Presence Of Phloroglucinol Research Articles

Application of carbon dots synthesized with amino acid as precursor in the detection of phloroglucinol

Nitrogen doped carbon dots (CDs) were successfully applied in the detection of phloroglucinol. CDs were hydrothermally synthesized from L-phenylalanine (L-phe) and L-leucine (L-leu) in alkaline aqueous solution. The morphology, size and functional groups of CDs were characterized by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The average size of CDs was 4.26 ± 1.77 nm. The main elements of CDs were C, N, O and functional groups were carboxyl, hydroxyl, pyridine and amino groups. CDs showed bright blue fluorescence with the quantum yield (QY) at 12.82 % (λex = 337 nm and λem = 437 nm). In the presence of phloroglucinol (PG), however, the fluorescence of CDs was quenched significantly. It could be ascribed from hydrogen bonds formed between PG and functional groups on the surface of CDs. There was a good linear response between the fluorescence quenching of CDs and the concentration of PG in the range of 0.5–150 μM with the limit of detection (LOD) at 12 nM. The strategy can be used to detect PG qualitatively and quantitatively with the advantages of low cost, high sensitivity, fast response and high specificity.

Oligomerization mechanism of epigallocatechin-3-O-gallate during autoxidation

The autoxidation of tea catechins by dissolved oxygen proceeds in pH-neutral aqueous solutions, and the major products are oligomers. However, the reaction mechanisms have not been clarified. In this study, the autoxidation of (−)-epigallocatechin-3-O-gallate (1) was examined. The autoxidation with β-cyclodextrin, which includes the A-ring of 1, significantly suppressed oligomer production and increased the formation of products generated by the oxidative cleavage of the B-ring, indicating the participation of the A-ring in the oligomerization. Further, the autoxidation of 1 in the presence of phloroglucinol, a mimic of the catechin A-ring, yielded products via the nucleophilic addition of phloroglucinol to the B-ring quinone of 1. These results indicated that the oxidative A–B ring couplings accounted for the major oligomerization mechanism.

Highly galloylated and A-type prodelphinidins and procyanidins in persimmon (Diospyros kaki L.) peel

The structure of persimmon peel proanthocyanidins (PPPAs) was characterized. After acid catalysis of PPPAs in the presence of phloroglucinol, five reaction products such as (epi)gallocatechin-phloroglucinol ((E)GC-ph) etc. were identified by HPLC-DAD-ESI/MS. FT-IR analysis confirmed that all of the compounds exhibited a 2, 3-cis configuration. Therefore, the extension units in PPPAs were EGC, epigallocatechin gallate (EGCG), epicatechin, and epicatechin gallate and only EGCG was detected as the terminal unit. PPPAs contained 25.21% of procyanidins and 74.79% of prodelphindins and had a high degree of 3-O-galloylation (>74.79%). The mean degree of polymerization of PPPAs was calculated to be 10.18. MALDI-TOF MS analysis showed that A-type linkage and galloylation existed commonly in PPPAs.

Pomegranate Fruit and Juice (cv. Mollar), Rich in Ellagitannins and Anthocyanins, Also Provide a Significant Content of a Wide Range of Proanthocyanidins.

Proanthocyanidins (PAs) were characterized in husk, membranes, arils, and seeds of 'Mollar de Elche' pomegranate cultivar using liquid chromatography-electrospray ionization-ion-trap tandem mass spectrometry after acid catalysis in the presence of phloroglucinol (phloroglucinolysis). PAs were also evaluated in commercial juice and in the juice made in the laboratory by pressing fresh arils. Pomegranate PAs were composed of a very rich mixture of flavan-3-ols: catechin, epicatechin, gallocatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate. Catechins and gallocatechins were the most abundant units, while galloylated flavanols were detected at lower concentrations. The highest PA content was found in husk (1840.5 μg/g), followed by seeds (316.2 μg/g) and membranes (51.4 μg/g). With regard to the pomegranate edible parts, arils showed concentrations of 34.4 μg/g and pressed aril juice of 21.3 mg/L, lower than those found in other traditional PA-containing food and beverages, such as apple, grapes, chocolate, red wine, or tea. Higher concentrations similar to those found in red wine were observed in commercial whole fruit pressed pomegranate juice (146.9 mg/L). These PA concentrations represented between 0.1 and 7% of the total polyphenol content in the different pomegranate samples, which were richer in ellagitannins and anthocyanins. The mean degree of polymerization (1.2-2.1) revealed that PAs in Mollar pomegranate samples are constituted mainly of monomers and dimers, which may influence in better absorption and bioavailability of these compounds.

