Qualitative analysis and HPLC isolation and identification of procyanidins from Vicia faba

The soluble proanthocyanidins of the coloured seed coats of Vicia faba L. were isolated and separated by solvent partition. The chemical characteristics of the proanthocyanidins were elucidated by total oxidation and partial degradation in the presence of phloroglucinol followed by HPLC analysis. The native extract of proanthocyanidins contained (+)-gallocatechin, (-)-epigallocatechin, (+)-catechin and (-)-epicatechin units. Oligomeric procyanidins were purified by chromatography on Sephadex LH-20 and the accessible compounds were isolated by RP-HPLC using a Licrospher Li 100 Column. The structures of the purified oligomeric procyanidins were elucidated using a procedure involving TLC, UV spectroscopy, ESI-MS and HPLC analysis of the products from the phloroglucinol reaction. The major condensed tannins of Vicia faba comprise six compounds identified as two A-type procyanidin dimers, the procyanidin dimers B1, B2 and B3, and a procyanidin trimer.     

The relationship between antiglycation activity and procyanidin and phenolic content in commercial grape seed products

Eight commercial grape seed products (GSPs) were assessed for their inhibition of the formation of advanced glycation end-products in vitro. All 8 commercial GSPs included in this study were potent inhibitors of advanced glycation end-product formation with IC(50) values ranging from 2.93 to 20.0?μg/mL. Total procyanidin content ranged from 60% to 73%. HPLC-DAD-ELSD results indicate that (+)-catechin, (-)-epicatechin, procyanidin B1, and procyanidin B2 were predominant and ubiquitously present in all the products under study, while gallic acid and procyanidin B4 were present in relatively minor amounts. The IC(50) values correlated with total phenolic content, and multiple regression analysis indicated that IC(50) is a linear function of the concentration of gallic acid and procyanidins B1, B2, and B4. Based on this study, GSPs have the potential to complement conventional diabetes medication toward disease management and prevention.     

Modulation of MPP+ uptake by procyanidins in Caco-2 cells: involvement of oxidation/reduction reactions

It is becoming increasingly evident that the absorption of certain nutrients and drugs and their effects are largely influenced by the concomitant ingestion of other substances. As various xeno- and endobiotics belong to the class of organic cations, the aim of this work was to study the modulation of the intestinal apical uptake of organic cations by diet procyanidins.Five procyanidin fractions with different structural complexity were obtained after fractionation of a grape seed extract. The effect of these compounds on 1-methyl-4-phenylpyridinium (MPP⁺) uptake was evaluated in Caco-2 cells.Apical uptake of ³H-MPP⁺ by Caco-2 cells was increased by a 60 min exposure to 600 μg ml⁻¹ of procyanidin fractions, that increase being positively related with procyanidins structural complexity. It was verified that ³H-MPP⁺ uptake increased with preincubation time. It was speculated that procyanidins were oxidized during preincubation, this change could interfered with transport activity. Tested oxidizing agents showed that the redox state of the transporter could affect its activity. Additionally, trans-stimulation experiments showed that catechin and fraction I (the simpler fraction) can use the same transporter as MPP⁺. The results are compatible with the hypothesis of these compounds being competitive inhibitors of MPP⁺ transport.In conclusion, procyanidins are capable to modulate MPP⁺ apical uptake in Caco-2 cells, this transport being most probably modulated through oxidation-reduction phenomena. Interactions between these compounds and drugs present in the diet may affect their absorption and bioavailability. Both the concentration and complexity of the procyanidin compounds should be taken into account in medical practice.     

Downregulated Lipid Metabolism in Differentiated Murine Adipocytes by Procyanidins from Defatted Grape Seed Meal

Recent reports have proposed possible anti-obesity mechanisms for antioxidants involving increased energy expenditure, pre-adipocyte differentiation and proliferation, decreased lipogenesis and increased lipolysis, and fat oxidation. The aim of this study was to examine and to confirm the anti-obesity effect of the oligomeric and polymeric procyanidin fractions from defatted grape seeds. The lipid metabolism-related mRNA level in the mouse preadipocytes, 3T3-L1 cells, was determined to evaluate the anti-obesity effect of the phenolic fractions from a grape seed meal. Lipid accumulation was reduced by 19% of the control level by the procyanidin fraction originating from the grape seed meal. Emerging from the effect of the treatment on HSL and LPL mRNA expression, lipolytic enzyme activity was not involved in the anti-obesity effects of CPE and FPP from the defatted grape seed meal. We tested and confirmed in this study the effect of the biological activities of oligomeric and polymeric procyanidins from the defatted grape seed meal. It is suggested from the results of this brief study that further studies would be desirable to focus on the anti-obesity effect of the purified extracts of a defatted grape seed meal.     

