACs), but the advances that are taking place in the last few years point to a significant development of the subject in the near future.

Procyanidin B2 gallate regulates TNF-α production from T cells through inhibiting glycolytic activity via mTOR-HIF-1 pathway.

Procyanidins are polyphenols with antioxidant, anti-obesity, and anti-inflammatory properties. Procyanidin B2 (PCB2) gallate; specifically, PCB2 3,3″-di-O-gallate (PCB2DG), inhibits cytokine production in T cells. However, the molecular interactions and partners of PCB2DG underlying this suppression of cytokine production are unclear. The present study aimed to elucidate mechanisms underlying regulation of tumor necrosis factor (TNF)-α production by PCB2DG. We found that production of TNF-α and glycolytic activity in activated CD4+ T cells were suppressed by PCB2DG treatment. The inhibition of TNF-α production was found to be mediated by mammalian target of rapamycin (mTOR) and hypoxia inducible factor 1 (HIF-1) pathway, as PCB2DG suppressed the expression of HIF-1α, p-mTOR, and p-p70S6K (a downstream of the mTOR complex, mTORC1). Moreover, suppression of TNF-α production was mediated by regulation of the glycolytic enzyme lactate dehydrogenase at the posttranscriptional level. These results suggest that PCB2DG regulates TNF-α production by inhibiting glycolytic activity via the mTOR-HIF-1 pathway.

Inhibition of ß-amyloid-induced neurotoxicity by planar analogues of procyanidin B3.

Reactive oxygen species (ROS) are known to be produced during the amyloid beta (Aß) aggregation process. Both ROS production and Aß fibril formation can result in nerve cell injury. Proanthocyanidins are oligomers of catechin that can act as inhibitors of Aß aggregation. Procyanidin B3 (Cat-Cat), the dimer of (+)-catechin, can easily cross the blood-brain barrier. Previously, we synthesized two derivatives of Cat-Cat, namely Cat-PCat and PCat-PCat, in which the geometry of one or both catechin molecules in Cat-Cat was constrained to be planar. The antioxidative activities of Cat-PCat and PCat-PCat were found to be stronger than that of Cat-Cat, with PCat-PC at exhibiting the most potent activity. These compounds are predicted to protect against Aß-induced neurotoxicity via inhibition of Aß aggregation as well as by antioxidative effects toward Aß-induced intracellular ROS generation. PCat-PCat exhibited the most potent neuroprotective effects against Aß-induced cytotoxicity, which resulted from inhibition of ß-sheet structure formation during the Aß aggregation process. PCat-PCat may be a promising lead compound for the treatment of Alzheimer's disease.

A new approach to procyanidins synthesis with potent anti-adipogenic effects.

Convenient structure modification of (+)-catechin (1) induced by nonthermal dielectric barrier discharge (DBD) plasma treatment afforded three novel methylene-linked flavan-3-ol oligomers, methylenetetracatechin (2), methylenetricatechin (3), and methylenedicatechin (4), together with two known catechin dimers, bis 8,8'-catechinylmethane (5) and bis 6,8'-catechinylmethane (6). The structures of the three new catechin oligomers 2-4 with methylene bridges were elucidated by detailed 1D- and 2D-NMR analysis, and the absolute configurations were established by the observation of circular dichroism (CD). The novel products 2 and 3 showed significantly enhanced anti-adipogenic capacities against both pancreatic lipase and differentiation of 3T3-L1 preadipocytes compared to the parent (+)-catechin.

A semisynthetic approach for the simultaneous reaction of grape seed polymeric procyanidins with catechin and epicatechin to obtain oligomeric procyanidins in large scale.

Polymeric procyanidins (PPCs) were the major constituents of procyanidins, while they have poor bioactivity. To better utilize PPCs, a semisynthetic approach for converting PPCs to oligomeric procyanidins (OPCs) was proposed. Grape seed PPCs were simultaneously reacted with catechin (C) and epicatechin (EC) under acid condition. Combining response surface methodology (RSM) and single-factor experiments, an optimized semisynthetic condition was confirmed with the ratio of PPCs with C and EC of 1:1:1, temperature of 40 °C, reaction time of 20 min and 0.1 M methanolic HCl. High-speed counter-current chromatography (HSCCC) was adopted to obtain three fractions from semisynthetic products and preparative-HPLC was used to isolate individual procyanidins. Thirteen B-type procyanidins including monomers, dimers and trimers were got with high yield of 0.8-17.8 mg from 200 mg semisynthetic products and high purity over 91%. The developed semisynthesis combined with separation method was efficient to obtain individual OPCs in preparative scale.

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.

Synthesis and characterization of proanthocyanidins-functionalized Ag nanoparticles.