Comprehensive characterization by LC-DAD-MS/MS of the phenolic composition of seven Quercus leaf teas

A complete characterization of the phenolic profile of leaves infusions from seven Mexican Quercus species was developed using different LC-DAD-MS/MS methodologies. The main families of phenolic compounds identified and quantified were: hydrolyzable tannins and flavonol glycosides, based on their fragmentation patterns and UV spectra, proanthocyanidins analyzed after acid-catalysis in the presence of phloroglucinol, and phenolic acids evaluated using UPLC-triple quadrupole mass spectrometer (QqQ). White oak species showed the largest amount of total phenols (830–2956mg/L) with hydrolyzable tannins as the predominant group (60–96%), mainly vescavaloninic acid, vescalagin, and castalagin. Red species (total phenolics 129–280mg/L), showing proanthocyanidins as the dominant family, consisted of units of catechin, gallocatechin and in less amount epicatechin gallate and epigallocatechin gallate and larger percentages of phenolic acids (10–19%).

3 - Peroxidasin-Catalysed Oxidative Modifications of Proteins in the Extracellular Matrix

Peroxidasin has been recently identified as a mammalian heme peroxidase that catalyses the formation of an essential crosslink in collagen IV in the extracellular matrix (ECM). This crosslink was identified as sulfilimine bond between methionine and hydroxylysine resuidues in the non-collagenous domain. Heme peroxidases catalyse the oxidation of a variety of substrates by hydrogen peroxide (H2O2). These include bromide (Br–) to give hypobromous acid (HOBr), which is proposed to be responsible for the collagen IV crosslink. However, the specificity of peroxidasin and the exact mechanism of sulfilimine formation are not completely understood. We investigated peroxidasin activity and the cross-linking of collagen IV in isolated ECM from cultured epithelial cells. We found that formation of the sulfilimine crosslink requires both H2O2 and Br–. It did not form in presence of other halides such as chloride, iodide or the pseudohalide thiocyanate, with both of the latter inhibiting the reaction. Acetaminophen (paracetamol), thioxanthine, and 4-aminobenzoic acid hydrazide, which all inhibit myeloperoxidase, had minimal effect on the peroxidasin-dependent crosslink. The specificity and inhibitory profile of peroxidasin is therefore different from myeloperoxidase. HOBr has been shown to form sulfilimine bonds in peptides containing Met and Lys, but it also undergoes other reactions with proteins, including the formation of a specific product bromotyrosine (Br-Tyr)(1). Using LC-MS, we detected Br-Tyr in the noncollagenous domain of collagen IV isolated from cultured cells, and to a lesser extent in the rest of matrix and the cell extract. Br-Tyr levels increased in cells grown in the presence of Br–, and decreased in presence of phloroglucinol, a potent inhibitor of cross-linking. Cross-linking was already maximal without added Br–, suggesting that the reaction is selective only at low peroxidasin activity. These results indicate that cross-linking of collagen IV requires Br–, and is susceptible to modulation by endogenous and exogenous substances. Furthermore, peroxidasin-mediated oxidative modification of proteins in the ECM might not be limited to formation of the sulfilimine crosslink. 1. Ronsein GE, Winterbourn CC, Di Mascio P, Kettle AJ. Cross-linking methionine and amine residues with reactive halogen species. Free radical biology & medicine. 2014;70:278-87.

A Fast and Robust UHPLC-MRM-MS Method to Characterize and Quantify Grape Skin Tannins after Chemical Depolymerization.

