Sea buckthorn (Hippophae rhamnoides) proanthocyanidins inhibit in vitro enzymatic hydrolysis of protein

Abstract: Interactions of phenolics with other food constituents and digestive enzymes are likely to have interference with the digestion and bioavailability of food and phenolics. In this study the effect of sea buckthorn proanthocyanidins on in vitro digestion of protein was evaluated. Optimization of the extraction conditions showed that maximum recovery of sea buckthorn proanthocyanidins was obtained with acidified acetone; water mixture (60% to 70%, v/v). Crude proanthocyanidin extracts thus prepared were purified using sephadex gel column chromatography and their average degree of polymerization and the effects on enzymatic hydrolysis of bovine serum albumin as influenced by their protein precipitation capacities were studied. Average degree of polymerization of proanthocyanidins in berry pulp, kernel, seed coat, and leaves was 7.4, 5.6, 8.2, and 10.6, respectively. The EC50 values for the protein precipitation by the PA of berry pulp, kernel seed coat, and leaves were 44.2, 44.1, 65.8, and 39.8 μg, respectively. Relative enzymatic hydrolysis of the protein‐proanthocyanidin complexes was 44.1% to 60.3% for pepsin and 57.5% to 67.7% for trypsin. Interactions of sea buckthorn proanthocyanidins with food proteins and digestive enzymes might alter the protein digestibility and phenolic bioavailabilty.     

Phenolics,fibre and fibre digestibility in the crop residue from bird resistant and non-bird resistant sorghum varieties

The relationship between phenolics and parameters of in-vitro digestibility of neutral-detergent fibre (NDF) in leaves and stems from the crop residue of bird resistant (BR) and non-bird resistant (NBR) sorghum varieties was determined. The phenolics studied were lignin, insoluble proanthocyanidins, soluble red pigments and soluble phenolics as measured by absorbance (280 nm)and precipitation with ytterbium. Parameters of the digestibility of NDF studied were 48-h digestibility, potentially digestible NDF, rate of digestion and indigestible NDF as determined by non-linear regression analysis. Leaves from BR varieties were higher in most phenolics than leaves from NBR varieties. Digestibility of NDF at 48 h, potentially digestible NDF and rate of NDF digestion was lower in leaves from BR varieties. The indigestible NDF was higher in BR varieties. Phenolics in leaves were negatively correlated with digestibility of NDF at 48 h, potentially digestible NDF and rate of NDF digestion and positively correlated with indigestible NDF. Phenolics in stems had non-significant correlations with parameters of the digestibility of NDF except for a positive correlation between lignin and indigestible NDF. These results indicate that breeding for bird resistance in sorghum would lead to lower nutritive value of the crop residue.     

Comparison of analytical methods for measuring proanthocyanidins in wines and their relationship with perceived astringency

The concentration of proanthocyanidins from twenty red wines from cv. Cabernet Sauvignon, five rosé wines from cv. Cabernet Sauvignon and five white wines from cv. Sauvignon Blanc was quantified using four analytical methodologies, and their relationship with the perceived astringency was investigated. Proanthocyanidin concentrations were determined by a methylcellulose precipitation assay, a protein precipitation assay and two colourimetric methods (Bate‐Smith and vanillin assay). The four methodologies showed high repeatability but differed widely in proanthocyanidin concentrations. The methylcellulose and protein precipitation assays could not quantify proanthocyanidins in rosé and white wines. The protein precipitation assay gave the lowest concentration of proanthocyanidins in all of the red wines. The methylcellulose precipitation assay (r = 0.7725; r² = 0.59) and the protein precipitation assay (r = 0.6828; r² = 0.47) showed a strong correlation with the perceived astringency compared with the colourimetric methods. The strong correlation of the methylcellulose precipitation method with the perceived astringency could be a useful tool to estimate red wine astringency.     

