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Abstract

Lupin seed globulin proteins form complexes with flavonoids, predominantly apigenin C-glycosides. Enzymes typical for the gastrointestinal tract were used to hydrolyze lupin seed globulins. Release of native flavonoids as a result of the proteolysis reaction was observed. Different analytical methods such as size exclusion chromatography, HPLC-MS, and fluorescence spectroscopy (steady-state fluorescence, fluorescence anisotropy, fluorescence lifetimes) were used for a detailed characterization of this phenomenon. Flavonoids liberated from lupin globulin proteins as a result of pancreatin-catalyzed digestion were bound by γ-conglutin resistant to this enzyme. Two possible mechanisms of this interaction may be suggested: hydrogen bonding between oligosaccharide chains of glycoproteins and the sugar moieties of the flavonoid glycosides or electrostatic attraction between positively charged γ-conglutin and flavonoids partially ionized at pH 7.5.

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... The existing literature offers limited research on the digestibility of lupin proteins using physiologically relevant digestion models that can be directly compared with the findings of our study. The majority of studies have investigated lupin digestibility only in the presence of simple digestive juices containing enzymes (such as pepsin, trypsin, chymotrypsin or pancreatin) and not employing more complex simulated digestive fluids and bile salts (Capraro et al., 2009;Czubinski et al., 2012Czubinski et al., , 2014Czubinski et al., , 2016. Capraro et al. (2009) and Czubinski et al. (2012Czubinski et al. ( , 2014 showed that lupine globulins exposed to pepsin, chymotrypsin, or a double digestion model (pepsin followed by pancreatin) were totally digested, with only some smeared bands at low molecular weights detected in the case of using pepsin or chymotrypsin. ...
... The majority of studies have investigated lupin digestibility only in the presence of simple digestive juices containing enzymes (such as pepsin, trypsin, chymotrypsin or pancreatin) and not employing more complex simulated digestive fluids and bile salts (Capraro et al., 2009;Czubinski et al., 2012Czubinski et al., , 2014Czubinski et al., , 2016. Capraro et al. (2009) and Czubinski et al. (2012Czubinski et al. ( , 2014 showed that lupine globulins exposed to pepsin, chymotrypsin, or a double digestion model (pepsin followed by pancreatin) were totally digested, with only some smeared bands at low molecular weights detected in the case of using pepsin or chymotrypsin. However, pancreatin and trypsin when used exclusively did not result in complete hydrolysis of lupine globulins, which could be due to the high specificity of their cleavage sites (Capraro et al., 2009;Czubinski et al., 2012Czubinski et al., , 2014Czubinski et al., , 2016. ...
... Capraro et al. (2009) and Czubinski et al. (2012Czubinski et al. ( , 2014 showed that lupine globulins exposed to pepsin, chymotrypsin, or a double digestion model (pepsin followed by pancreatin) were totally digested, with only some smeared bands at low molecular weights detected in the case of using pepsin or chymotrypsin. However, pancreatin and trypsin when used exclusively did not result in complete hydrolysis of lupine globulins, which could be due to the high specificity of their cleavage sites (Capraro et al., 2009;Czubinski et al., 2012Czubinski et al., , 2014Czubinski et al., , 2016. Such studies suffer from scarce relevance, as the overall complexity of the real physiological conditions are not taken into consideration. ...
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Despite the growing interest in lupin as a source of protein, due to its potential to decrease the environmental footprint of food supply, its allergenic properties have not been fully explored. This study focused on lupin's potential to induce allergic reactions in food‐sensitized individuals. The antigenic properties of lupin proteins were investigated after exposing lupin protein isolate as well as lupin flours to an in vitro digestion. The effect of thermal treatment, on the digestibility and immunoreactivity of the proteins was also investigated. Protein breakdown was analyzed by SDS‐PAGE, but also by evaluating the distribution of peptide sizes and the extent of hydrolysis via quantifying the free N‐terminal amino acid content. Immunogenicity was assessed by Western blot analysis using a commercial antibody. The individual or combined effects of the food matrix and processing were noted after gastric digestion of the lupin flours, as some of the protein fractions showed resistance toward pepsin activity and caused responses to the commercial antibodies, particularly the fractions likely associated with α‐conglutin (20 kDa) and γ‐conglutin (17 kDa). Gastric digestion of lupin flours also revealed the possibility of formation of neo‐allergens, which warrants future identification. However, after the intestinal phase, lupin proteins underwent complete hydrolysis, with no observed responses toward the specific antibody used. Nevertheless, these findings underscore the need for further research employing sera from allergic patients to lupin in order to avoid potential false‐negative results.