A rapid, sensitive, and selective analysis method using ultra high performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ-MS) has been developed for the characterization and quantification of grape skin flavan-3-ols after acid-catalysed depolymerization in the presence of phloroglucinol (phloroglucinolysis). The compound detection being based on specific MS transitions in Multiple Reaction Monitoring (MRM) mode, this fast gradient robust method allows analysis of constitutive units of grape skin proanthocyanidins, including some present in trace amounts, in a single injection, with a throughput of 6 samples per hour. This method was applied to a set of 214 grape skin samples from 107 different red and white grape cultivars grown under two conditions in the vineyard, irrigated or non-irrigated. The results of triplicate analyses confirmed the robustness of the method, which was thus proven to be suitable for high-throughput and large-scale metabolomics studies. Moreover, these preliminary results suggest that analysis of tannin composition is relevant to investigate the genetic bases of grape response to drought.

Preparative HSCCC isolation of phloroglucinolysis products from grape seed polymeric proanthocyanidins as new powerful antioxidants

Polymeric proanthocyanidins isolated from a grape seed phenolic extract were hydrolysed in the presence of phloroglucinol into monomer catechins and their nucleophile derivatives. Each of the phloroglucinolysis products was successfully separated and isolated in large amount by semi-preparative HSCCC technique under the optimized conditions based on a selection of suitable solvent system. The optimized solvent system consisted of n-hexane–ethyl acetate–water (1:80:80, v/v/v) with a combination of head–tail and tail–head elution modes. By only one-step HSCCC separation, the purity of each obtained phloroglucinolysis product, including monomer catechins and their nucleophile derivatives was above 76%, verified by UPLC. The structures of these products were tentatively identified by UPLC based on their retention time and further confirmed by MS and 1H NMR analysis. Furthermore, by DPPH, ABTS and FRAP assays, it was verified that all these phloroglucinolysis products possessed strong antioxidant activities, being catechin-nucleophile derivatives more powerful than free catechins.

Polyphenolic characterisation of old local apple varieties from Southeastern European region

Ancient apple cultivars grown in local areas have so far been largely unexplored and might represent an important source of polyphenols. In this work, the phenolic profile in the flesh and peel of thirteen old apple varieties from the Balkan region was studied by liquid chromatography with ultraviolet–visible diode array detector and mass spectrometry detectors (ion trap, quadrupole time-of-flight); 26 compounds were identified. Proanthocyanidins were detected as dimers to heptamers and they were further analysed after acid-catalysis in the presence of phloroglucinol (degree of polymerisation 3.8–10.3). Epicatechin represented more than 85% of constitutive units. Proanthocyanidins were the major class of polyphenols in flesh and peel (>70% of total polyphenols), phenolic acids were mainly distributed in the flesh (6–25%); flavonols (1–13%) and dihydrochalcones (1–10%) in the peel, and anthocyanins (1–7%) in the peel of red coloured apples. “Ljubeničarka” showed unique red-coloured flesh (unusual for apples) due to the presence of anthocyanins in the flesh. Old apple varieties are rich in phenolic compounds, in quantities even higher than those found in commercial varieties, and might constitute the basis for further breeding.

Effect of Polymerization on Antioxidant and Xanthine Oxidase Inhibitory Potential of Sea Buckthorn (H. rhamnoides) Proanthocyanidins

Inhibitory potential of sea buckthorn (Hippophae rhamnoides L) seed proanthocyanidins against oxidative stress and xanthine oxidase activity was evaluated. Composition of antioxidant proanthocyanidins was profiled by analyzing the cleavage products obtained by the acid catalyzed hydrolysis in the presence of phloroglucinol. Catechin, epicatechin, gallocatechin, and epigallocatechin were found as the extension and terminal subunits of proanthocyanidins with an average degree of polymerization (ADP) of 14.7. Seed proanthocyanidins showed considerably high antioxidant and xanthine oxidase inhibitory potentials. Antioxidant and xanthine oxidase inhibitory capacity evaluation of proanthocyanidin fractions with varying ADP showed that proanthocyanidins with lower molecular size were more effective as superoxide anion (ADP ≤ 4.2) and hydroxyl radical (ADP ≤ 5.9) scavengers and xanthine oxidase (ADP ≤ 3.1) inhibitors. ADP of the studied proanthocyanidin fractions did not show significant influence on their DPPH and ABTS radical scavenging and ferric reduction capacities.