Anti-oxidative characterisation of NaN3-induced common bean mutants

The contents of total phenolics, total anthocyanins, total proanthocyanidins and antioxidant activities were measured using 2,2-diphenyl-1-picrylhydrazyl radicals scavenging activity, ferric reducing antioxidant power, lipid peroxidation inhibition ability and ABTS⁻ radical-scavenging activity assays. These were compared in the seed coats of common bean (Phaseolus vulgaris L.) cultivar Hwachia and its eighteen NaN₃-induced mutants. NaN₃-induced mutants generally accumulated more total phenolics (19% more), total anthocyanins (65% more) and total proanthocyanidins (4% more) than Hwachia (containing 29.94 mg g⁻¹ total phenolics, 0.31 mg g⁻¹ total anthocyanins and 12.94 mg g⁻¹ total proanthocyanidins, respectively). Anthocyanidins including delphinine, cyanidin and pelargonidin were detectable in seed coat, each with different levels depending on the tested accessions. Significant correlations (average of 0.847 across all comparisons, p < 0.01) were found between the tested antioxidant activities, total phenolics, total anthocyanins and total proanthocyanidins. Mutants SA-11-2, SA-13-2 and SA-34-2 that are enriched with antioxidants may be useful in food and other applications.     

Phenolics and in-vitro degradability of protein and fibre in West African Browse

Relationships among soluble phenolics, soluble and insoluble proanthocyanidins (PAC), lignin, N, neutral-detergent fibre (NDF), and in-vitro degradability of protein and NDF were determined in 72 West African fodder trees and shrubs. Species were collected in the semi-arid (Niger), sub-humid (Nigeria) and humid/sub-humid (Benin) zones. Variation among species in chemical composition and in-vitro degradability of protein and NDF was large. Zones did not differ in mean content of phenolic compounds. Protein degradability was negatively correlated with soluble phenolics (r = ?0.34, P < 0.01) and soluble PAC (r = ?0.47, P < 0.001). NDF was positively correlated with soluble PAC (r = 0.44, P < 0.001), insoluble PAC (r = 0.28, P < 0.05) and lignin (r = 0.76, P < 0.001). NDF degradability was negatively correlated with soluble PAC (r = ?0.40, P < 0.001) and lignin (r = ?0.59, P < 0.001). Chemical composition and in-vitro degradability along with field observations can provide useful criteria for determining the nutritive value of browse species.     

Proteolytic regulation of the extent of dietary proteins with skin grape proanthocyanidin and anthocyanidin's interactions

During technological processing, proanthocyanidins and anthocyanidins could be partly lost due to the complexation phenomena, affecting food and beverage nutritional properties, organoleptic properties and health-promoting potentials. A common issue is encountered when processing food and beverage which is binding of phenolics to dietary proteins. The present investigation aims at evaluating the proteolysis contribution, using pure protease (Pepsin, 3000 units g⁻¹), to protein–anthocyanidin and protein–proanthocyanidin interactions. Bovine serum albumin (BSA), ovalbumin (OVA) and casein (CAS) dietary protein models were used. High-performance liquid chromatography coupled to diode array detector (HPLC-DAD) and size exclusion chromatography analyses proved that pepsin treatment significantly (P > 0.05) decreased the ratio of flavonoids’ interaction with tested proteins . The proteolysis reduced anthocyanidin interactions with CAS, OVA and BSA by 64.88%, 57.37% and 42.87% respectively. Similarly, proanthocyanidins interaction with CAS, OVA and BSA were reduced by 34.23%, 13.74% and 2.39% respectively. This study provides the basis to develop innovative technologies to limit protein–flavonoid complexation during food and beverage processing.     