... The nutritional key role of lupin seeds in unquestionable, due to the massive presence of macro and micro nutrients. Among these nutrients, proteins play a relevant role considering their amino acid composition which can easily be balanced in the diet (Czubinski et al., 2012). However, recently, it has been acknowledged that food proteins are not only a source of constructive and energetic compounds as the amino acids, but also they may play bioactive roles themselves and/or can be the precursors of biologically active peptides with various physiological functions (Arnoldi et al., 2015). ...
... This protein undergoes quaternary assembly in a pH-dependent manner (Capraro et al., 2010) and is able to bind divalent metal cations, such as Zn 2+ and Ni 2+ , which at the same time promote the refolding process after acidic treatment of c-conglutin (Duranti et al., 2002). Furthermore, the protein is resistant to pancreatin and trypsin proteolysis (Czubinski et al., 2012(Czubinski et al., , 2014. The ability to bind insulin, which appears to be strongly affected by the ionic strength is another important property of c-conglutin (Magni et al., 2004). ...
... The ability to bind insulin, which appears to be strongly affected by the ionic strength is another important property of c-conglutin (Magni et al., 2004). Recent studies have demonstrated unique flavonoid-binding properties of c-conglutin (Czubinski et al., 2012(Czubinski et al., , 2014. ...
Article
Lupin seed protein γ‐conglutin positively modulates lipid and glucose homoeostasis by interacting with peripheral tissues such as muscles, liver or pancreas. By contrast, the influence of γ‐conglutin on adipose tissue functions has not been elucidated so far. Since adipose tissue is involved in controlling glucose and lipid metabolism we hypothesised that γ‐conglutin may affect energy homoeostasis by interacting with adipocytes including fat tissue formation. Therefore, the aim of this study was to characterise the influence of γ‐conglutin on adipogenesis using 3T3‐L1 preadipocytes. We found that γ‐conglutin had no effects on viability and proliferation of 3T3‐L1 preadipocytes. By contrast, γ‐conglutin suppressed the expression of adipogenic markers (Pparγ, C/ebpα, Fabp4 and leptin) during differentiation process. Furthermore, 3T3‐L1 preadipocytes differentiated with γ‐conglutin showed reduced intracellular lipid content comparing with control cells. In conclusion, these results showed that γ‐conglutin suppresses differentiation of 3T3‐L1 preadipocytes into mature fat cells. The effects of γ‐conglution on the differentiation of 3T3‐L1 preadipocytes into mature adipocytes were studied. The results revealed that γ‐conglution suppressed lipid accumulation and expression of adipogenic geneses indicating its suppressive effects on the differentiation of 3T3‐L1 preadipocytes.
... The measurement of fluorescence parameters (fluorescence emission, fluorescence anisotropy, fluorescence lifetime) is a useful technique providing information on the molecule microenvironment and is often used in investigations of polyphenol-protein interactions and in binding affinity studies ( Czubinski et al., 2012Czubinski et al., , 2014Stojadinovic et al., 2013;Shen et al., 2014;He et al., 2015;Vesic et al., 2015;Bose, 2016;Sun et al., 2016). According to the literature data, excitation at 240-280 nm of proteins induced the emission of fluorescence in the range of 340-350 nm as a result of the presence of aromatic amino acids, and a decrease in this fluorescence emission range can be attributed to ligand binding ( Rawel et al., 2002a,b;Liu et al., 2010;Czubinski et al., 2014). ...
... Changes in protein tertiary structure Rawel et al. Lupin flavonoids, vitexin Noncovalent complex formation Czubinski et al. (2012Czubinski et al. ( , 2014) Fluorescence of synthetic fluorescent probes Soy glycinin (SG) and soy trypsin inhibitor (STI) ...
... Chicken egg lysozyme Fisetin, morin Protein-phenolic compound complex formation, determination of binding sites binding energy and H-bonding distance Roy et al. (2015) polyphenols by proteins is associated with an increase in anisotropy value. This method may be used to study the changes in protein-polyphenol interactions, for example during digestion ( Czubinski et al., 2012Czubinski et al., , 2014). The reduced mobility of the phenolic compounds bound to the protein molecule usually results in enhanced fluorescence intensity or blue shift of polyphenol fluorescence emission maximum ( Czubinski et al., 2012). ...
