Search

Erophaca baetica is a legume endemic to the Mediterranean region. Although the fruits and seeds are large, the presence of the “locoism" which produces the alkaloid, swainsonine has prevented its use as animal feed or for human nutrition. Their protein content and chromatographic profile, amino acid composition, fatty acid composition, and polyphenol contents have been determined in order to explore the potential of the E. baetica seeds as a source of dietary protein with functional components. The protein content was found to be 36% (w/w), and an amino acid analysis revealed a deficiency in sulphur amino acids, tryptophane, and lysine. The low lysine content is probably due to the abundance of alkaloids metabolically derived from this amino acid. Oleic and linoleic acids are the major fatty acids in the seeds. The antioxidant activity of polyphenol extracts was higher than the activity of the polyphenols extracted from most edible legume seeds. Henee, E. baetica seeds represent a promising source of functional and nutritional components on the condition that the anti-nutritional alkaloids are previously removed. [ABSTRACT FROM AUTHOR]

Research Background: Authentic Mexican cheeses have potential health benefits, although there are few studies on their bioactive components. In this study, we analysed soluble extracts from añejo cheese from Zacazonapan (Mexico), obtained from two dairies (A and B) that used milk from cows of different breeds and differed in processing., Experimental Approach: The soluble extracts of Zacazonapan añejo cheese during ripening (0, 30, 95 and 180 days) were used to determine proximate composition, amino acid composition, peptide profile, molecular mass profile, antioxidant and antihypertensive activities., Results and Conclusions: The molecular mass of the released protein fragments ranged from 0.10 to 22.43 kDa. During ripening, amino acids such as Pro, His, Tyr, Trp, Met, Cys, Ala, Gly, Leu and Val were present, which are associated with antioxidant activity. The inhibition of β-carotene bleaching was below 50 % at all ripening times. A significant difference between the cheese samples was observed only after 180 days. Amino acids associated with angiotensin-I converting enzyme (ACE-I) inhibition were found in the extracts (Trp, Phe, Tyr, Pro, Lys and Arg). The highest activity was observed in cheese ripened for 180 days (IC 50 of cheese A and B was 0.38 and 0.42 mg/mL, respectively)., Novelty and Scientific Contribution: These results suggest that Zacazonapan añejo cheese is a potential source of antioxidant and antihypertensive peptides, which are influenced by the dairy factor (milk source and production technique) and ripening time. This is relevant as there are no reports of these bioactivities in this type of cheese., Competing Interests: CONFLICT OF INTEREST Authors declare no conflict of interest., (Authors.)

Zantaz honey is a monofloral variety produced from the melliferous plant Bupleurum spinosum (Apiaceae), a shrub that grows mainly in the Atlas Moroccan Mountains. Determination of the polyphenol composition revealed that methyl syringate accounts for more than 50% of total polyphenols, which represents a very useful parameter for the characterization of this monofloral honey. Epicatechin, syringic acid and catechin are also abundant. Caco-2 and THP-1 cells were used for determination of antioxidant and antiproliferative activities in Zantaz honey, respectively. All six commercial samples that were used for these studies exhibited antioxidant activity and inhibited cell proliferation. Interestingly, these activities had a positive correlation mainly with the content in methyl syringate and gallic acid. The recognition of health promoting activities in Zantaz honey should increase its commercial value, which would have a positive economic impact on the poor rural communities of Morocco where it is produced., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Vicia palaestina Boiss. is an annual herb that grows in dry areas of eastern Mediterranean countries. It belongs to section Cracca subgenus Vicilla, which is characterized by having a high content in the non-protein amino acid canavanine. The seeds from some of these vetches are also rich in lectins. The purification and characterization of a single-chain lectin from the seeds of V. palaestina is described here. This lectin was the most abundant protein in albumin extracts. It has affinity for the glycoconjugate N-acetylgalactosamine and inhibits proliferation of the cancerous Caco-2 and THP-1 cell lines. In addition to their high nutritional value, the seeds from V. palaestina represent a source of lectins with health promoting and pharmacological potential because of their antiproliferative activity., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

