Oligomeric procyanidins in apple polyphenol are main active components for inhibition of pancreatic lipase and triglyceride absorption

Inhibitory effects of apple polyphenol extract (AP) and procyanidin contained in AP on in vitro pancreatic lipase activity and in vivo triglyceride absorption in mice and humans were examined. AP and procyanidin considerably inhibited in vitro pancreatic lipase activity. However, polyphenols, except for procyanidin, in AP (i.e., catechins, chalcones, and phenol carboxylic acids) showed weak inhibitory activities on pancreatic lipase. Procyanidins separated by normal-phase chromatography according to the degree of polymerization were also examined. Inhibitory effects of procyanidins increased according to the degree of polymerization from dimer to pentamer. On the other hand, pentamer or greater procyanidins showed maximal inhibitory effects on pancreatic lipase. These results suggested that with respect to in vitro pancreatic lipase inhibition, the degree of polymerization was an important factor and oligomeric procyanidin mainly contributed. Next, we performed a triglyceride tolerance test in mice and humans. Simultaneous ingestion of AP and triglyceride significantly inhibited an increase of plasma triglyceride levels in both models. These results suggested that the oligomeric procyanidins contained in AP inhibited triglyceride absorption by inhibiting pancreatic lipase activity in mice and humans.     

Procyanidin trimers to pentamers fractionated from apple inhibit melanogenesis in B16 mouse melanoma cells

The effects of apple polyphenols on melanogenesis in B16 mouse melanoma cells were investigated. The inhibitory effect of apple polyphenols was stronger than that of arbutin or kojic acid. Three polyphenol fractions (phenolic acid derivatives, procyanidins and other flavonoids) were isolated, and the procyanidins were fractionated according to the degree of polymerization using normal-phase chromatography. The procyanidin trimer-to-pentamer fractions were found to have the most pronounced effect on melanogenesis. Furthermore, each procyanidin fraction inhibited mushroom tyrosinase. No correlation between the degree of procyanidin polymerization and tyrosinase inhibitory activity was observed. Nevertheless, these observations suggest that procyanidins are effective inhibitors of tyrosinase.     

Rapid preparation of procyanidins B2 and C1 from Granny Smith apples by using low pressure column chromatography and identification of their oligomeric procyanidins

Research in the field of procyanidins is always hindered by the lack of procyanidin standards, and the preparation of procyanidins, especially in large scale, remains difficult and time-consuming. Commercial sources of procyanidin standards are scarce. In this study, a rapid preparation method of procyanidins by using low-pressure column chromatography was developed. Procyanidins in Granny Smith apples were extracted with boiled water and purified on an ADS-17 macroporous resin column to obtain a Granny Smith apple procyanidin extract (GSE). GSE was fractionated according to its degree of polymerization on a Toyopearl TSK HW-40s column. Procyanidins B2 (epicatechin-(4beta-8)-epicatechin) and C1 (epicatechin-(4beta-8)-epicatechin-(4beta-8)-epicatechin) were prepared without HPLC separation. Oligomeric procyanidins from Granny Smith apples were also identified by liquid chromatography-electrospray ionization-mass spectrometry.     

Isolation and structural elucidation of some procyanidins from apple by low-temperature nuclear magnetic resonance

Procyanidin fractions from apple were separated according to the degree of polymerization using normal phase chromatography. Evaluation of physiological functionalities of procyanidins requires individual structural determination. However, it is difficult to elucidate the structure of procyanidins, in particular those with (+)-epicatechin (1) or (-)-catechin (2) units, and determine whether the interflavanoid bonds are 4beta-->8 or 4beta-->6 without cleavage and acetylation. Structural determination used LC-MS and low-temperature NMR. Nine procyanidins were separated by preparative HPLC consisting of three well-known procyanidins [procyanidin B1 (3), procyanidin B2 (4), and procyanidin C1 (5)] and six new procyanidins [epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin (6); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-catechin (7); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-epicatechin (8); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-catechin (9); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-epicatechin (10); and epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin (11)]. Compounds 6-11 were detected for the first time as apple constituents.     