Polyphenolic Constituents of Cynomorium songaricum Rupr. and Antibacterial Effect of Polymeric Proanthocyanidin on Methicillin-Resistant Staphylococcus aureus

Oligomeric and polymeric flavan-3-ols were obtained by chromatographic fractionation of extracts from Cynomorium songaricum Rupr. The structure of the polymeric constituent, cynomoriitannin, was characterized using spectral and chemical data. Results from acid-catalyzed degradation indicated that cynomoriitannin is a polymeric proanthocyanidin predominantly composed of epicatechin, together with low proportions of epicatechin-3-O-gallate and catechin as extension units. The terminal unit was chiefly composed of catechin, with an admixture of epicatechin. Size exclusion chromatographic analysis demonstrated a mean polymerization degree of 14. Two new phloroglucinol adducts (cynomoriitannin-phloroglucinol adducts A and B) obtained by acid-catalyzed degradation of cynomoriitannin in the presence of phloroglucinol were characterized using spectral analyses. Six oligomeric flavan-3-ols were also identified as follows: procyanidin B3, catechin-(6'-8)-catechin, catechin-(6'-6)-catechin, epicatechin-(4β-8)- epicatechin-(4β-8)-catechin, epicatechin-(4β-6)-epicatechin-(4β-8)-catechin, and arecatannin A1, respectively. These flavan-3-ols were isolated from C. songaricum. This is the first time that this procedure has been described. The antibacterial activity of the fractions and constituents was tested against methicillin-resistant Staphylococcus aureus (MRSA). The crude acetone-water (7:3) extract had moderate activity against MRSA. Cynomoriitannin was the most effective of the plant constituents against MRSA.

Phenolic compound content of fresh and dried figs (Ficus carica L.)

Eighteen fig cultivars commonly grown in the south-eastern Spain were collected in two crops (spring and summer) and their polyphenolic profile was determined. Fruit from the first crop generally showed higher phenolic values than those from the second. Higher concentrations of total phenolics were found in skin than in flesh. LC–UV-DAD/ESI-MSn analysis of the figs pointed to a high anthocyanin content, mainly cyanidin-3-rutinoside, flavonols such as quercetin-rutinoside, phenolic acids such as chlorogenic acid and flavones like luteolin 6C-hexose-8C-pentose and apigenin-rutinoside. The c-glycosides have not been previously described in figs. Moreover, to compare intact proanthocyanidins and proanthocyanin cleavage products, an acid-catalysis was made in the presence of phloroglucinol (phloroglucinolysis) and the mean degree of polymerisation was calculated. Finally, three cultivars of dried figs were also studied.

Polyphenols from Ginkgo biloba

By Sephadex LH-20 gel chromatography of an extract from Gingko biloba leaves, polymeric proanthocyanidins were eluted after the fractions of flavonol glycosides and biflavone glycosides. A purified proanthocyanidin polymer accounted for 86.6% of the total proanthocyanidins, and for 37.7% of the total antioxidant activity of this leaf extract. For structure elucidation, the polymer was submitted to acidic depolymerization in the presence of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were determined on the basis of 1D-and reverse 2D-NMR (HSQC, HMBC) spectra of their peracetylated derivatives, MALDI-TOF-MS and CD-spectroscopy. The observations resulting from the degradation with phloroglucinol were confirmed by 13C-NMR spectroscopy of the polymer. The mean molecular weight of the polymeric fraction was estimated to be 9–10 flavan-3-ol-units.