Vicia ervilia is an ancient crop from the Mediterranean Region. It may represent a useful source of proteins for food and animal feed, as well as bioactive components. Seed samples from 39 populations of V. ervilia have been analyzed. Polyphenol contents ranged from 0.09% to 0.19%. Luteolin, kaempferol, apigenin, and quercetin were the major aglycones. The total free amino acid content of the seeds was 0.05% to 0.19% in which canavanine represented 9% to 22%. The protein content was 24.1%. The amino acid composition indicated a high content in acidic amino acids and a deficit in sulphur amino acids. V. ervilia seeds proved to be a good substrate for the preparation of protein isolates. The seed extracts inhibited the proliferation of Caco-2 colon tumor cells, simultaneously, exerting antioxidative effects. Hence, seeds of V. ervilia could represent a source of high-value food and feed components, as well as functional components. PRACTICAL APPLICATIONS: Vicia ervilia (bitter vetch) (Leguminosae) is an ancient crop from the Mediterranean Region. Although it was still grown in many Mediterranean countries at the beginning of the twentieth century, other crops that provide higher and more consistent yield later replaced it. However, V. ervilia seeds may represent a useful source of proteins for human nutrition and animal feeding, and a source of bioactive components with health-promoting properties. Our results show that the seeds of V. ervilia could, indeed, represent a source of high-value food and feed components, as well as functional, health-promoting components. This may result in a revalorization of this neglected crop. The availability of numerous populations in seed banks guarantees the preservation of a genetic diversity in V. ervilia that could be used for the production of new varieties with better nutritional and functional characteristics., (© 2020 Wiley Periodicals LLC.)

Three olive modified pectin extracts have been produced by heat and acid treatment of the major by-product of olive oil production. Their effect on proliferation of the colon carcinoma Caco-2 and the leukemia monocytic THP-1 cell lines has been studied in order to determine possible anti-tumor properties. All extracts inhibited proliferation at some of the concentrations ranging from 1 to 10 mg ml -1 . Interestingly none of the extracts inhibited the growth of confluent Caco-2 cells, showing the specificity of the antiproliferative effect for the transformed Caco-2 phenotype. All the extracts inhibited agglutination of red blood cells by galectin-3, a lectin involved in tumor growth, metastasis, and immune cell regulation that has been proposed as a mediator of the anti-tumor effects of modified pectins. In addition, activation of caspase-3 in THP-1 cells indicates that treatment with the pectin-rich extracts triggers apoptosis. These results point to a possible use as health-promoting food ingredients or supplements.

Chickpea is a grain legume widely consumed in the Mediterranean region and other parts of the world. Chickpea seeds are rich in proteins but they also contain a substantial amount of free amino acids, especially arginine. Hence chickpea may represent a useful source of free amino acids for nutritional or pharmaceutical purposes. Arginine is receiving great attention in recent years because it is the substrate for the synthesis of nitric oxide, an important signaling molecule involved in numerous physiological and pathological processes in mammals. In this work we describe a simple procedure for the purification of arginine from chickpea seeds, using nanofiltration technology and an ion-exchange resin, Amberlite IR-120. Arginine was finally purified by precipitation or crystallization, yielding preparations with purities of 91% and 100%, respectively. Chickpea may represent an affordable green source of arginine, and a useful alternative to production by fermentation or protein hydrolysis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The role of the peptides-phenolic compounds (PC) interaction on the antioxidant capacity profile (ACP) of protein hydrolysates from rapeseed (Brassica napus) was studied in 36 hydrolysates obtained from a PC-rich and PC-reduced protein substrate. The latent profile analysis (LPA), with data of seven in vitro methods and one assay for cellular antioxidant activity (CAA), allowed identifying five distinctive groups of hydrolysates, each one with distinctive ACP. The interaction of peptides with naturally present PC diminished in vitro antioxidant activity in comparison with their PC-reduced counterparts. However, CAA increased when peptides-PC interaction occurred. The profile with the highest average CAA (62.41 ± 1.48%), shown by hydrolysates obtained by using alcalase, shared typical values of Cu(2+)-catalysed β-carotene oxidation (62.41 ± 0.43%), β-carotene bleaching inhibition (91.75 ± 0.22%) and Cu(2+)-chelating activity (74.53 ± 0.58%). The possibilities for a sample to exhibit ACP with higher CAA increased with each unit of positively charged amino acids, according to multinomial logistic regression analysis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A method for determination of the non-protein amino acid l-α-amino-γ-(guanidinooxy)-n-butyric acid (L-canavanine) and other free amino acids in Vicia disperma is presented. Seed extracts were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography. Calibration curves showed very good linearity of the response. The limit of detection and quantification were 0.15 and 0.50 μM, respectively. The method has a high intra- (RSD=0.35%) and inter-repeatability (RSD=2.86%), and a remarkable accuracy with a 99% recovery in spiked samples. The method is very easy to carry out and allows for ready analysis of large number of samples using very basic HPLC equipment because the derivatized samples are very stable and have very good chromatographic properties., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A method for determination of β-cyano-L-alanine, γ-glutamyl-β-cyano-L-alanine and other free amino acids in Vicia sativa is presented. Seed extracts were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography. Calibration curves showed very good linearity of the response. The limit of detection and quantification was 0.15 and 0.50 μM, respectively. The method has high intra- (RSD = 0.28-0.31%) and interrepeatability (RSD = 2.76-3.08%) and remarkable accuracy with a 99% recovery in spiked samples. The method is very easy to carry out and allows for ready analysis of large number of samples using very basic HPLC equipment because the derivatized samples are very stable and have very good chromatographic properties. The method has been applied to the determination of γ-glutamyl-β-cyano-L-alanine, β-cyano-L-alanine, and common free amino acids in eight wild populations of V. sativa from southwestern Spain.