Article
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Interactions between the different compounds present in foods are common and have influence on the nutritional and functional properties of food products. Among a wide range of these interactions, the formation of complexes between proteins and phenolic compounds seems to be the most important issue. Complexation of the phenolic compounds with proteins can be analysed considering several aspects. These complexes might strongly affect nutritional potential of polyphenols by masking their antioxidant capacity, and on the other hand might have influence on the structure of proteins which may cause their precipitation or decrease susceptibility to digestion. The complexity of protein–phenolic compound interactions is a challenge for food analysts and forced researchers to establish a wide range of analytical methods, allowing determination of complexes formation. The main aim of this review is to give researchers an overview of the currently used methods that can be applied to study the interactions between proteins and phenolic compounds.
... Apigenin is able to reduce cancer cell glucose uptake, it inhibits remodelling of the extracellular matrix, inhibits cell adhesion molecules that participate in cancer progression , and opposes chemokine signaling pathways that direct the course of metastasis into other locations (Lefort & Blay, 2013). Apigenin glycosides may bind to proteins within plant sources and be released as those proteins are broken down as part of the digestive process (Czubinski et al., 2012). Interactions between the different compounds present in foods are common and can simultaneously influence nutritional and functional properties of food products. ...
... Complexation of phenolic compound with proteins can be considered in several aspects. Those complexes might strongly affect the nutritional potential of polyphenols by masking their antioxidant capacity, and on the other hand, causes protein precipitation or lack of protein digestion susceptibility (Arts et al., 2002; Czubinski et al., 2012). The food matrix in which phenolic compounds occur can significantly promote the formation of flavonoid–protein complexes. ...
... 2.3. Protein extractions and c-conglutin purification Protein extraction was carried out according to the procedure described by Czubinski et al. (2012). Briefly, albumins were removed from defatted lupin flour using extraction with water pH 8.0 containing 10 mM CaCl 2 , 10 mM MgCl 2 (34 ml per 1 g of lupin flour) for 4 h. ...
... 10 Noteworthy, γ-conglutin is able to form a static complex with native lupin seed flavonoids. 11,12 The mechanism for this interaction uses electrostatic attraction between the positively charged protein and flavonoids partially ionised at pH 7.5. 13 Unfortunately, the importance of this phenomenon for the plant and the protein has not been determined so far. ...
... The flavonoids were identified based on characteristic UV-visible spectra, a specific retention time and comparisons with reference lupin seed samples where phenolic compounds were identified by mass spectrometry. 11,22 The quantitative content of flavonoids was determined using the vitexin (determination of apigenin derivatives; y = 2619x; r 2 = 0.9963) or apigenin (determination of apigenin; y = 3181x; r 2 = 0.9919) standard curves. ...
Article
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BACKGROUND Basic 7S globulins, a group of proteins commonly found in legumes, undergo the intriguing phenomenon of release from the seeds into hot water. γ‐Conglutin is a representative of this group of proteins found in lupin seeds. The physiological significance and the molecular mechanism of the selective release of γ‐conglutin from lupin seeds remain unknown. Therefore, the presented study aimed to determine changes in the functionality of this protein in response to the high temperature occurring during lupin seed incubation. RESULTS It was confirmed that the main protein fraction released from the seeds during high‐temperature incubation was γ‐conglutin. The incubation condition favours the occurrence of this protein in a monomeric form, and the temperature used corresponds to its midpoint unfolding temperature. Subsequent analysis carried out on the γ‐conglutin monomer revealed changes in its functionality after heat shock. The thermally treated protein shows a considerable increase in its interaction strength with flavonoids. Moreover, the inhibitory activity against glycoside hydrolases was enhanced when γ‐conglutin monomer was exposed to specific temperatures. CONCLUSION The results of the present study provide a potential explanation of the physiological relevance of γ‐conglutin and shed new light on a possible mechanism of its activation upon specific heat treatment. This knowledge will help characterise homologous proteins, which are commonly found in other legumes and undergo a similar heat‐induced secretion phenomenon. © 2021 Society of Chemical Industry.