Optimized ultra-high-pressure-assisted extraction of procyanidins from lychee pericarp improves the antioxidant activity of extracts

To establish optimal ultra-high-pressure (UHP)-assisted extraction conditions for procyanidins from lychee pericarp, a response surface analysis method with four factors and three levels was adopted. The optimum conditions were as follows: 295 MPa pressure, 13 min pressure holding time, 16.0 mL/g liquid-to-solid ratio, and 70% ethanol concentration. Compared with conventional ethanol extraction and ultrasonic-assisted extraction methods, the yields of the total procyanidins, flavonoids, and phenolics extracted using the UHP process were significantly increased; consequently, the oxygen radical absorbance capacity and cellular antioxidant activity of UHP-assisted lychee pericarp extracts were substantially enhanced. LC-MS/MS and high-performance liquid chromatography quantification results for individual phenolic compounds revealed that the yield of procyanidin compounds, including epicatechin, procyanidin A2, and procyanidin B2, from lychee pericarp could be significantly improved by the UHP-assisted extraction process. This UHP-assisted extraction process is thus a practical method for the extraction of procyanidins from lychee pericarp.     

Antioxidant properties of catechins and proanthocyanidins: Effect of polymerisation, galloylation and glycosylation

A range of catechins and oligomeric procyanidins was purified by high performance liquid chromatography (HPLC) from grape seed, apple skin, lentil and almond flesh. Catechins, galloylated epicatechin, glycosylated catechin, procyanidin dimers, galloylated dimers, trimer, and tetramer species were all identified, purified and quantified by HPLC, LC-MS and NMR. The antioxidant properties of these compounds were assessed using two methods: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; (b) scavenging of the radical cation of 2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). Antioxidant activity in the lipid phase decreased with polymerisation in contrast with antioxidant action in the aqueous phase which increased from monomer to trimer and then decreased from trimer to tetramer. Galloylation of catechin and dimeric procyanidins decreased lipid phase and increased aqueous phase antioxidant activity. Glycosylation of catechin demonstrated decreased activity in both phases.     

Antioxidant properties of catechins and proanthocyanidins: Effect of polymerisation,galloylation and glycosylation

A range of catechins and oligomeric procyanidins was purified by high performance liquid chromatography (HPLC) from grape seed, apple skin, lentil and almond flesh. Catechins, galloylated epicatechin, glycosylated catechin, procyanidin dimers, galloylated dimers, trimer, and tetramer species were all identified, purified and quantified by HPLC, LC-MS and NMR. The antioxidant properties of these compounds were assessed using two methods: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; (b) scavenging of the radical cation of 2,2′-azinobis(3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). Antioxidant activity in the lipid phase decreased with polymerisation in contrast with antioxidant action in the aqueous phase which increased from monomer to trimer and then decreased from trimer to tetramer. Galloylation of catechin and dimeric procyanidins decreased lipid phase and increased aqueous phase antioxidant activity. Glycosylation of catechin demonstrated decreased activity in both phases.     

葡萄籽中原花青素提取方法优化处理

溶剂提取法是提取葡萄籽原花青素的常用方法 ,然而 ,在不同提取条件下 ,提取效果并不一致。利用水、甲醇、乙醇、丙酮及它们的水溶液提取葡萄籽中的原花青素 ,而后用铁盐催化比色法测原花青素含量 ,考察了提取剂的浓度、粉碎度对提取的影响。在优化处理的基础上 ,获得了有效的提取条件 :葡萄籽粉碎度 10 0目 ,提取剂 70 %甲醇水溶液。     

Procyanidins from the seeds of Vitis amurensis

Three procyanidins, procyanidin B-5 3'-O-gallate, vitisinol, and amurensisin, were isolated from the seeds of Vitis amurensis, whose structures were elucidated on the basis of spectral and chemical evidence. Vitisinol and amurensisin contain a spiro-type biflavanyl linkage and a biphenyl-lactone partial structure, respectively.     