Background: Bean seeds are an inexpensive source of protein. Anthracnose disease caused by the fungus Colletotrichum lindemuthianum results in serious losses in common bean (Phaseolus vulgaris L.) crops worldwide, affecting any above-ground plant part, and protein dysfunction, inducing the synthesis of proteins that allow plants to improve their stress tolerance. The aim of this study was to evaluate the use of beans damaged by anthracnose disease as a source of peptides with angiotensin-converting enzyme (ACE-I)-inhibitory activity., Results: Protein concentrates from beans spoiled by anthracnose disease and from regular beans as controls were prepared by alkaline extraction and precipitation at isolelectric pH and hydrolysed using Alcalase 2.4 L. The hydrolysates from spoiled beans had ACE-I-inhibitory activity (IC(50) 0.0191 mg protein mL(-1)) and were very similar to those from control beans in terms of ACE-I inhibition, peptide electrophoretic profile and kinetics of hydrolysis. Thus preparation of hydrolysates using beans affected by anthracnose disease would allow for revalorisation of this otherwise wasted product., Conclusion: The present results suggest the use of spoiled bean seeds, e.g. anthracnose-damaged beans, as an alternative for the isolation of ACE-I-inhibitory peptides to be further introduced as active ingredients in functional foods., (© 2012 Society of Chemical Industry.)

The nutritional quality of the protein in the leaves of 11 Asphodeline (Liliaceae) species was investigated by the determination of the amino acid composition and calculation of several nutritional parameters. The average protein content was 4.7% and ranged from 2.5% in Asphodeline damascena ssp. rugosa to 8.2% in A. turcica. The most abundant essential amino acids were Thr (5.7%), Val (6.0%), Ile (4.7%), and Trp (2.1%). The amino acid composition of Asphodeline peshmeniana was well equilibrated according to Food and Agriculture Organisation standards, but Lys and sulphur amino acids were at limiting concentrations in all the other taxa. Determination of the protein efficiency ratio and biological value revealed that the protein in the leaves of Asphodeline species is of high nutritional quality. Hence, the Asphodeline leaves that are typically used in Turkey for the preparation of salads, represent a good source of protein with high levels of several essential amino acids and a good nutritional value. Analysis of the similarity based on the amino acid composition indicated the existence of different clusters that are consistent with the taxonomical classification, area of distribution, and morphological similarities of the Asphodeline species., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bean protein isolate and phaseolin were hydrolysed using pepsin and pancreatin, and the resulting hydrolysates were filtered through a 1kDa cut-off membrane and fractionated by size exclusion chromatography. Three fractions corresponding to MW 0.7-1.0kDa, 0.43-0.7kDa and <0.43kDa (A1, A2, and A3 for protein isolate fractions, and B1, B2, and B3 for phaseolin fractions) were assayed for antioxidant and metal chelating activity and they were also subjected to amino acid and SDS-PAGE analysis. Fractions A1 and B1 had the highest copper chelating activity (78% and 82%, respectively), while iron chelating activity was the highest in fractions A1 and B3 (36% and 16%, respectively). Fractions A2 and B3 had the highest antioxidant activity as determined by inhibition of reducing power and β-carotene bleaching, while the highest ABTS radical scavenging activity was found in A3 and B3. Thus, fractions coming from the isolate and phaseolin had similar activities except for iron chelation, suggesting that phaseolin is the major contributor to the antioxidant and copper chelating activities of the hydrolysed protein isolate., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