... 3 Secondly, the protein exhibits different digestion susceptibility in the gastrointestinal tract, and recent studies highlighted its insensitivity to pancreatin as well as trypsin actions. [4][5][6] The ability to bind insulin is another important feature of this lupin seed protein. 7 Additionally, it was proved that -conglutin-insulin complex stability is strongly affected by ionic strength. ...
... Recent studies have demonstrated that -conglutin is also capable of forming a stable complex with native lupin flavonoids, i.e. apigenin C-glycosides. 5,6 The unique lupin protein shares a high level of homology with glycoside hydrolases inhibitor proteins, although it lacks any kind of inhibitory activity against plant cell degradation enzymes. [11][12][13] Simultaneously, -conglutin also displays a less pronounced structural similarity to pepsin-like aspartic proteases but it is proteolytically dysfunctional. ...
Article
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BACKGROUND Protein post‐translational modifications are a key element for the functional diversity of the proteome. The modifications generally refer to the addition of functional groups to certain proteins, however, proteolytic cleavage is also one of the relevant events during proteins maturation. γ‐Conglutin is a unique protein fraction present in lupin seeds that is marked by numerous unusual properties. This protein fraction undergoes very complex post‐translational maturation. Unfortunately, the precise mechanism of γ‐conglutin post‐translational processing has not been fully understood, yet. RESULTS Two independent methods were used to study γ‐conglutin post‐translational cleavage processing. Edman N‐terminal sequencing indicates that the signal peptide is processed at Tyr34 while α‐ and β‐subunits cleavage takes place between Ser295‐Ser296. High‐resolution mass spectrometry (MS) revealed a great diversity of N‐terminal sequences of γ‐conglutin α‐subunit. However, most abounded peptides also began from Tyr34. MS analyses additionally confirmed the subunits cleavage place between two serine residues. CONCLUSIONS The results indicate that the proteolytic processing of γ‐conglutin signal peptide is not precise. On the other hand, the post‐translational cleavage between α‐ and β‐subunits of γ‐conglutin is very conserved. Interestingly, the results also indicate that proteolytic processing that leads to the formation of two subunits of γ‐conglutin is not fully appeared leaving a certain amount of the protein in an uncut form.
... Considering that polyphenols may strongly interact with proteins either by covalent or non-covalent binding, a still open issue is the possible role of polyphenols in the observed effects. Unfortunately, literature does not report any analytical data on the concentration of polyphenols in the materials used in these clinical studies, although a recent paper has shown that lupin seed globulins form stable complexes with flavonoids and in particular with apigenin C-glucosides H and I (Czubinski et al., 2012). In fact, a study has shown that the digestion of the lupin protein with pepsin releases these apigenin derivatives that may be easily identified by mass spectrometry (Czubinski et al., 2012). ...
... Unfortunately, literature does not report any analytical data on the concentration of polyphenols in the materials used in these clinical studies, although a recent paper has shown that lupin seed globulins form stable complexes with flavonoids and in particular with apigenin C-glucosides H and I (Czubinski et al., 2012). In fact, a study has shown that the digestion of the lupin protein with pepsin releases these apigenin derivatives that may be easily identified by mass spectrometry (Czubinski et al., 2012). This important issue would certainly deserve a detailed investigation. ...
... 2,4 Moreover, γ-conglutin shows insensitivity to pancreatic proteolysis and, at the same time, is capable of binding flavonoids released during in vitro hydrolysis from other lupin seed proteins. [5][6][7][8] The formation of a static complex between γ-conglutin and vitexin (apigenin C-glycoside) was already described in a previous study by the authors. 8 The formation of such complexes between γ-conglutin and many other phenolic compounds has also been observed. ...
Article
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BACKGROUND Several different factors underlie the molecular mechanisms of phenolic compound‐protein interactions. They include the environmental conditions. In the case of γ‐conglutin, pH conditions translate directly into the adoption of two distinct oligomeric assemblies, i.e. hexameric (pH 7.5) or monomeric (pH 4.5). This paper reports research on the pH‐dependent oligomerization of γ‐conglutin in terms of its ability to form complexes with a model flavonoid (vitexin). RESULTS Fluorescence‐quenching thermodynamic measurements indicate that hydrogen bonds, electrostatic forces, and van der Waals interactions are the main driving forces involved in the complex formation. The interaction turned out to be a spontaneous and exothermic process. Assessment of structural composition (secondary structure changes and arrangement/dynamics of aromatic amino acids), molecular size, and the thermal stability of the different oligomeric forms showed that γ‐conglutin in a monomeric state was less affected by vitexin during the interaction. CONCLUSION The data show precisely how environmental conditions might influence phenolic compound‐protein complex formation directly. This knowledge is essential for the preparation of food products containing γ‐conglutin. The results can contribute to a better understanding of the detailed fate of this unique health‐promoting lupin seed protein after its intake. © 2023 Society of Chemical Industry.