Cellulase inhibition by tannins in the testa of field beans (Vicia faba)

The testa of field bean varieties with coloured flowers contained 4–8 % tannin (measured as total phenolics) compared with less than 0.6 % in the testa of white flowered varieties. The tannin present in whole beans was almost entirely contained in the testa. The presence of high levels of tannin reduced in vitro digestibility and solubility in cellulase solutions of the testa and addition of high tannin testa decreased the solubility of cotyledon protein in acid pepsin. Water extracts of the testa from coloured-flower varieties inhibited the action of fungal cellulase and reduced the in vitro digestibility of white flowered testa and cellulose. A highly significant relationship (r= ?0.93***) was found between the tannin content and solubility in cellulase of testa for a range of 24 varieties. It is concluded that the effect of testa tannins on in vitro digestibility can be mainly attributed to their effect on cell wall digesting enzymes rather than an effect on protein solubility per se.     

Estimation of the monomeric phenolics of ciders

A method is described for the separation and quantitative determination of the monomeric phenolics of ciders. These are fractionally eluted from a silica-gel column with 2-methylpropan-2-ol/chloroform mixtures of increasing 2-methylpropan-2-ol content. The u.v. spectrum of each fraction is obtained in the range 250-400 nm. Fractions containing hydroxycinnamic acid esters of quinic acid are first irradiated with u.v. light to equilibrate cis and trans forms. The concentration of each of the phenolics is calculated from extinctions at appropriate wavelengths. Ciders from some cider-apple cultivars contained in order of decreasing concentration 5-chlorogenic acid, 4-p-coumarylquinic acid, catechins, phloridzin and phloretin xyloglucoside. In one cultivar catechins were undetectable while in others they exceeded the p-coumarylquinic acid. The contribution of the phenolics to the tannin content is discussed.     

Effect of drying conditions on tannin,fibre and lignin levels in mature oak (Quercus incana) leaves

Oak (Quercus incana Roxb) leaves are rich in tannins and produce adverse effects when fed to livestock. The effects of shade and sun drying and oven drying at 60°C and 90°C for different lengths of time (24 to 72 h) on total phenolics, condensed tannins, protein precipitation capacity, degree of polymerisation, specific activity (protein bound per unit tannins), fibre-linked proanthocyanidins, acid and neutral detergent fibres and lignin levels in mature oak leaves were studied. Removal/inactivation of tannins was not observed on wilting or drying under several conditions. Similarly the drying conditions had no effect on fibre composition. The adverse effects on feeding fresh or dried oak leaves are expected to be of similar magnitude.     

Tannin levels in leaves of some oak species at different stages of maturity

Total phenolics, condensed tannins, degree of polymerisation, protein precipitation capacity, protein precipitable phenolics and specific activity (protein bound per unit tannins) were determined in the leaves of four oak species at different stages of maturation (4 days old to 1 year old). The content of total extractable phenols was higher in younger leaves in Quercus ilex Linn, Quercus semecarpifolia Sm and Quercus serrata Roxb, whereas in Quercus glauca Thumb the content was higher in the mature leaves. In all species studied, condensed tannins increased with maturation. Protein precipitation capacity had a trend similar to that of total phenols. In Q serrate and Q semecarpifolia the apparent degree of polymerisation increased, and the content of protein precipitable phenolics and specific activity decreased as the leaves matured. The decrease in protein precipitation capacity with maturation in these two species may be explained by both the decrease in the content and the change in the nature of phenols capable of binding proteins. Protein precipitation capacity was not detectable in Q ilex leaves. Protein precipitation capacity in the mature leaves decreased in the order of Q serrata > Q semecarpifolia > Q glauca > Q ilex.     