The erythrocyte agglutinating activity of polyphenol extracts from six grain legumes was investigated. Polyphenols are amphipathic molecules that can bind to proteins and lipids through hydrophobic and polar interactions, leading to agglutination of liposomes and bacteria. The extracts from four of the six legumes that were studied caused erythrocyte agglutination at concentrations in the μM range. Soybean extracts had the highest activity, followed by the extracts from lentils, broad bean, and chickpea. As a good representative of these legumes, binding of the polyphenols extracted from lentils to erythrocytes was investigated in more detail, showing that agglutination was mediated by binding of 84% of the polyphenols present in the incubation, which corresponds to 2.42 μg bound polyphenols/mg erythrocytes, and a maximum polyphenol binding of 96% according to Lineweaver-Burk plots. The relatively high concentrations that are required for agglutination justify that polyphenols more probably do not agglutinate erythrocytes in vivo, but the possibility still exists that in vivo binding without agglutination could occur, which could have some effects on the metabolism and health-promoting properties of polyphenols., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The goal of this research was the characterisation of Vicia faba (broadbean) protein isolates and related fractions in order to determine whether this grain legume could be used for production of high quality protein products and other fractions rich in functional components. Alkaline extraction of the defatted seed flour, followed by precipitation at the isoelectric pH, yielded a 92% protein isolate with a high oil absorption capacity. The contents of the favism-inducing glycosides, vicine and convicine, in the isolate were reduced by more than 99% as compared to the original flour, although the amino acid composition was similar to that of the flour. Some of the by-products of protein isolate production may also be of interest from a nutritional and functional point of view. Thus, the oil resulting from hexane extraction of the flour is rich in unsaturated fatty acids, and polyphenols (resulting from extraction of the defatted flour with acetone) showed a high ABTS radical-scavenging activity. In addition, the solid residue (resulting from protein solubilisation) was high in fibre and showed good water absorption. These results show good nutritional and functional properties in V. faba protein isolates and related fractions, which may favour the revalorisation of this traditional bean crop., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Antioxidant and chelating activities were determined in protein hydrolysates that were produced by treating a protein isolate of a non-toxic genotype of Jatropha curcas with the protease preparation alcalase., Results: 50 min protein hydrolysate with a degree of hydrolysis of 31.7% showed highest antioxidant and chelating activity. These activities were also determined in six peptidic fractions that were separated by gel filtration chromatography of the 50 min hydrolysate. The lower-molecular-weight peptidic fractions had the highest antioxidant and chelating activities, which correlated with a higher content in antioxidant and chelating amino acids such as tyrosine and histidine., Conclusion: Results show that J. curcas represents a good source of bioactive peptides. This may be important for the revalorization of defatted J. curcas flour, a by-product resulting form oil extraction for biodiesel production. This is especially important in Third World and developing countries such as Mexico., (Copyright © 2011 Society of Chemical Industry.)

Plant protein hydrolysates are a source of bioactive peptides. There are peptides that decrease the micellar cholesterol solubility from bile acids and therefore may reduce in vivo cholesterol absorption. The presence of these peptides in sunflower protein hydrolysates has been studied. Sunflower protein hydrolysates produced with alcalase plus flavourzyme or with pepsin plus pancreatin inhibited in some degree the cholesterol incorporation to micelles. Protein hydrolysates generated after 30 min of hydrolysis with alcalase, and after 30 min of hydrolysis with pepsin, were the inhibitoriest of the cholesterol incorporation to micelles. The average amino acid hydrophobicity of inhibitory peptides in cholesterol micelles was higher than the observed in the corresponding protein hydrolysates. This high hydrophobicity probably favours their inclusion in the lipid micelles. In vivo, this inhibition may translate in a decrease of cholesterol absorption. Reported results show that a combination of different characteristics such as peptide size or hydrophobicity may be responsible of the inhibitory activity of generated peptides.