Impact of Processing on the Noncovalent Interactions between Procyanidin and Apple Cell Wall

Procyanidins can bind cell wall material in raw product, and it could be supposed that the same mechanism of retention of procyanidins by apple cell walls takes place in cooked products. To evaluate the influence of cell wall composition and disassembly during cooking on the cell walls' capacity to interact with procyanidins, four cell wall materials differing in their protein contents and physical characteristics were prepared: cell wall with proteins, cell wall devoid of protein, and two processed cell walls differing by their drying method. Protein contents varied from 23 to 99 mg/g and surface areas from 1.26 to 3.16 m(2)/g. Apple procyanidins with an average polymerization degree of 8.7 were used. The adsorption of apple procyanidins on solid cell wall material was quantified using the Langmuir isotherm formulation. The protein contents in cell wall material had no effect on procyanidin/cell wall interactions, whereas modification of the cell wall material by boiling, which reduces pectin content, and drying decreased the apparent affinity and increased the apparent saturation levels when constants were expressed relative to cell wall weight. However, boiling and drying increased apparent saturation levels and had no effect on apparent affinity when the same data were expressed per surface units. Isothermal titration calorimetry indicated strong affinity (K(a) = 1.4 × 10(4) M(-1)) between pectins solubilized by boiling and procyanidins. This study higllights the impact of highly methylated pectins and drying, that is, composition and structure of cell wall in the cell wall/procyanidin interactions.     

Polyphenolic profiles of Basque cider apple cultivars and their technological properties

The polyphenolic compositions of 31 Basque cider apple cultivars were determined in pulp, peel, and juice by high-performance liquid chromatography with diode array detection analysis of crude extracts and after thiolysis. Total polyphenols are distributed in a wide concentration range depending on the cultivar. Procyanidins are the class of polyphenols that present major concentrations in apple. Their average degrees of polymerization range from 4 to 8 depending on the cultivar. Apple cultivars were technologically classified into bitter and nonbitter categories using different classification systems obtained by applying several pattern recognition techniques, such as principal component analysis, K-nearest neighbors, soft independent modeling of class analogy, partial least-squares, and multilayer feed-forward-artificial neural networks, to apple pulp, peel, or juice data (individual polyphenol concentrations, total procyanidin content, and the average degree of polymerization of procyanidins). Bitter apple cultivars present higher contents of flavan-3-ols and/or dihydrochalcones than nonbitter cultivars. Detailed knowledge of the polyphenolic profile of each apple cultivar affords information about their susceptibility to oxidation, their sensory properties (bitterness, astringency), and their possible influence on the characteristics and quality of the final product (juice, cider) when apples are processed.     

Isolation and structure elucidation of procyanidin oligomers from Saskatoon berries (Amelanchier alnifolia)

Proanthocyanidin oligomers with different degrees of polymerization were isolated from Saskatoon berries (Amelanchier alnifolia) by means of gel adsorption and normal-phase liquid chromatography. The proanthocyanidins were identified using electrospray ionization mass spectrometry, nuclear magnetic resonance spectroscopy, and thiolytic degradation coupled with reversed-phase liquid chromatography. The results established that Saskatoon berries contain proanthocyanidins from dimers through heptamers and higher polymers. Saskatoon proanthocyanidins are essentially of procyanidin type, consisting mainly of epicatechin units linked by B-type bonds. The simple procyanidin profile of Saskatoon berries allowed the procyanidins to be separated precisely according to their degrees of polymerization. In the future they can be used as standard compounds for qualitative and quantitative analysis of procyanidins as well as for elucidation of the biological activities of proanthocyanidins.     

Procyanidin-rich extract from grape seeds prevents cataract formation in hereditary cataractous (ICR/f) rats

Antioxidants such as vitamin C, vitamin E, and carotenoids have been reported to prevent the progression of experimentally induced cataracts. However, little is known of the effect of procyanidins, a powerful antioxidant, on cataract formation. This paper investigates the anticataract activity of grape seed extract (GSE, which contains 38.5% procyanidins) in hereditary cataractous rats (ICR/f rats). The ICR/f rats were fed a standard diet containing 0 or 0.213% GSE [0.082% procyanidins in the diet (w/w)] for 27 days. The GSE significantly prevented and postponed development of cataract formation by evaluation of slit lamp observations of the rats' eyes. Lens weight and malondialdehyde concentration in the lens and plasma cholesteryl ester hydroperoxide (ChE-OOH) level induced by CuSO4 were significantly lower in the GSE group compared with the control group. The rats were also fed for 14 days either the diet containing 0.085% procyanidin dimer to tetramer fraction (0.085% as the procyanidins), the diet containing 0.090% procyanidin pentamer to heptamer fraction (0.085% as the procyanidins), or the diet containing 0.093% procyanidin oligomers more than decamer fraction (0.085% as the procyanidins). The ChE-OOH levels in the procyanidin pentamer to heptamer and procyanidin oligomers more than decamer groups were significantly lower than in the procyanidin dimer to tetramer group. These results suggested that procyanidins and their antioxidative metabolites prevented the progression of cataract formation by their antioxidative action. The larger molecular procyanidins in the GSE might contribute this anticataract activity.     