... In the case of γ-conglutin, this protein occurs as a monomer at acidic pH and form a hexameric assembly when environmental conditions change to alkaline (Capraro et al., 2010;Czubiński et al., 2015). The main research interest of γ-conglutin is associated with the fact that this protein binds divalent metal ions (Duranti et al., 2001), is insensitive to pancreatin proteolysis (Capraro et al., 2009;Czubiński et al., 2012Czubiński et al., , 2014, binds to insulin (Czubinski, 2021a,b;Magni et al., 2004), binds native lupin flavonoids (Czubiński et al., , 2014, and is able to control glycemia in mammals (Capraro et al., 2011;Terruzzi et al., 2011). Thus, studying the properties of this protein can provide valuable information that can expand our knowledge about molecular factors determining the interaction between polyphenols and proteins. ...
Article
The molecular basis underlying the interaction between proteins and phenolic compounds are still not fully understood. The specific structural properties of proteins, as well as phenolics, strongly determine the complex formation. In this work, the interactions between γ-conglutin, a unique lupin seed protein, and twenty-one different phenolic compounds representing six different classes of phenolics that differ in their structures were investigated. The interactions were studied based on a fluorescence quenching experiment, and the determined binding constant (Ka) ranged from 6.88 × 10² (protocatechuic acid) to 5.06 × 10⁶ (hesperidin), while the number of binding sites of phenolic compound molecules to the protein (n) was on average 1.14 ± 0.16. Within the analysed compounds, phenolic acids interacted the weakest with the protein, while flavonoids showed considerable higher affinity strength to γ-conglutin. Notably, flavanones and flavones were the phenolics that formed the complex with markedly higher values of Ka and n (1-3 orders of magnitude). Additionally, principal component analysis allowed to formulate the general regularities of phenolic compounds preferences for γ-conglutin. Finally, the obtained results also indicate that phenolic compounds' binding preferences for γ-conglutin can result from their native occurrence in lupin seeds.
... For example, apigenin forms a complex with globulin proteins of Lupin seeds. It is released in the gastrointestinal tract after proteolysis reaction (Czubinski et al., 2012;Lefort & Blay, 2013). In a study, it was suggested that apigenin and apigenincontaining compounds are absorbed into the bloodstream by diffusion, both active and passive. ...
Article
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Numerous diseases such as cancer, diabetes, cardiovascular, neurodegenerative diseases, etc. are linked with overproduction of reactive oxygen species (ROS) and oxidative stress. Apigenin (5,7,4′‐trihydroxyflavone) is a widely distributed flavonoid, responsible for antioxidant potential and chelating redox active metals. Being present as glycosides or polymers, the apigenin degrades to variable amount in the digestive tract; during processing, its activity is also reduced due to high temperature or Fe/Cu addition. Although its metabolism remains elusive, enteric absorption occurs sufficiently to reduce plasma indices of oxidant status. Delayed clearance in plasma and slow liver decomposition enhance its systematic bioavailability. Antioxidant mechanism of apigenin includes: oxidant enzymes inhibition, modulation of redox signaling pathways (NF‐kB, Nrf2, MAPK, and P13/Akt), reinforcing enzymatic and nonenzymatic antioxidant, metal chelation, and free radical scavenging. DPPH, ORAC, ABTS, and FRAP are the major in vitro methods for determining the antioxidant potential of apigenin, whereas its protective effects in whole and living cells of animals are examined using in vivo studies. Due to limited information on antioxidant potential of apigenin, its in vitro and in vivo antioxidant effects are, therefore, discussed with action mechanism and interaction with the signaling pathways. This paper concludes that apigenin is a potent antioxidant compound to overcome the difficulties related to oxidative stress and other chronic diseases.