The growth of mammalian cells in vitro requires the use of rich culture media that are prepared by combining serum with specific nutrient formulations. Serum, the most expensive component of culture media, provides a complex mixture of growth factors and nutrients. Protein hydrolysates that can support in vitro cell growth and eliminate or reduce the need to use serum have been obtained from different sources. Here we describe the use of two food grade proteases to produce a chickpea protein hydrolysate that has been added to cell culture medium in order to determine whether it can be used as a substitute for serum. Medium containing the hydrolysate has been tested using two human cells lines: the monocytic THP-1 cell line which grows in suspension, and the epithelial Caco-2 cell line which grows as a monolayer. The chickpea protein hydrolysate was a good substitute for serum in the first case, but did not allow growth of Caco-2 cells. Supplementation of culture media with this inexpensive and safe hydrolysate would greatly reduce the cost of cell culture.

The fractioning of some components and their immobilization of Flavourzyme, a commercial protease/aminopeptidase preparation, has been investigated to improve its specificity and stability. Adsorption of Flavourzyme on two ionic exchangers yielded two fractions with endoprotease activity and one fraction containing aminopeptidase activity. The use of an amine agarose gel has made it possible to purify a 43 kDa protein with only endoprotease activity. Immobilization of this endoprotease and the original Flavourzyme preparation onto glyoxyl-agarose provided derivatives that were more thermostable than their soluble counterparts. Tests using immobilized Flavourzyme and immobilized purified endoprotease for the hydrolysis of chickpea proteins showed that both preparations can be used for the production of protein hydrolysates and compare very favorably with the original crude Flavourzyme in terms of reducing the production of free amino acids. This was especially so in the case of immobilized endoprotease, which produced only 0.2% free amino acids. Keeping free amino acids content low is very important in protein hydrolysates for nutritional use to avoid excessive osmotic pressure.

Copper-chelating peptides were purified from sunflower protein hydrolysates by affinity chromatography using immobilized copper. A variety of protein hydrolysates were obtained by incubation with the proteases Alcalase and Flavourzyme for different periods of time. Chelating activity was indirectly determined by measuring the inhibitory effect of hydrolysates on the oxidation of beta-carotene by copper. Copper-binding peptides purified from the two hydrolysates that inhibited oxidation by copper the most contained 25.4 and 42.0% histidine and inhibited beta-carotene oxidation 8 and 3 times more than the original hydrolysates, which had 2.4 and 2.6% histidine, respectively. Thus, histidine content is not the only factor involved in antioxidant activity, and probably other factors such as peptide size and amino acid sequence are also important. This work shows that affinity chromatography can be used for the purification of copper-chelating peptides and probably other metals of nutritional interest such as calcium, iron, and zinc. In addition to their antioxidant potential, chelating peptides are of nutritional interest because they increase bioavailability of minerals.

Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

Brassica carinata protein isolates were hydrolyzed using the digestive enzymes trypsin, chymotrypsin, and carboxypeptidase A in order to obtain hydrolyzates with a high Fischer's ratio. The proteases were immobilized using two glyoxyl-agarose supports of different porosity, 4 and 10% agarose gels, in order to evaluate the effect of substrate diffusion into the support containing the enzyme on the hydrolytic process. Reaction time, substrate concentration, and the enzyme to substrate ratio were optimized in an attempt to increase the Fischer's ratio in the resulting hydrolyzates. Gel filtration chromatography of a hydrolyzate with a degree of hydrolysis of 36% yielded a fraction that represented 31% of the total hydrolyzed proteins and had a Fischer's ratio of 28.3 with a phenylalanine + tyrosine content below 1.5%. This material could be used for preparing special diets when there is a need to increase the supply of branched amino acids and/or reduce the intake of aromatic amino acids.

A lung extract rich in angiotensin converting enzyme (ACE) and pure ACE were immobilized by reaction with the activated support 4 BCL glyoxyl-agarose. These immobilized ACE derivatives were used for purification of ACE inhibitory peptides by affinity chromatography. The immobilized lung extract was used to purify inhibitory peptides from sunflower and rapeseed protein hydrolysates that had been obtained by treatment of protein isolates with alcalase. The ACE binding peptides that were retained by the derivatives were specifically released by treatment with the ACE inhibitor captopril and further purified by reverse-phase C18 HPLC chromatography. Inhibitory peptides with IC50 50 and 150 times lower than those of the original sunflower and rapeseed hydrolysates, respectively, were obtained. The derivative prepared using pure ACE was used for purification of ACE inhibitory peptides from the same type of sunflower protein hydrolysate. ACE binding peptides were released from the ACE-agarose derivatives by treatment with 1 M NaCl and had an IC50 a little higher than those obtained using immobilized extract and elution with captopril. Affinity chromatography facilitated the purification of ACE inhibitory peptides and potentially other bioactive peptides present in food proteins.