Preparation of Ag nanoparticles with high antibacterial activities and low adverse effects is urgently needed to apply to the medical and consumer products. In this study, a facile, one-step hydrothermal method has been developed to synthesize proanthocyanidins-functionalized Ag nanoparticles in aqueous solution. UV absorption spectra measurement indicated that the obtained Ag nanomaterials are mainly spherical in shape. FTIR results implied that the poly flavonoids moiety of proanthocyanidins have specific interactions with the surface of Ag nanoparticles. HR-TEM and XRD determination revealed that the Ag nanoparticles had a shell-core structure and the Ag core possessed a highly crystalline structure. Antibacterial tests demonstrated that the proanthocyanidins-functionalized-Ag nanoparticles have high bactericidal efficiency against both bacterial and fungus. Further taking into account their excellent stability, the proanthocyanidins-functionalized Ag nanoparticles in present study have the potential to be applied to the products required for the long-acting antibacterial action.

Synthesis and Evaluation of Antimicrobial and Antibiofilm Properties of A-Type Procyanidin Analogues against Resistant Bacteria in Food.

Natural A-type procyanidins have shown very interesting biological activities, such as their proven antiadherence properties against pathogenic bacteria. In order to find the structural features responsible for their activities, we describe herein the design and synthesis of six A-type procyanidin analogues and the evaluation of their antimicrobial and antibiofilm properties against 12 resistant bacteria, both Gram positive and Gram negative, isolated from organic foods. The natural A-type procyanidin A-2, which had known antiadherence activity, was also tested as a reference compound for the comparative studies. Within the series, analogue 4, which had a NO2 group on ring A, showed the highest antimicrobial activity (MIC of 10 µg/mL) and was one of the best molecules at preventing biofilm formation (up to 40% decreases at 100 µg/mL) and disrupting preformed biofilms (up to 40% reductions at 0.1 µg/mL). Structure-activity relationships are also analyzed.

Regioselective Synthesis of Procyanidin B6, A 4-6-Condensed (+)-Catechin Dimer, by Intramolecular Condensation.

Proanthocyanidins, also known as condensed tannins or oligomeric flavonoids, are found in many edible plants and exhibit interesting biological activities. Herein, we report a new, simple method for the stereoselective synthesis of procyanidin B6, a (+)-catechin-(4-6)-(+)-catechin dimer, by Lewis acid-catalyzed intramolecular condensation. The 5-O-t-butyldimethylsilyl (TBDMS) group of 5,7,3'4'-tetra-O-TBDMS-(+)-catechin was regioselectively removed using trifluoroacetic acid, leading to the "regio-controlled" synthesis of procyanidin B6. The 5-hydroxyl group of the 7,3',4'-tri-O-TBDMS-(+)-catechin nucleophile and the 3-hydroxyl group of 5,7,3',4'-tetra-O-benzylated-(+)-catechin electrophile were connected with an azelaic acid. The subsequent SnCl4-catalyzed intramolecular condensation proceeded smoothly to give the 4-6-condensed catechin dimer. This is the first report on the complete regioselective synthesis of a 4-6-connected oligomer without modifying the 8-position.

Enhanced radical scavenging activity of a procyanidin B3 analogue comprised of a dimer of planar catechin.

Proanthocyanidins are oligomers of catechins that exhibit potent antioxidative activity and inhibit binding of oxidized low-density lipoprotein (OxLDL) to the lectin-like oxidized LDL receptor (LOX-1), which is involved in the onset and development of arteriosclerosis. Previous attempts aimed at developing proanthocyanidin derivatives with more potent antioxidative activity and stronger inhibition for LOX-1 demonstrated the synthesis of a novel proanthocyanidin derivative (1), in which the geometry of one catechin molecule in procyanidin B3 was constrained to a planar orientation. The radical scavenging activity of 1 was 1.9-fold higher than that of procyanidin B3. Herein, we synthesized another procyanidin B3 analogue (2), in which the geometries of both catechin molecules in the dimer were constrained to planar orientations. The radical scavenging activity of 2 was 1.5-fold higher than that of 1, suggesting that 2 may be a more effective candidate than 1 as a therapeutic agent to reduce oxidative stress induced in arteriosclerosis or related cerebrovascular disease.

Challenges and complexity of functionality evaluation of flavan-3-ol derivatives.

Flavan-3-ol derivatives are common plant-derived bioactive compounds. In particular, (-)-epigallocatechin-3-O-gallate shows various moderate biological activities without severe toxicity, and its health-promoting effects have been widely studied because it is a main ingredient in green tea and is commercially available at low cost. Although various biologically active flavan-3-ol derivatives are present as minor constituents in plants as well as in green tea, their biological activities have yet to be revealed, mainly due to their relative unavailability. Here, I outline the major factors contributing to the complexity of functionality studies of flavan-3-ol derivatives, including proanthocyanidins and oligomeric flavan-3-ols. I emphasize the importance of conducting structure-activity relationship studies using synthesized flavan-3-ol derivatives that are difficult to obtain from plant extracts in pure form to overcome this challenge. Further discovery of these minor constituents showing strong biological activities is expected to produce useful information for the development of functional health foods.