... Trp was exposed to the environment and surface hydrophobicity was increased. For pH 3.8 spectra, the complex of other two polyphenols and proteins had a blue shift toward the short wave, showed that the molecule of gliadin aggregated, and Trp was tightly wrapped and hydrophobicity was weakened [8]. All the three polyphenols had strong quenching effect on the protein. ...
Article
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The effect of interactions between polyphenolic substances (Chlorogenic acid, epicatechin and gallic acid) and gliadin under different pH (pH 3.8/7.0) were analyzed by fluorescence spectrum. The results showed that adding polyphenols could induce static fluorescence quenching of gliadin solution at pH 3.8 and 7.0. Fluorescence quenching spectrum of chlorogenic acid-gliadin complex at pH 3.8 presented a red shift toward long wave, while other phenols spectra showed blue shifts toward the short wave. The binding ability showed a significant difference between acid condition and neutral condition, and the quenching constants (Kq and KSV) of all three polyphenols at pH 3.8 were lager than pH 7.0. Compared to epicatechin and gallic acid, pH value had a marked effect on binding ability of chlorogenic acid with gliadin.
... Sugarcane flavonoids may interact with protein molecule and be eliminating protein those are broken during the digestion process 26 . Apigenin derived from sugarcane has been used to treat various diseases such as inflammatory, neuralgia, and shingles 27 . ...
... Soy protein isolate-κcarrageen Quercetagetin CD -Interaction between the quercetagetin and protein was through the hydrophobic interaction hydrogen bonding [119] Zein-CAS NPs Curcumin DSC FTIR Zein-CAS NPs interacts with curcumin via hydrogen bonding and hydrophobic interactions [120] Lupin seed globulin Flavonoid (apigenin glycosides) SSF FL -Lupin seed globulins bind with phenolic compounds through electrostatic attraction or hydrogen bonding. Pepsin digestion caused release of apigenin glycosides (mainly 7-O-β-apiofuranosyl-6,8-di-C-β-glucopyranoside) [121] c. Hydroxycinnamic acid and chlorogenic acids Soy protein isolate HCA (caffeic, ferulic acids), and CHA (chlorogenic acids) from green coffee ITC MDS -Significant proportion of HCA and CHA were bound to proteins through electrostatic, hydrogen bonds and hydrophobic interactions -pH affected binding affinity of ligand to soy protein, reduced as pH beyond pI (4.5) ...
Article
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In the food industry, proteins are regarded as multifunctional systems whose bioactive hetero-polymeric properties are affected by physicochemical interactions with the surrounding components in formulations. Due to their nutritional value, plant proteins are increasingly considered by the new product developer to provide three-dimensional assemblies of required structure, texture, solubility and interfacial/bulk stability with physical, chemical or enzymatic treatment. This molecular flexibility allows them to form systems for the preservation of fresh food, retention of good nutrition and interaction with a range of microconstituents. While, animal- and milk-based proteins have been widely discussed in the literature, the role of plant proteins in the development of functional foods with enhanced nutritional profile and targeted physiological effects can be further explored. This review aims to look into the molecular functionality of plant proteins in relation to the transport of bioactive ingredients and interaction with other ligands and proteins. In doing so, it will consider preparations from low- to high-solids and the effect of structural transformation via gelation, phase separation and vitrification on protein functionality as a delivery vehicle or heterologous complex. Applications for the design of novel functional foods and nutraceuticals will also be discussed.
... creating complexes with proteins. 5,6 The two main protein fractions found in lupin seeds are globulins and albumins, and the quantitative ratio between these protein groups is 9:1, respectively. 7 Albumins are water-soluble proteins that fulfill enzymatic and regulatory functions. ...