The assay of angiotensin-converting enzyme (ACE) inhibition by food-derived peptides is usually carried out by using soluble ACE in a batch process. The purification of this enzyme from tissues is not an easy task, and the resulting preparation loses activity very fast. In addition, ACE commercial preparations are very expensive. In this work the immobilization of ACE, through lysine amino groups, to 4% beads cross-linked (4 BCL) glyoxyl-agarose is described. The amount of immobilized enzyme increased with increasing concentrations of enzyme and with incubation time until a saturation point was reached at 50 mg protein/mL gel and 3.5 hours, respectively. The IC50 values for a noncompetitive sunflower peptide inhibitor were similar for the soluble (30.56 microM) and immobilized (32.7 microM) enzymes. An immobilized derivative was obtained that was 60 times more stable than the soluble enzyme at 60 degrees C. This procedure yields a derivative that can be reused and has increased thermal stability compared to that of the soluble enzyme. Thus, ACE immobilization is a good alternative to using soluble freshly prepared or commercial preparations because of economical and practical reasons.

Pulses should be part of a healthy diet, and it is also becoming clear that they have health-promoting effects. Nevertheless, most studies on the bioactive or health-promoting properties of pulses have been carried out using soybeans. We have studied cell growth-regulating properties, which may be responsible for anti-cancer properties, in chickpea seeds. Chickpea seeds are a staple in the traditional diet of many Mediterranean, Asian, and South and Central American countries. In addition, chickpea seeds have industrial applications since they can be used for the preparation of protein concentrates and isolates. The cell lines Caco-2 (epithelial intestinal) and J774 (macrophages) have been exposed to chickpea seed extracts and protein preparations in order to screen the different chickpea fractions for effects on cell growth. Both cell growth-promoting and cell growth-inhibiting effects were found. Most interestingly, a fraction soluble in ethanol and acetone specifically and almost completely inhibited the growth of Caco-2 cells exhibiting a cancerous phenotype. It is concluded that chickpea seeds are a source of bioactive components and deserve further study for their possible anti-cancer effect.

Sunflower protein isolates and the proteases pepsin and pancreatin were used for the production of protein hydrolysates that inhibit angiotensin-I converting enzyme (ACE). Hydrolysates obtained after 3 h of incubation with pepsin and 3 h with pancreatin were studied. An ACE inhibitory peptide with the sequence Phe-Val-Asn-Pro-Gln-Ala-Gly-Ser was obtained by G-50 gel filtration chromatography and high-performance liquid chromatography C18 reverse phase chromatography. This peptide corresponds to a fragment of helianthinin, the 11S globulin from sunflower seeds, which is the main storage protein in sunflower. These results show that sunflower seed proteins are a potential source of ACE inhibitory peptides when hydrolyzed with pepsin and pancreatin.

Reactions between lipid peroxidation products and proteins in foods have detrimental nutritional effects, most importantly, losses of essential amino acids. One of the major products of the reaction of malondialdehyde and alkanals with amino groups are 4-substituted 1,4-dihydropyridine-3,5-dicarbaldehyde derivatives. The product of the reaction of lysine with malondialdehyde and acetaldehyde, N,N'-di-(4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde)lysine (MDDL), has been synthesized and used for in vitro and in vivo bioavailability studies. Release of free lysine did not occur in incubations of MDDL with tissue homogenates. After oral administration of radioactively labeled MDDL, radioactivity was only recovered in feces. Radioactivity was not incorporated into hepatic microsomes after intraperitoneal administration, which would have indicated release of available lysine. These results show that MDDL is a form of unavailable lysine, because it is not metabolized to free lysine and cannot be absorbed from the gut. Thus, formation of this derivative in foods would result in loss of available lysine.