Condensed Tannin Accumulation and Composition in Skin of Shiraz and Cabernet Sauvignon Grapes during Berry Development

Condensed tannins derived from the grape berry play a significant role in wine astringency, bitterness, color stability, and aging potential. Understanding the regional, varietal, and seasonal variability in winegrape tannin composition throughout berry development is essential for developing vineyard strategies to manage and manipulate winegrape tannin to meet winery specifications. Current knowledge of tannin accumulation and composition in grape berries is largely based on a single variety and geographical region, with most studies investigating seed tannin. To explore the variation in tannin accumulation and composition, Shiraz and Cabernet Sauvignon grape skin from two growing seasons in the Sunraysia region of southeast Australia were measured by acid-catalyzed cleavage in the presence of phloroglucinol. High-performance liquid chromatography was used to quantify the subunit composition, total tannin content, and average polymer length throughout berry development. The pattern of tannin accumulation and composition were similar for both varieties and seasons, with tannin content highest at fruit set before declining toward veraison. The pattern of accumulation and composition seen in grape skin from this study was different to that previously reported in Shiraz from the cooler growing region of McLaren Vale in South Australia where tannin content was highest at veraison. Results indicate that the differences between the pattern of tannin accumulation and composition in Sunraysia and McLaren Vale may be due to regional influences, which had greater impact on tannin composition than varietal or seasonal variability. Further research is required to determine the full extent of variation between regions and to determine the environmental factors that can be used to influence the pattern of tannin accumulation.

Characterisation of highly polymerised prodelphinidins from skin and flesh of four cashew apple ( Anacardium occidentale L.) genotypes

Tannins were extracted by acetone/water 60:40 from skin and flesh of four cashew apple genotypes from Brazil and Bénin (West Africa), and separated from monomeric phenols. Tannins were submitted to acid-catalysed degradation in the presence of phloroglucinol and the products were analysed by HPLC–DAD/ESI–MS. Both skin and flesh tannins contained high percentages of (-)-epigallocatechin and (-)-epigallocatechin- O-gallate, followed by minor quantities of (-)-epicatechin and (-)-epicatechin-3- O-gallate; 100% of the compounds were the 2,3- cis configuration. Skin tannins were half as galloylated (∼20%) than flesh tannins (∼40%). Their weight-average molecular weight ( M w) was high.

Berry Integrity and Extraction of Skin and Seed Proanthocyanidins during Red Wine Fermentation

In this study, microscale fermentations were conducted on Vitis vinifera L. cv. Merlot. Five treatments were established varying from 0-100% crushed fruit (25% increments x 5 replicates). Caps were kept submerged throughout the experiment, and fermentation temperatures were maintained at 25 degrees C. Samples were collected throughout fermentation and from the free run and press wine at the time of pressing. Proanthocyanidins were determined by acid-catalyzed depolymerization in the presence of phloroglucinol, followed by reversed phase, high performance liquid chromatography (RP-HPLC). Total proanthocyanidin extraction increased with time in all treatments. In addition, crushing increased the rate at which proanthocyanidins were extracted. When the extraction of skin and seed proanthocyanidins were monitored separately, skin proanthocyanidin extraction rate exceeded that for seed proanthocyanidins and followed a Boltzmann sigmoid extraction model. The highest proanthocyanidin concentration for skin (435 mg/L) and seed (344 mg/L) was observed for the 75% crushed fruit treatment at the time of pressing (17 days). The highest skin proanthocyanidin proportion (79%) was observed for the 75% crushed fruit treatment on day 9 with a total proanthocyanidin concentration of 439 mg/L. For all treatments, skin proanthocyanidin extraction reached a plateau concentration prior to pressing, with the plateau concentration increasing with crushing. Seed proanthocyanidin concentration increased throughout maceration.