Characterization of oligomeric procyanidins and identification of quercetin glucuronide from lotus ( Nelumbo nucifera Gaertn.) seedpod

Procyanidins are a class of polyphenols in the plant kingdom. Lotus ( Nelumbo nucifera Gaertn.) seedpods, the inedible part of lotus and a byproduct during the production of lotus seeds, were found to be a new source rich in procyanidins. Detailed information about oligomeric procyanidins in lotus seedpods remains unknown. In this study, lotus seedpods were extracted using 60% aqueous methanol and characterized with phloroglucinolysis and liquid chromatography (mass spectrometry with an electrospray ionization source). The results indicate that the oligomeric and polymeric fraction had a mean degree of polymerization of 3.2 and 15.4, respectively, and consisted of (+)-catechin (m/z 289), gallocatechin or epigallocatechin (m/z 305), quercetin glycoside (m/z 463), quercetin glucuronide (m/z 477), procyanidin dimers (m/z 577.1), proanthocyanidin dimer gallate (m/z 593.3), prodelphinidin dimers (m/z 609.1), procyanidin trimers (m/z 865.1), etc. Quercetin glucuronide was further purified using flash chromatography and identified as quercetin-3-O-β-glucuronide by determining its exact mass using ion-trap time-of-flight mass spectrometry and 1H and 13C nuclear magnetic resonance, 1H-detected heteronuclear single-quantum coherence, and 1H-detected heteronuclear multiple-bond correlation analyses.     

Polyphenols are intensively metabolized in the human gastrointestinal tract after apple juice consumption

Polyphenols are secondary plant compounds showing anticarcinogenic effects both in vitro and in animal experiments and may thus reduce the risk of colorectal cancer in man. The identification of polyphenol metabolites formed via their passage through the small intestine of healthy ileostomy subjects after apple juice consumption is presented. Identification and quantification of polyphenols and their metabolites were performed using HPLC-DAD as well as HPLC-ESI-MS/MS. Total procyanidin content (TPA) was measured, and additionally the mean degree of polymerization (DPm) of the procyanidins was determined in the apple juice and ileostomy effluents. As products of polyphenol metabolism, D-(-)-quinic acid and methyl esters of caffeic acid and p-coumaric acid are liberated from the corresponding hydroxycinnamic acid esters. 1-Caffeoylquinic acid and 3-caffeoylquinic acid were determined as products of isomerization. Phloretin 2'-O-glucoside (phloridzin) and phloretin 2'-O-xyloglucoside were metabolized into the corresponding aglycons phloretin and phloretin 2'-O-glucuronide and all were found in the ileostomy effluent. Ninety percent of the consumed procyanidins were recovered in the ileostomy effluent and therefore would reach the colon under physiologic circumstances. The DP m was reduced (DP m of apple juice=5.7) and varied depending on the time point of excretion. The gastrointestinal passage seems to play an important role in the colonic availability of apple polyphenols.     

Thiolysis-HPLC characterization of apple procyanidins covering a large range of polymerization states

Procyanidins from the cortex of two cider apple varieties (Malus domestica; Kermerrien and Avrolles) were extracted by solvents. After a solid-phase extraction step, they were fractionated by normal- or reversed-phase HPLC at the semipreparative scale to obtain a series of purified fractions covering a wide range of polymerization states. Freeze-dried fractions were characterized by reversed-phase HPLC following thiolysis. Elution on normal-phase HPLC gave oligomeric procyanidins fractions with (average degree of polymerization) values varying from 2 to 8, whereas polymeric fractions ( values varying from 7 to 190) were obtained by reversed-phase HPLC. Constitutive units were mainly (-)-epicatechin with a proportion above 95% for all fractions. Thiolysis yields were wholly homogeneous with an average value of 75%, which indicates that the efficiency of the reaction did not depend on the polymerization state of the procyanidin fractions.     

Inhibition of trypsin by condensed tannins and wine

Phenolic compounds are abundant vegetable secondary metabolites in the human diet. The ability of procyanidin oligomers and wine polyphenols to inhibit trypsin activity was studied using a versatile and reliable in vitro method. The hydrolysis of the chromogenic substrate N-benzoyl-d,l-arginine-p-nitroanilide (BApNA) by trypsin was followed by spectrophotometry in the presence and absence of condensed tannins and wine. A clear relationship between the degree of polymerization of procyanidins and enzymatic inhibition was observed. Trypsin activity inhibition was also detected in several types of wine. In general, the inhibition increased with the concentration of phenolic compounds in wines. These results may be relevant when considering these compounds as antinutritional factors, thereby contributing to a reduced absorption of nutrients.     