The lipid peroxidation product malondialdehyde is mostly bound to proteins in foods as an N-2-propenal derivative that is released as N-epsilon-(2-propenal)lysine by digestive enzymes. N-2-Propenals have been identified as the major forms of malondialdehyde in urine. To determine whether available lysine can be released from the N-2-propenals of lysine in vivo, two preparations containing N-epsilon-(2-propenal)lysine and N-alpha-(2-propenal)lysine or N,N'-di-(2-propenal)lysine were synthesized using radioactively labeled lysine and were administered to rats by gastric intubation and intraperitoneal injection. Both preparations were absorbed from the digestive tract, although not as efficiently as free lysine, but most of the radioactivity was excreted in urine. The radioactive label was also readily excreted after intraperitoneal injection. It is concluded that the N-2-propenals of lysine are fairly stable in vivo, so that, although they are absorbed from the gut, most of the absorbed material is not metabolized and is readily excreted as nonavailable lysine.

The respiratory burst in alveolar macrophages is enhanced in vitro by pre-exposure to nontoxic concentrations of hydroperoxides before stimulation by an agonist, which may represent a feed-forward regulatory mechanism. Tricyclodecan-9-yl-xanthate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), suppresses this priming of the respiratory burst by pre-exposure to H(2)O(2) in NR8383 alveolar macrophages (up to 100 microM D609, 400 nmol of H(2)O(2) added to 5 x 10(6) cells 15 min before stimulation with ADP). D609 has potential as an antioxidant due to its dithiocarbonate functional group that allows it to slowly react with H(2)O(2) and rapidly reduce cytochrome c, which interferes with a common assay for the respiratory burst. Nonetheless, the antioxidant properties of D609 do not account for its inhibition of priming of the respiratory burst by H(2)O(2). Reduction of nitro blue tetrazolium is the basis for an assay for superoxide production with which D609 does not interfere. With this assay, it was found that D609 does not inhibit the respiratory burst per se, but prevents its enhancement by pre-exposure to H(2)O(2). Consistent with a role of diacylglycerol generation by phospholipase C, this enhancement was mimicked by pre-exposure to phorbol ester. In contrast with priming, receptor-mediated stimulation of the respiratory burst depends on the better characterized phosphatidylinositol-specific phospholipase C. Priming of the respiratory burst by H(2)O(2) joins the list of inflammatory responses that are inhibited by D609. Nevertheless, the results herein indicate that caution should be exercised in the interpretation of the effects of D609 to consider both antioxidant effects and inhibition of PC-PLC.

Previous investigation showed that preincubation within a range of nontoxic H(2)O(2) concentrations enhanced subsequently stimulated superoxide production by rat alveolar macrophages in response to various stimuli. In the present study, the NR8383 rat alveolar macrophage cell line was used to further investigate the priming effect of H(2)O(2). Using nitroblue tetrazolium, which formed an insoluble formazan when reduced by superoxide, modulation of the respiratory burst was visualized in a cell population exposed to a concentration gradient of H(2)O(2) before stimulation. This model system illustrates how H(2)O(2) may constitute a signaling molecule for a feed-forward regulation of the respiratory burst during inflammation. n-Butanol, which allows consumption of phosphatidic acid by the transphosphatidylation reaction, and propanolol, which inhibits phosphatidic acid phosphohydrolase, were used to investigate the possible involvement of phospholipase D in this phenomenon. These two agents were found to inhibit the basal adenosine diphosphate-stimulated respiratory burst. Inhibition of the H(2)O(2)-enhanced respiratory burst was equally or slightly less effective when expressed as percentage of controls. Furthermore, phospholipase D was not activated by H(2)O(2) concentrations that enhance superoxide production. Thus, phospholipase D does not mediate the enhancement of the respiratory burst by H(2)O(2), although it may be activated by high concentrations of this hydroperoxide.

Regulation of the free intracellular calcium concentration, [Ca2+]i, plays a major role in physiological signal transduction. Many of the essential enzymes in signaling cascades are Ca(2+)-dependent, as are numerous proteins that participate in the regulated function. Oxidative stress, which for many years was considered synonymous with cell and tissue injury, has more recently been demonstrated to alter signal transduction in both positive and negative directions. The realization that hydrogen peroxide and lipid hydroperoxides are produced as part of normal metabolism has led to the proposal that these oxidants function as second messengers. Exposure to environmental and other agents that produce hydroperoxides or the addition of exogenous hydroperoxides also causes elevation of [Ca2+]i in some cells. At sublethal exposure to hydroperoxides, the elevation in [Ca2+]i can either alter or mimic physiological stimulation. In addition to endoplasmic reticulum, mitochondria, and the extracellular space, the phospholipid- and Ca(2+)-binding proteins known as annexins constitute a Ca2+ pool from which this ion may be released under situations of oxidative stress. In this article, the source and consequences of Ca2+ elevation are reviewed with an emphasis on studies done with alveolar macrophages. These phagocytes, which modulate much of the physiological and immunological function of the lung, are susceptible targets for environmental oxidants.