Identification of Proanthocyanidins from Litchi (Litchi chinensis Sonn.) Pulp by LC-ESI-Q-TOF-MS and Their Antioxidant Activity

Content of total proanthocyanidins as well as total phenolics, flavonoids, antioxidant activities were evaluated for litchi (Litchi chinensis Sonn.) pulp of 32 cultivars. One cultivar, Hemaoli, showed the highest total proanthocyanidins and total phenolics, and DPPH or ABTS radical scavenging activities. ESI-MS and NMR analysis of the Hemaoli pulp crude extracts (HPCE) showed that procyandins composed of (epi)catechin unites with degree of polymerization (DP) of 2–6 were dominant proanthocyanidins in HPCE. After the HPCE was fractionated by a Sephadex LH-20 column, 32 procyanidins were identified by LC-ESI-Q-TOF-MS in litchi pulp for the first time. Quantification of individual procyanidin in HPCE indicated that epicatechin, procyanidin B2, procyanidin C1 and A-type procyanidin trimer were the main procyanidins. The radical scavenging activities of different fractions of HPCE as well as six procyanidins standards were evaluated by both DPPH and ABTS assays. HPCE fractions showed similar antioxidant activities with those of Vc and six individual procyanidins, the IC₅₀ of which ranged from 1.88 ± 0.01 to 2.82 ± 0.10 μg/ml for DPPH assay, and from 1.52 ± 0.17 to 2.71 ± 0.15 μg/ml for ABTS assay. Such results indicate that litchi cultivars rich in proanthocyanidins are good resources of dietary antioxidants and have the potential to contribute to human health.     

Grape and Grape Seed Extract Capacities at Protecting LDL against Oxidation Generated by Cu 2+ , AAPH or SIN-1 and at Decreasing Superoxide THP-1 Cell Production. A Comparison to other Extracts or Compounds

A large body of evidence supports the key role of oxidized low-density lipoprotein in atherosclerosis. The aim of this study was to compare the capacity of natural polyphenols (PP) from Vitis vinifera and Olea europea at protecting LDL against oxidation brought about by Cu 2+ , oxygen-centered radical-generating AAPH, or peroxynitrite-generating SIN-1 in vitro systems, or at impairing superoxide production in promonocyte cells (THP-1) conveniently differentiated into adherent macrophages. PP were either from the whole grape (fraction A) containing mainly procyanidins, (epi)-catechin and anthocyanins, or from grape seed extracts (fractions B and C) consisting of tannins and procyanidin oligomers with a higher content in B than in C, or from a grape skin extract (fraction D) consisting mainly of anthocyanins, or from a hydrosoluble olive mill wastewater PP extract (fraction E) containing hydroxytyrosol and oleuropein. Chlorogenic acid (F) and catechin (G) were taken as archetypes of PP preventing oxidation partly as copper scavenger and as radical scavenger only, respectively. All grape fractions were efficient towards Cu 2+ system (equally or more efficient than F), whereas they were rather poorly efficient towards AAPH and SIN-1 (less efficient than G but as efficient as F). Among the PP fractions, B was the most effective at protecting LDL in the SIN-1 system and at impairing THP-1 superoxide production. Taken together, these data suggest that the PP fraction from grape seed rich in procyanidins achieves the best compromise between the direct and indirect (i.e. cell-mediated) types of action in protecting LDL against oxidation, strengthening the need for improving the knowledge of its bioavailability in humans.     

Changes in grape seed polyphenols during fruit ripening

The quantity and characterization of extracted flavan-3-ol monomers and procyanidins was determined in seeds from Vitis vinifera cv. Cabernet Sauvignon berries, over the course of ripening and at different levels of vine water status. The per berry extractive yield of all polyphenols decreased with maturity, and followed second-order kinetics. The flavan-3-ol monomers decreased most rapidly, followed by the procyanidin extension units and finally, the terminal units. The relative proportion of procyanidin extension units did not vary with maturity. During fruit ripening, the mean degree of polymerization of extracted procyanidins is unchanged when analyzed intact by HPLC, but decreases by thiolytic degradation. The proportion of extracted procyanidins resistant to acid catalyzed thiolysis increased with maturity. Changes in vine water status affected polyphenol amounts, indicating that cultural practices can be used to influence composition. Oxidation of the seed polyphenols during fruit ripening, could explain these observations.     