The inhibitory effects of wine phenolics on lysozyme activity against lactic acid bacteria

The lysozyme of hen's egg white is used in winemaking to control spontaneous lactic acid bacteria (LAB). A total of eight LAB strains, isolated from grape must and wine, were used to assess the inhibitory effects of wine phenolics on lysozyme activity. The presence of phenolics, extracted from grape pomace, in growth medium reduced the mortality rate due to the lysozyme activity. This effect was especially clear in the case of strains belonging to Lactobacillus uvarum, Pediococcus parvulus and Oenococccus oeni, which are more sensitive to lysozyme than L. plantarum and L. hilgardii strains. Cell lysis assays carried out on four strains sensitive to lysozyme and Micrococcus lysodeikticus ATCC 4698, used as a reference strain, confirmed the inhibition of grape pomace phenolics on the muramidase. There was no interference from non-flavonoids, flavanols and flavonol compounds, when they were tested individually, on the lysozyme activity against the strains. Anthocyanins extracted from grape skins slightly inhibited the activity only against M. lysodeikticus. However, proanthocyanidins extracted from seed berries, strongly inhibited the lysozyme. In this extract, dimers were the predominant oligomers of flavan-3-ol. The study demonstrated that the effectiveness of lysozyme against LAB in red winemaking is related to the amount of low molecular weight proanthocyanidins that are released when the grapes are macerating.     

Effect of condensed tannins prepared from several forages on the in vitro precipitation of ribulose-1,5-bisphosphate carboxylase (Rubisco) protein and its digestion by trypsin (EC 2.4.21.4) and chymotrypsin (EC 2.4.21.1)

A series of in vitro experiments was undertaken to determine the extent to which Sephadex LH-20 treated extracts from a range of temperate forages precipitated ribulose-1,5-bisphosphate carboxylase (Rubisco) and affected the enzymatic hydrolysis of Rubisco protein by trypsin and chymotrypsin at a range of pH values. Rubisco was chosen because it represents the principal dietary protein for ruminants fed fresh forages. Condensed tannins (CT) or proanthocyanidins (PA) are routinely purified by chromatography using Sephadex LH-20 as a matrix. However, these extracts contained non-CT phenolics together with PA so the term ‘CT extract’ was preferred to ‘PA’ to describe the extracts. The in vitro precipitation of Rubisco provided a means to compare the reactivity of the CT extracts. The amount of CT extract required to precipitate all the Rubisco in 10 μg of total soluble leaf protein from white clover (Trifolium repens) when this protein was incubated with CT extracts of Lotus corniculatus, L pedunculatus and sainfoin (Onobrychis viciifolia) was similar, with between 25 and 50 μg of extract required. The CT extract of sulla (Hedysarum coronarium) also precipitated all the Rubisco, however this only occurred with 50 μg of the extract. The CT extract of dock (Rumex obtusifolius) precipitated all the Rubisco when 5 μg of extract or greater was incubated with total soluble leaf protein. However, the differences between the reactivity of all these CT extracts at a range of pH values appeared to be small. Condensed tannin extracts of L corniculatus and L pedunculatus partially inhibited the hydrolysis of Rubisco by trypsin and chymotrypsin to a similar extent, but the extent of the inhibition was affected by pH. The inhibition was greater at pH 6·0 than 7·0, whilst at pH 8·0, CT extracts had little or no affect on trypsin and chymotrypsin. It was concluded that, although the precipitation of Rubisco provided an ideal method for comparing CT extracts, reactivity alone was unlikely to account for the differences in nutritive value that occur with forages containing CT. © 1998 SCI.     

Antioxidant actions and phenolic and vitamin C contents of common Mauritian exotic fruits