Total synthesis of proanthocyanidin A1, A2, and their stereoisomers.

The first novel stereoselective synthesis of naturally occurring A1 (1) and A2 proanthocyanidins (2) has been achieved. The key synthetic steps involved (a) the formation of a coupled product (13 or 14) between an open chain C-ring C-4 hydroxyethoxy analogue of either (+)-catechin or (-)-epicatechin with 5,7,3',3'-tetra-O-benzyl-(+)-catechin/-(-)-epicatechin in the presence of bentonite clay K-10, (b) removal of benzyl protecting groups under mild catalytic hydrogenation conditions to form the desired A-type compound in situ as a mixture of diastereomers via ketal/oxonium ion/carbonium ion formation, and (c) separation of the diasteromers via silica gel column chromatography. The structures of A1 and A2 proanthocyanidins were unequivocally established by analytical comparison to the natural products. Following this methodology, an additional six diastereomers of proanthocyanidins A1 and A2 have been synthesized. A plausible mechanism for the formation of the A-type linkage in proanthocyanidins has been proposed.

Synthesis, characterisation and antioxidant features of procyanidin B4 and malvidin-3-glucoside stearic acid derivatives.

The acylation of procyanidin B4 with a saturated fatty acid chloride containing 18 carbon atoms was studied in order to obtain procyanidin B4 3-O-di-stearic acid conjugate. This compound was structurally characterised by mass spectrometry and 1D and 2D NMR techniques. Derivatization of malvidin-3-glucoside using stearoyl chloride in acetonitrile was also performed yielding mono-, di- and tri-stearic ester derivatives. The novel derivatives obtained revealed significant antioxidant activity, although lower than the respective precursors. However, the chemical modification of anthocyanins and procyanidins (water soluble pigments) to more lipophilic compounds has the advantage of increased bioavailability in biological matrices, and to potentiate their application in food matrices and cosmetic products.

Conformational equilibria in selected A-type trimeric procyanidins.

A-type procyanidin trimers cinnamtannin B-1, cinnamtannin D-1, lindetannin, and aesculitannin B were studied in terms of their conformation and interaction with four solvents: methanol, acetone, DMSO and pyridine. The experiments demonstrated that for each trimer there are two principal conformers observable in the NMR. The ratio of the conformers (rotamers) depends on the structure of a given trimer as well as on the solvent used for NMR measurements. The DFT calculations (B3LYP/6-31G(d,p)) proved the presence of two main conformers to be the result of a steric hindrance that prevents free rotation along the B-type interflavan bond. An analysis of the solvent-procyanidin interactions showed that the strong electron donating solvents, pyridine and DMSO, favor different conformers from methanol and acetone, which prefer the lowest-energy gas phase conformer. These findings are in line with predictions of DFT/M06-2X calculations with the inclusion of the thermal corrections. The variations in the rotamer ratios in the studied solvents correlate with the solvent's capacity to induce local changes in the electron density of the particular procyanidin trimer.

Annulation approach to doubly linked (A-type) oligocatechins: syntheses of (+)-procyanidin A2 and (+)-cinnamtannin B1.

The first stereoselective syntheses of doubly linked (A-type) oligocatechins, (+)-procyanidin A2 and (+)-cinnamtannin B1, have been achieved. Ethylenedioxy-bridged flavans served as excellent platforms, thus allowing annulation with nucleophilic catechin units in a stereoselective manner. An additional key was the new synthetic approach to selectively protected nucleophilic catechin, thus enabling regioselective construction of the key dioxabicyclo skeleton of the A-type oligocatechins.

Semisynthetic preparation and isolation of dimeric procyanidins B1-B8 from roasted hazelnut skins (Corylus avellana L.) on a large scale using countercurrent chromatography.

Dimeric procyanidins B1-B8 were produced via semisynthesis from a polymeric proanthocyanidin fraction of hazelnut skins (Corylus avellana L.). This polymeric fraction was found to consist mostly of (+)-catechin and (-)-epicatechin as upper units. Therefore, according to the choice of nucleophile agent, it is possible to semisynthesize dimeric procyanidins B1, B3, B6, and B7 with (+)-catechin and B2, B4, B5, and B8 with (-)-epicatechin. The semisynthetic mixtures were separated on a preparative scale using high-speed countercurrent chromatography (HSCCC) and low-speed rotary countercurrent chromatography (LSRCCC). C4 → C8 linked dimeric procyanidins B1-B4 were isolated in amounts of 350-740 mg. To the best of the authors' knowledge this is the first study isolating dimeric procyanidins B1-B8 in large amounts with countercurrent chromatography. Moreover, the dimeric prodelphinidins B1, B2, and B3 and their structural elucidation by (1)H NMR spectroscopy without derivatization are described for hazelnuts as natural compounds for the first time.