Impact of noncovalent interactions between apple condensed tannins and cell walls on their transfer from fruit to juice: studies in model suspensions and application

The adsorption of procyanidins (condensed tannins) on cell-wall material was quantified by bringing into contact solutions of procyanidins and suspensions of cell-wall material. A model was developed on the basis of the Langmuir isotherm formulation and a factorial experimental design. The parameters that influenced the adsorption were the concentration and molecular weight of the procyanidins, the ionic strength of the solution, the temperature, and the apple cell-wall concentration. The model was applied to partitioning of procyanidins from apple between juice and mash. The parameters to be taken into account are the composition of the apples and, specifically, (i) the concentration and molecular weight of the procyanidins, (ii) their acidity and pH as a determinant of the ionic strength, and (iii) their cell-wall content and the temperature at pressing. To estimate the ability of the model to relate procyanidin concentrations in the juice to their concentration in the apple, apples of three varieties of widely different procyanidin compositions were pressed in conditions that prevent oxidation. In these conditions, yields in the juice were >80% for phenolic acids or catechin monomers but <50% for procyanidins, with the lowest rates obtained for the higher polymers in accordance with the model.     

Technological classification of basque cider apple cultivars according to their polyphenolic profiles by pattern recognition analysis

The polyphenolic compositions of 31 Basque cider apple cultivars were determined in pulp, peel, and juice by high-performance liquid chromatography--diode array detection analysis of crude extracts and after thiolysis. Data sets, consisting of individual polyphenol concentrations, total procyanidin content, and the average degree of polymerization of procyanidins, were evaluated by multivariate chemometric techniques, to develop decision rules for classifying apple cultivars technologically into bitter and nonbitter categories. A preliminary study of the data structure was performed by cluster analysis and principal component analysis in each apple material. Bitter apple varieties presented higher contents of flavan-3-ols and/or dihydrochalcones than nonbitter cultivars. Different classification systems for the two categories on the basis of the chemical data were obtained applying several supervised pattern recognition procedures, such as linear discriminant analysis, K-nearest neighbors, soft independent modeling of class analogy, partial least-squares, and multilayer feed forward artificial neural networks. Excellent performance in terms of recognition and prediction abilities for both categories (100% of hits) was achieved in every case (pulp, peel, or juice). Polyphenolic profiles of apple pulp, peel, or juice provide enough information to develop classification criteria for establishing the technological group of apple cultivars (bitter or nonbitter).     

NMR, ESI/MS, and MALDI-TOF/MS analysis of pear juice polymeric proanthocyanidins with potent free radical scavenging activity

The structure of a polymeric proanthocyanidin fraction isolated from pear juice was characterized by NMR, ESI/MS, and MALDI-TOF/MS analyses, and its antioxidant activity was investigated using the DPPH free radical scavenging method. The results obtained from 13C NMR analysis showed the predominance of signals representative of procyanidins. Typical signals in the chemical shift region between 70 and 90 ppm demonstrated the exclusive presence of epicatechin units. The results obtained through negative ESI/MS analysis showed singly and doubly charged ions corresponding to the molecular mass of procyanidins with a degree of polymerization up to 22. The spectra obtained through MALDI-TOF/MS analysis revealed the presence of two series of tannin oligomers. Supporting the observations from NMR spectroscopy, the first series consists of well-resolved tannin identified as procyanidin polymers units with chain lengths of up to 25. A second series of monogalloyl flavan-3-ols polymers with polymerization degree up to 25 were also detected. This is the first mass spectrometric evidence confirming the existence of galloylated procyanidin oligomers in pear fruits. Within each of these oligomers, various signals exist suggesting the presence of several oligomeric tannins. The antioxidant properties of the polymeric fraction were investigated through reduction of the DPPH free radical, and the results obtained showed that the polymeric fraction exhibited a higher antioxidant power compared to those of (+)-catechin and B3 procyanidin dimer.     

HPLC method for the quantification of procyanidins in cocoa and chocolate samples and correlation to total antioxidant capacity.

Monomeric and oligomeric procyanidins present in cocoa liquors and chocolates were separated and quantified in four different laboratories using a normal-phase high-performance liquid chromatography (HPLC) method with fluorescence detection. Procyanidin standards through decamers were obtained by extraction from cocoa beans, enrichment by Sephadex LH-20 gel permeation chromatography, and final purification by preparative normal-phase HPLC. The purity of each oligomeric fraction was assessed using HPLC coupled to mass spectrometry. A composite standard was then prepared, and calibration curves were generated for each oligomeric class using a quadratic fit of area sum versus concentration. Results obtained by each of the laboratories were in close agreement, which suggests this method is reliable and reproducible for quantification of procyanidins. Furthermore, the procyanidin content of the samples was correlated to the antioxidant capacity measured using the ORAC assay as an indicator for potential biological activity.     