Grape and grape seed extract capacities at protecting LDL against oxidation generated by Cu2+, AAPH or SIN-1 and at decreasing superoxide THP-1 cell production. A comparison to other extracts or compounds

A large body of evidence supports the key role of oxidized low-density lipoprotein in atherosclerosis. The aim of this study was to compare the capacity of natural polyphenols (PP) from Vitis vinifera and Olea europea at protecting LDL against oxidation brought about by Cu 2+ , oxygen-centered radical-generating AAPH, or peroxynitrite-generating SIN-1 in vitro systems, or at impairing superoxide production in promonocyte cells (THP-1) conveniently differentiated into adherent macrophages. PP were either from the whole grape (fraction A) containing mainly procyanidins, (epi)-catechin and anthocyanins, or from grape seed extracts (fractions B and C) consisting of tannins and procyanidin oligomers with a higher content in B than in C, or from a grape skin extract (fraction D) consisting mainly of anthocyanins, or from a hydrosoluble olive mill wastewater PP extract (fraction E) containing hydroxytyrosol and oleuropein. Chlorogenic acid (F) and catechin (G) were taken as archetypes of PP preventing oxidation partly as copper scavenger and as radical scavenger only, respectively. All grape fractions were efficient towards Cu 2+ system (equally or more efficient than F), whereas they were rather poorly efficient towards AAPH and SIN-1 (less efficient than G but as efficient as F). Among the PP fractions, B was the most effective at protecting LDL in the SIN-1 system and at impairing THP-1 superoxide production. Taken together, these data suggest that the PP fraction from grape seed rich in procyanidins achieves the best compromise between the direct and indirect (i.e. cell-mediated) types of action in protecting LDL against oxidation, strengthening the need for improving the knowledge of its bioavailability in humans.     

Procyanidin B2 has anti- and pro-oxidant effects on metal-mediated DNA damage

Procyanidin B2 (epicatechin-(4beta-8)-epicatechin), which is present in grape seeds, apples, and cacao beans, has antioxidant properties. We investigated the mechanism of preventive action of procyanidin B2 against oxidative DNA damage in human cultured cells and isolated DNA. Procyanidin B2 inhibited the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in the human leukemia cell line HL-60 treated with an H2O2-generating system. In contrast, a high concentration of procyanidin B2 increased the formation of 8-oxodG in HL-60 cells. Experiments with calf thymus DNA also revealed that procyanidin B2 decreased 8-oxodG formation by Fe(II)/H2O2, whereas procyanidin B2 induced DNA damage in the presence of Cu(II), and H2O2 extensively enhanced it. An electron spin resonance spin trapping study utilizing 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) demonstrated that procyanidin B2 decreased the signal of M4PO-OH from H2O2 and Fe(II), whereas procyanidin B2 enhanced the signal from H2O2 and Cu(II). As an antioxidant mechanism, UV-visible spectroscopy showed that procyanidin B2 chelated Fe(II) at equivalent concentrations. As a pro-oxidant property, we examined DNA damage induced by procyanidin B2, using 32P-labeled DNA fragments obtained from genes relevant to human cancer. Our results raise the possibility that procyanidin B2 exerts both antioxidant and pro-oxidant properties by interacting with H2O2 and metal ions.     

Isolation and structure elucidation of tetrameric procyanidins from unripe apples (Malus pumila cv. Fuji) by NMR spectroscopy

Procyanidins are plant secondary metabolites widely consumed and known to have various physiological functions, but their bioavailability and mechanism of action are still unclear especially for larger oligomers. One of the reasons is scarce information about the detailed structure of oligomeric procyanidins. As for apple, structures of procyanidin components larger than trimers are scarcely known. In this study, 11 tetrameric procyanidins including two known compounds were isolated from unripe apples (Malus pumila cv. Fuji) and identified by NMR spectroscopic analysis and phloroglucinol degradation. As a result, the detailed structural diversity of tetrameric procyanidins in apple was established.     

An analytical survey of the polyphenols of seeds of varieties of grape (Vitis vinifera) cultivated in Greece: implications for exploitation as a source of value-added phytochemicals

Seed samples from 12 white and 25 red international and Hellenic native grape varieties (Vitis vinifera) were screened for their polyphenolic composition. The polyphenols determined were mainly of low molecular weight, including gallic acid, catechin, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate and the procyanidins B1 and B2. Average values of total content for white and red varieties (376 and 388 mg/100 g seeds, respectively) were very similar. Comparable results were observed with respect to the individual polyphenol content with seeds from red varieties being, in general, slightly richer. The predominant flavanol monomer in white and red varieties was catechin (which accounted for 50.5 and 49.3%, respectively, of the total content), whilst gallic acid and epigallocatechin were the constituents showing the lowest content, respectively. The data obtained are discussed with regard to the exploitation of grape seeds as a low-cost source of value-added phytochemicals.     