Effect and analysis of phenolic compounds during somatic embryogenesis induction in Feijoa sellowiana Berg

The effect of phenolic compounds on somatic embryogenesis in Feijoa sellowiana was analysed. The results showed that caffeic acid (140-560 microM) significantly increased somatic embryogenesis induction compared with the control. The presence of phloridzin, even at lower concentrations (11.5 microM), or caffeic acid or phloroglucinol at concentrations greater than 140.0 and 197.5 microM, respectively, inhibited somatic embryo development beyond the globular stage. When somatic embryos were transferred to the germination medium, the highest rates of germination (81.9%) were obtained with embryos induced in the presence of phloroglucinol (79.0 microM). At all concentrations tested, somatic embryos induced in medium containing phloroglucinol germinated at higher rates than those induced in the presence of caffeic acid. Histological and ultrastructural studies showed that somatic embryos were formed in close association with phenolic-rich cells which, in more advanced stages of development, formed a zone isolating the embryo from the maternal tissue. A comparative analysis of total phenolic content indicated that phenolics reached a peak by the third week of culture, independently of the medium used. However, after that period, the amount of phenolic compounds was significantly higher in explants cultured in the presence of phloroglucinol than in those cultured in the control or in caffeic acid-containing medium. Attempts to identify the type of phenolic compounds showed that flavan-3-ols and gallic acid derivatives were mainly produced in phloroglucinol-containing medium, whereas flavanones and dihydroflavonols were also present in medium containing caffeic acid. Flavones were the main phenols detected in the control. The ways in which phenolic compounds may affect somatic embryogenesis are discussed.

Antioxidant oligomeric proanthocyanidins from Cistus salvifolius

The purified proanthocyanidin oligomers of Cistus salvifolius herb extract accounted for 78% of the total proanthocyanidins and 73% of the total antioxidant activity of this extract. To elucidate the structure of the oligomer, it was depolymerized by acid catalysis in the presence of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were determined on the basis of 1D- and reverse 2D-NMR (HSQC, HMBC) experiments of their peracetylated derivatives, MALDI-TOF-MS and CD spectroscopy. These observations resulting from the degradation with phloroglucinol were confirmed by 13C NMR spectroscopy of the oligomer. The mean molecular weight of the higher oligomeric fraction was estimated to be 5–6 flavan-3-ol-units.

Structural Identification and Distribution of Proanthocyanidins in 13 Different Hops

Ten newly isolated hop proanthocyanidin oligomers and flavan-3-ol monomers from 13 different hops have been identified as gallocatechin, gallocatechin-(4alpha-->8)-catechin, gallocatechin-(4alpha-->6)-catechin, catechin-(4alpha-->8)-gallocatechin, catechin-(4alpha-->6)-gallocatechin, afzelechin-(4alpha-->8)-catechin, catechin-(4alpha-->8)-catechin-(4alpha-->8)-catechin, epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin, catechin-(4alpha-->8)-gallocatechin-(4alpha-->8)-catechin, and gallocatechin-(4alpha-->8)-gallocatechin-(4alpha-->8)-catechin, together with seven previously isolated oligomers, namely, catechin, epicatechin, epicatechin-(4beta-->8)-catechin, epicatechin-(4beta-->8)-epicatechin, catechin-(4alpha-->8)-catechin, catechin-(4alpha-->8)-epicatechin, and epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin. These compounds were subjected to acid-catalyzed degradation in the presence of phloroglucinol or by partial or complete acid-catalyzed degradation and reaction with benzyl mercaptan followed by desulfurization. The resultant adducts when compared to authentic samples by high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry served to identify the precursors. The composition of proanthocyanidins from 13 different hops was similar, but the concentration of individual compounds showed some differences, which indicated that hop proanthocyanidin profiles are affected by geographic origin and are variable depending on the cultivars.

Qualitative Determination of Proanthocyanidin Cleavage Products After Acid-Catalyzed Degradation in the Presence of Excess Phloroglucinol by Cyclodextrin-Modified Micellar Electrokinetic Chromatography

A micellar electrokinetic chromatographic (MEKC) method has been established for the identification and determination of acid-catalyzed proanthocyanidin cleavage products in the presence of phloroglucinol. Using fused-silica capillaries (50/57 cm, 22 kV) with phosphate buffer containing sodium dodecyl sulfate as the surfactant and β-cyclodextrin as modifier, structurally similar phloroglucinol adducts and flavan-3-ols were fully separated.