Lipid peroxidation was induced in freshly isolated rat hepatocytes by incubation in the presence of Fe3+, resulting in accumulation of thiobarbituric acid reactive substances. Analysis of lipid classes revealed that the levels and fatty acid compositions of the two major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), remained unchanged but the levels of triacylglycerols (TAG) were significantly reduced, and some of their polyunsaturated fatty acids were selectively lost as the result of oxidant treatment. Acyl turnover in PC and PE as determined by 18O incorporation from H2 (18)O-containing media remained largely unchanged during oxidant treatment, while some increased turnover of the saturated fatty acids in TAG was observed. We hypothesize that constitutive recycling of membrane phospholipids rather than selective in situ repair eliminates peroxidized species of PC and PE. TAG could serve as an expendable fatty acid reserve, providing a limited but very dynamic pool of polyunsaturated fatty acids for the resynthesis of phospholipids.

ADP-complexed Fe3+ has been used in various in vitro systems and in intact cells to induce lipid peroxidation. During studies on the effects of oxidative stress on lipid metabolism we observed a significant increase in de novo glycerolipid synthesis in Fe3+/ADP-treated rat hepatocytes as evidenced by increased [U-14C]glycerol incorporation. Here we show that this increase, largely due to enhanced triacylglycerol synthesis, is caused by ADP rather than Fe3+/ADP-induced oxidative stress. Hence, metabolic alterations due to treatment of intact cells by Fe3+/ADP must be interpreted with caution.

Peroxidative treatment of rat heart mitochondria results in a gradual increase of the apparent molecular weight of the adenine nucleotide translocase (ANT) by up to 1.2 kDa. ANT isolated from mitochondria treated with 1 mM tert-butyl hydroperoxide and 5-40 microM Cu2+ for 1 h at 37 degrees C exhibited a progressive loss of lysine, cysteine, arginine, and valine residues compared to native ANT. N-Ethylmaleimide, dithiothreitol, and the specific inhibitor of ANT, carboxyatractyloside (CAT), inhibited the peroxidation-induced molecular weight shift without inhibiting lipid peroxidation, which is believed to be the primary cause of the observed ANT modification. Bongkrekic acid, which stabilizes ANT in a conformation different from that brought about by CAT, did not inhibit the ANT molecular weight shift. Dithiothreitol, as well as CAT, was found to protect ANT against most of the losses of amino acid residues, indicating that alteration of sulfhydryl residues is required for chemical modification of, not only cysteine, but also lysine, arginine, and valine. We conclude that the peroxidative modification of ANT is conformation-dependent and involves chemical modification of cysteine as a critical step.

We have previously reported that treatment of isolated rat heart mitochondria with the free radical-generating system Cu2+/tert-butylhydroperoxide produces striking changes in the adenine nucleotide translocase (ANT) of the inner membrane. These changes include a small increase in apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by its disappearance from the polypeptide profile upon further oxidant treatment (Zwizinski and Schmid (1992) Arch. Biochem. Biophys. 294, 178-183). In order to characterize its peroxidative modification in more detail, we have purified rat heart ANT and prepared polyclonal antibody against it. Using this antibody, we have observed that increasing oxidant treatment results in a gradual increase in the ANT protein's apparent molecular weight by up to 1 kDa. The progressive nature of the molecular weight shift, which parallels the generation of thiobarbituric acid reactive substances, supports the hypothesis that this phenomenon may be the result of covalent addition of increasing amounts of lipid peroxidation products. Strong oxidative treatment of cardiac mitochondria also causes fragmentation and polymerization of the ANT protein. However, Western blot analysis showed that a major portion of the original ANT survives even extensive oxidation as a distinct, modified protein. Therefore, the almost complete disappearance of ANT from Coomassie-stained gels appears to be the result of cross-linking and fragmentation reactions, as well as a decreased efficiency of the Coomassie staining. Because a measurable molecular weight shift of ANT occurs at the mildest oxidative treatment tested (resulting in the production of only 1.1 nmol malondialdehyde/mg protein), it may be relevant as a parameter of myocardial ischemia-reperfusion injury.