Seventeen commonly consumed exotic fruits from Mauritius were analysed for their antioxidant capacity, total phenolics, proanthocyanidins, flavonoids and vitamin C content. Two independent methods were used to evaluate the antioxidant potential of total fruit extracts. The antioxidant activities of the fruits ranged from 1 to 47 µmol Trolox equivalent antioxidant capacity (TEAC) g^?1 fresh weight and from 0.3 to 34 micro/mol g fresh weight (FRAP) g^?1 fresh weight. Total phenolics in the fruits ranged from 118 to 5638 µg g^?1 fresh weight, proanthocyanidins from 7 to 2561 µg g^?1 fresh weight, flavonoids from 21 to 712 µg g^?1 fresh weight and vitamin C content from 8 to 1426 µg g^?1 fresh weight. There were strong correlations between antioxidant activity (assessed by both TEAC and FRAP) and total phenolics and proanthocyanidins. Flavonoids seemed to contribute less to the antioxidant potential of the fruits, while very poor correlations were observed between ascorbate content and antioxidant activity. The highest antioxidant capacities were observed in red and yellow Psidium cattleianum Sabine ‘Chinese guava’, sweet and acid Averrhoa carambola L ‘starfruit’, Syzygium cumini L Skeels ‘jamblon’ and white Psidium guajava L ‘guava’. These fruits were also characterised by high levels of total phenolics. Mauritian exotic fruits are thus a significant source of phenolic antioxidants, which may have potential beneficial effects on health. Copyright © 2003 Society of Chemical Industry     

Degradation of cell walls of forages by sequential treatment with sodium hydroxide and a commercial cellulase preparation

Cell walls of barley straw (whole sample and stem), Italian ryegrass (shoot) and lucerne (mature stem) were treated sequentially with NaOH followed by a commercial ‘cellulase’ preparation and, in other experiments, with the cellulase followed by the alkali. The phenolic compounds released by NaOH were determined and related to increases in the degradability of the walls as measured with cellulase. Two sequential treatments of the cell walls of barley straw increased degradability from 11% on treatment with cellulase alone to 87%, compared with increases from 72 to 99% and from 19 to 54% for the ryegrass and lucerne walls respectively. The first alkali treatment released ferulic and p-coumaric acids from the walls of barley and ryegrass together with other u.v. absorbing compounds, whereas the compounds released by the second alkali treatment did not include these two acids. The treatment with alkali did not release either ferulic acid or p-coumaric acid from the walls of lucerne and the amounts of other u.v. absorbing compounds that were released were much smaller.     

Changes in tannin content,polymerisation and protein precipitation capacity in oak (Quercus incana) leaves with maturity

Changes in the content of total phenolics, condensed tannins, proanthocyanidins and flavan-4-ols, the degree of polymerisation, the protein precipitation capacity, the protein-precipitable phenolic content and the specific activity (protein: tannin in the complex) were determined in Quercus incana leaves with maturity. Total phenolic content and protein precipitation capacity decreased whereas the levels of condensed tannins, proanthocyanidins, flavan-4-ols and degree of polymerisation increased with maturation. The protein-precipitable phenolic content was highest in young leaves and decreased as leaves matured. The specific activity decreased slightly. The results suggest that young leaves would be more toxic to livestock than mature leaves.     

Antioxidant activities of barley seeds extracts

The antioxidant activities of different polarity solvents extracts from barley were evaluated by various antioxidant assays, including reducing power, free radical scavenging and lipid oxidation inhibition. Those various antioxidant activities were compared to standard antioxidants, butylated hydroxyluene (BHT) and ascorbic acid (Vit. C). The properties of the extracting solvents significantly affected the total phenolics, proanthocyanidins and antioxidant activities of barley extract. The highest contents of total phenolics and proanthocyanidins were obtained from extraction with 70% acetone. For three different solvent extracts, the antioxidant activities were in this order: 70% acetone extract > 70% ethanol extract ? 70% methanol extract. Reducing powers of three extracts and their scavenging effects on 2, 2-diphenyl-1-picrylhydrazyl radicals were effective at amount of 200 μg. The 70% acetone extract of barley exhibited high antioxidant activity in linoleic acid system, which was not significantly (P < 0.05) different from BHT during the incubation time.     