Fractionation of polymeric procyanidins from lowbush blueberry and quantification of procyanidins in selected foods with an optimized normal-phase HPLC-MS fluorescent detection method

The polymeric procyanidins were fractionated from lowbush blueberry on a Sephadex LH-20 column. The degree of polymerization (DP) for the polymers was determined by thiolysis to be in a range of 19.9 to 114.1. Normal-phase HPLC analysis indicated that the polymeric procyanidins did not contain oligomeric procyanidins with DP < 10. The polymers eluted as a single peak at the end of the chromatogram. The normal-phase HPLC gradient was modified to improve the separation of procyanidin monomers through decamers and to elute all the polymers beyond those as a distinct peak. Monomers through decamers were quantified individually. All the polymers (DP > 10) were quantified using a mixture of purified polymers as an external standard. Polymers were found to be the dominant procyanidins in brown sorghum bran, cranberry, and blueberry. Thiolysis of the polymer peaks indicated that epicatechin was present as extension units in these foods, however, the composition of terminal units varied considerably between catechin and epicatechin, or an A-type dimer linkage in the case of cranberry.     

Inhibition of apple polyphenol oxidase activity by procyanidins and polyphenol oxidation products

The rate of consumption of dissolved oxygen by apple polyphenol oxidase in cider apple juices did not correlate with polyphenol oxidase activity in the fruits and decreased faster than could be explained by the decrease of its polyphenolic substrates. The kinetics parameters of a crude polyphenol oxidase extract, prepared from apple (Braeburn cultivar), were determined using caffeoylquinic acid as a substrate. Three apple procyanidin fractions of n 80, 10.5, and 4 were purified from the parenchyma of cider apples of various cultivars. Procyanidins, caffeoylquinic acid, (-)-epicatechin, and a mixture of caffeoylquinic acid and (-)-epicatechin were oxidized by reaction with caffeoylquinic acid o-quinone in order to form oxidation products. All the fractions were evaluated for their inhibitory effect on PPO activity. Native procyanidins inhibited polyphenol oxidase activity, the inhibition intensity increasing with n. The polyphenol oxidase activity decreased by 50% for 0.026 g/L of the fraction of n 80, 0.17 g/L of the fraction of n 10.5, and 1 g/L of the fraction of n 4. The inhibitory effect of oxidized procyanidins was twice that of native procyanidins. Oxidation products of caffeoylquinic acid and (-)-epicatechin also inhibited polyphenol oxidase.     

Study of the interaction of pancreatic lipase with procyanidins by optical and enzymatic methods

The interactions between porcine pancreatic lipase (PL) and grape seed procyanidins were studied by an enzymatic assay, fluorescence quenching, nephelometry, and dynamic light scattering (DLS). An inhibitory effect of grape seed procyanidins on lipase hydrolytic activity was found. Both the inhibition of lipase activity by procyanidins and the respective quenching of intrinsic protein fluorescence increased with the average degree of polymerization of the tested procyanidins. The association between procyanidins and enzyme involves a specific interaction as inferred from the fluorescence assays despite not changing significantly the tertiary structure of the protein. For all tested procyanidins it was shown, both by DLS and by nephelometry, that an increase in aggregation occurs up to a stoichiometric maximum after which further procyanidin addition causes a decrease in aggregation of aggregates. The maximum size of aggregates was shown to be closely related to the maximum overall aggregation. It was also shown that the inhibition of enzyme activity is to a large extent independent of the formation of aggregates.     

High-performance liquid chromatography separation and purification of cacao (Theobroma cacao L.) procyanidins according to degree of polymerization using a diol stationary phase

A new chromatographic approach for separating cacao procyanidins according to their degree of polymerization has been developed. It utilizes diol stationary phase columns operating in normal phase mode with a binary gradient of acidified acetonitrile and methanol-water. Performance of the diol stationary phase was evaluated on an analytical scale utilizing classical chromatographic conditions for the normal phase separation of procyanidins according to their degree of polymerization. The new separation approach was developed on an analytical scale but further extended to the preparative scale. These newly developed analytical and preparative high-performance liquid chromatography procedures were successfully applied to the separation, as well as isolation, of cacao procyanidins from unfermented cacao seeds. The degree of polymerization associated with each molecular weight fraction was determined by mass spectrometry.