Interactions between apple (Malus x domestica Borkh.) polyphenols and cell walls modulate the extractability of polysaccharides

Apple polyphenol (procyanidin)–cell wall interactions were investigated and their impact on polysaccharide extractability were determined. Native and oxidised procyanidins with average degrees of polymerisation of 13 and 55 were incubated with cell walls. The effect of polyphenol oxidation was evaluated according to two designs: polyphenols were chemically oxidised either before or during interaction. The extent of procyanidin binding to cell walls was assessed by the weight increase of procyanidin–cell wall complexes as compared to weights of cell walls alone. Pectins and hemicelluloses were subsequently extracted from cell walls and from cell wall–procyanidin adducts using a chelating agent (ammonium oxalate), a pectin lyase treatment and NaOH.Weight increases of complexes ranged from 20% to 29%. Weight gains increased in the following order: native, pre-oxidised, simultaneously oxidised and bound procyanidins, these different fractions were, respectively, bound to cell walls. In presence of native procyanidins, oxalate extracted less pectins, and those pectins had lower degrees of methylation, as compared to cell walls alone. When cell walls were incubated with oxidised and oxidising procyanidins, even less pectins with lower degree of methylation were extracted. Major findings indicated that procyanidins mainly bound to pectins as compared to other cell wall compounds: (1) the procyanidin adsorption to cell walls limited the depolymerisation of pectins supposedly induced by pectin lyase. Thus less pectins were extracted but their degree of methylation increased, indicative of products of lysis of pectin lyase. (2) Hemicelluloses extracted using NaOH (4 M) were more abundant in pectins when oxidised or oxidising procyanidins were complexed rather than non complexed to cell walls.     

葡萄籽中的原花色素

本文对葡萄籽中原花色素的组成结构、提取分离、生理活性进行综述。     

Metabolism of dietary procyanidins in rats

Procyanidins are major dietary polyphenols made of elementary flavan-3-ol (epi)catechin units. They have antioxidant properties and may contribute to health benefits in humans, but little is known about their metabolic fate. We compared here the metabolism of procyanidin dimer B3, trimer C2, and polymer isolated from willow tree catkins to that of catechin monomer in rats. These compounds were administered in the rat diet (0.1%, w/w) for 5 d and their metabolites estimated in 24 h urine. In rats fed procyanidins, neither parent compound nor catechin derivatives could be detected in contrast to animals fed catechin monomer, which excreted large amounts of catechin and its 3'-O-methylated form (25.7 +/- 0.6%). On the other hand, 16 metabolites of microbial origin were detected and identified as phenylvaleric, phenylpropionic, phenylacetic, and benzoic acid derivatives. Their total yields significantly decreased from the catechin monomer (10.6 +/- 1.1%) to the procyanidin dimer (6.5 +/- 0.2%), trimer (0.7 +/- 0.1%), and polymer (0.5 +/- 0.1%). Therefore, the degree of procyanidin polymerization has a major impact on their fate in the body characterized by a poor absorption through the gut barrier and a limited metabolism by the intestinal microflora as compared to catechin. This will have to be considered to explain the health effects of procyanidins. The contribution of their microbial metabolites should also be further investigated.     

Effects of polyphenols from grape seeds on oxidative damage to cellular DNA

Grape seed polyphenols have been reported to exhibit a broad spectrum of biological properties. In this study, eleven phenolic phytochemicals from grape seeds were purified by gel chromatography and high performance liquid chromatography (HPLC). The antioxidant activities of five representative compounds with different structure type were assessed by the free radical-scavenging tests and the effects of the more potent phytochemicals on oxidative damage to DNA in mice spleen cells were investigated. Procyanidin B4, catechin, epicatechin and gallic acid reduced ferricyanide ion and scavenged the stable free radical, alpha, alpha-diphenyl-beta-picrylhydrazyl (DPPH) much more effectively than the known antioxidant vitamin ascorbic acid, while epicatechin lactone A, an oxidative derivative of epicatechin, did not reduce ferricyanide ion appreciably at concentrations used and was only about half as effective on free radical-scavenging as epicatechin. Mice spleen cells, when pre-incubated with relatively low concentration of procyanidin B4, catechin or gallic acid, were less susceptible to DNA damage induced by hydrogen peroxide (H2O2), as evaluated by the comet assay. In contrast, noticeable DNA damage was induced in mice spleen cells by incubating with higher concentration (150 microM) of catechin. Collectively, these data suggest that procyanidin B4, catechin, gallic acid were good antioxidants, at low concentration they could prevent oxidative damage to cellular DNA. But at higher concentration, these compounds may induce cellular DNA damage, taking catechin for example, which explained the irregularity of dose-effect relationship.