Anti-Diabetic Potential of Phenolic Compounds: A Review

Starch is the main carbohydrate in human nutrition. Starch digestibility can vary from a rapid digestion to indigestibility. Therefore, postprandial glycaemic control in type 2 diabetics is of great interest in the context of worldwide health concerns. Although powerful synthetic inhibitors of starch digestive enzymes, such as acarbose, are available to control postprandial hyperglycemia, plant-based enzyme inhibitors are potentially safer. Natural enzyme inhibitors, such as wheat albumin, the Phaseolus vulgaris α-amylase inhibitor, and several phenolic compounds, have the potential to serve as a remedy against hyperglycemia-induced chronic diseases. The inhibition of α-amylase and α-glucosidase is mediated by different phenolics found in varieties of raspberry. Maltase inhibitory activities of chebulagic acid and chebulinic acid from fruit of Terminalia chebula are comparable to that of acarbose. The Nepalese herb Pakhanbhed (Bergenia ciliata) phenolics, (-)-3-O-galloylepicatechin and (-)-3-O-galloylcatechin, showed effective inhibition against starch digesting enzymes. In separate studies, oral administration of starch and maltose with persimmon (Diospyros kaki) leaf tea proanthocyanidins [containing (-)-epigallocatechin-3-O-gallate] and black/bitter cumin (Centratherum anthelminticum) seed phenolics, respectively, resulted in a significant and dose-dependent decrease in the blood glucose level in Wistar rats. Co-application of phenolics with synthetic enzyme inhibitors may reduce the effective dose of synthetic inhibitors required in the regulation of starch digestion. Several phenolic compounds might be useful functional food components and could contribute to manage both hyperglycemia and proper cellular redox status. Human dose-selecting studies and well-controlled long-term human studies would help to optimize the beneficial effects of phenolic compounds.     

Valorisation of sweet corn (Zea mays) cob by extraction of valuable compounds

The main objective of this study was to investigate the proximate, mineral and phytochemical compositions of sweet corn cob (SCC), often neglected and regarded as agricultural waste. Compositional analysis showed that more than 60% of SCC was composed of insoluble dietary fibre, with cellulose being the major constituent. Results also showed that SCC can be a good source of non-essential protein and minerals (phosphorus, potassium and manganese). SCC had a total phenolic content of 6.74 g GAE kg⁻¹ dry weight (DW), of which bound phenolics were predominant. The bound phenolics fraction showed the highest antioxidant capacity in all three antioxidant capacity assays (TEAC, FRAP and DPPH) and contained the highest amount of ferulic and p-coumaric acid. The main carotenoids present in SCC were β-carotene, zeaxanthin and lutein. This investigation shows that SCC can be a potential source of natural colorant (carotenoids), antioxidants (phenolics) and nutritional supplements (proteins and phytochemicals).     

Tannin levels in cassava,a comparison of methods of analysis

Low levels of tannins (proanthocyanidins) have been detected in dried cassava (Manihot esculenta Crantz) samples using standard methods of analysis based on extraction procedures. A direct vanillin assay method was compared with other direct methods of analysis based on acid hydrolysis and protein precipitation. The specificity of the methods is discussed in relation to the levels of tannins detected and their potential biological activity in freeze dried fresh and dried/processed cassava samples. The results are discussed with respect to the amount of indigestible material present in the samples. The results of these experiments indicate that tannins present in dried/processed cassava products may be a factor limiting their nutritional value.     

Determination of the tryptophan content of feedingstuffs with particular reference to cereals

Three procedures, commonly used for the determination of tryptophan, were found to be unsatisfactory for the assay of cereals. A method using p-dimethylaminobenzaldehyde was unsatisfactory because cereals dissolved slowly in the sulphuric acid reagent, produced turbid solutions and gave high blank values. Racemisation of L-tryptophan was found to be incomplete on hydrolysis with barium hydroxide; therefore, microbiological assays are invalidated. Neutralisation of the barium hydroxide with sulphuric acid or carbon dioxide resulted in considerable loss of tryptophan. A method is described based on hydrolysis with barium hydroxide, precipitation of barium sulphate from an acid solution and colorimetric analysis using p-dimethylaminobenzaldehyde. The procedure has proved useful for the routine analysis of cereals and other feedingstuffs.