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BACKGROUND Lupin‐based food, due to the high content of functional proteins and phenolic compounds, are widely used in human nutrition. Unfortunately, proteins and phenolic compounds can easily interact with each other which results in formation of complexes that affect properties of both components. Therefore, in this study, composition of the seeds storage proteins isolated from Lupinus albus and L. angustifolius and their interactions with native flavonoids were investigated. RESULTS Based on the chromatographic separations, six proteins fractions of lupin seeds storage proteins were identified. The results indicate that two dominant fractions, α‐conglutin and β‐conglutin, constitute up to 80% of all proteins present in the seeds. Three flavonoids interacting with the proteins were identified as apigenin C‐glycosides. The lowest flavonoids content was noted in the main storage proteins while in both lupin seeds species over 90% of flavonoids interacted with the proteins present in late‐embryogenesis abundant (LEA) protein fraction. CONCLUSIONS Protein–phenolic compound complexes can affect the digestibility of proteins and bioavailability of phenolic compounds, and thus the functional and nutritional properties of products derived from lupin seeds can be changed. Therefore, a better understanding of factors affecting the nutritional value of lupin seeds proteins and flavonoids is necessary to optimize the biological use of this plant for human nutrition. © 2019 Society of Chemical Industry
... Food proteins extracted from plants are important for human and animal nutrition particularly in developing countries where average protein intake is less than required [2]. The production of plant protein isolates is of growing interest to food industry because of the increasing applications of plant proteins in food markets, nutraceutical products, and functional foods [3,4]. It is known that sacha inchi seeds have a high content of oil (54%) and protein (27%) [5,6]. ...
Article
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Objective: The aim of this study was to evaluate the digestibility and anti-inflammatory activity in vitro of sacha inchi protein isolate. Methods: Proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gastric digestibility was evaluated with pepsin at different pHs and at different relation enzyme/substrate. Anti-inflammatory activity in vitro of sacha inchi protein isolate was evaluated using denatured protein method with egg albumin. Results: A yield of 20.88% of protein isolate of defatted sacha inchi flour at pH 4.0 with a 93.1% of protein was obtained. 11S globulins were resistant to gastric and duodenal digestion. Sacha inchi protein isolate at pH 4.0 (1000 μg/ml) presents 78.13% of anti-inflammatory activity in vitro. Conclusions: Sacha inchi seed is a good source of proteins. 11S globulins are resistant to pepsin and pancreatin hydrolysis. Sacha inchi protein isolate has high anti-inflammatory activity in vitro. © 2016, Innovare Academics Sciences Pvt. Ltd. All rights reserved.
... The production of plant protein isolates is of growing interest to the food industry because of the increasing applications of plant proteins in food markets, nutraceutical products, and functional foods. Presently, it is possible to find products derived from plant proteins isolate such as soybean, quinoa, amaranth lupin seed, and walnuts [3,4]. It is known that sacha inchi seeds have a high content of oil (54%) and protein (27%) [5]. ...
Article
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Objective: The aim of this study was to obtain protein isolate from sacha inchi using alkaline pH at different pHs of precipitation with water and salt and to analyze protein isolate with electrophoresis. Methods: Sacha inchi protein isolates were obtained using isoelectric precipitation method at different pHs. Proteins were analyzed using electrophoresis native-polyacrylamide gel electrophoresis (PAGE), one-dimensional, two-dimensional-sodium dodecyl sulfate-PAGE. Results: A yield of 20.88% of protein isolate of defatted sacha inchi flour at pH 4.0 with a 75.31% of protein was obtained. The yield of protein isolate using water and salt was similar. Polypeptides profile is between 14 and 70 kDa. Conclusions: Sacha inchi seed is a good source of proteins. Globulins and albumins were identified in the sacha inchi protein isolate in the presence of water and salt. © 2016, Innovare Academics Sciences Pvt. Ltd. All rights reserved.
... Apigenin normally exists in a glycosidic form in fruits and vegetables. These apigenin glycosides may bind to some plant source proteins to facilitate their storage and transport in plants, and be released when those proteins are hydrolysed in the digestive process (Czubinski et al., 2012). The glycosidic apigenin requires to be further deglycosylated by β-glucosidases, which normally exist in the bacteria colonizing the gut lumen (par-ticularly in the colon), to release the bioactive product apigenin to enter cells and have its effects (Nemeth et al., 2003). ...
Article
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Apigenin is a common dietary plant flavonoid widely distributed in vegetables and fruits. It exhibits chemopreventive activity against various cancer cells. In this study, we demonstrated that apigenin directly blocked heat shock protein 90 (Hsp90) and cell division cycle protein 37 (Cdc37) interaction using split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) assay. Apigenin inhibited complemented Renilla luciferase (RL) activity of NRL-Hsp90/Cdc37-CRL, while its analogues did not. Apigenin also inhibited NRL-Hsp90 and Cdc37(Ser13Ala)-CRL complementation. In addition, casein kinase II (CK2) specific inhibitor 4, 5, 6, 7-tetrabromobenzotriazole (TBB) did not affect NRL-Hsp90/Cdc37-CRL complementation, indicating that the inhibitory effect of apigenin on Hsp90/Cdc37 did not rely on CK2 activity. Moreover, apigenin blocked Hsp90/Cdc37 complex and induced kinase clients protein kinase B (Akt), cyclin-dependent-kinase 4 (CDK4) and matrix metalloproteinase-9 (MMP-9) degradation and, as a consequence, induced intracellular reactive oxygen species (ROS) accumulation and inhibited cell proliferation and migration in pancreatic cancer cells.
... Interestingly, the comparative assessment of the concentration of identified phenolics in hydrolysates produced by Alcalase at 120 min and Savinase at 90 min (Table 4) indicated that the latter treatment leads to a remarkable increase in hydroxycinnamic acid (ferulic acid), flavan-3-ol (catechin) and anthocyanin (pelargonidin-3-glucoside) families. Our results are consistent with a recent investigation showing the release of flavonoids (apigenin-C glycosides) bound by ionic bridges and hydrogen bonding to γ-conglutin from lupin (Czubinski et al., 2012). Higher concentration of hydroxycinnamic acids observed in Savinase hydrolysates obtained after 90 min can be also ascribed to the higher esterase activity found in the Savinase enzymatic preparation. ...
Article
A multitude of plant-derived bioactive compounds have shown significant promise in preventing chronic illnesses, with flavonoids constituting a substantial class of naturally occurring polyphenolic compounds. Apigenin, a flavone identified as 4',5,7-trihydroxyflavone, holds immense promise as a preventative agent against chronic illnesses. Despite its extensive research and recognized nutraceutical value, its therapeutic application remains underexplored, necessitating further clinical investigations. This review delves into the biological sources, nutraceutical prospects, chemistry, pharmacological insights, and health benefits of apigenin. Through multifaceted analytical studies, we explore its diverse pharmacological profile and potential therapeutic applications across various health domains. The manuscript comprehensively examines apigenin's role as a neuroprotective , anti-inflammatory compound, and a potent antioxidant agent. Additionally, its efficacy in combating cardiovascular diseases, anti-diabetic properties, and anticancer potential has been discussed. Furthermore, the antimicrobial attributes and the challenges surrounding its bioavailability, particularly from herbal supplements have been addressed. Available in diverse forms including tablets, capsules, solid dispersions, co-crystals, inclusion complexes and nano formulations. Additionally, it is prevalent as a nutraceutical supplement in herbal formulations. While strides have been made in overcoming pharmacokinetic hurdles, further research into apigenin's clinical effectiveness and bioavailability from herbal supplements remains imperative for its widespread utilization in preventive medicine.
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Seeds of ten Andean lupin (Lupinus mutabilis Sweet) ecotypes were collected from different regions of Peru and treated with an aqueous debittering method. Both untreated and treated seeds were analyzed by using LC-MS to investigate flavonoid profiles of different ecotypes and impact of debittering process on these compounds. Thirteen isoflavones (mainly as glycosides of genistein and methoxy-genistein) and eight flavones (glycosylated apigenins and methyl-luteolins) were characterized as the main flavonoids in the seed samples. The untreated lupin seeds contained 187-252 mg/100 g (dry weight) of flavonoids. The main difference among lupin ecotypes was observed in the levels of genistein-malonylhexoside, methoxy-genistein-malonylhexoside, and methyl-luteolin-malonylhexoside. After the debittering treatment, the total flavonoid content in the seeds was decreased to 125-203 mg/100 g dry weight, the aglycones of genistein, methoxy-genistein, and methyl-luteolin being the key distinguishing compounds of ecotypes. The aqueous treatment was effective in degrading flavonoid glycosides and releasing the corresponding aglycones.
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Interactions between plant‐based proteins (PP) and phenolic compounds (PC) occur naturally in many food products. Recently, special attention has been paid to the fabrication of PP–PC conjugates or complexes in model systems with a focus on their effects on their structure, functionality, and health benefits. Conjugates are held together by covalent bonds, whereas complexes are held together by noncovalent ones. This review highlights the nature of protein–phenolic interactions involving PP. The interactions of these PC with the PP in model systems are discussed, as well as their impact on the structural, functional, and health‐promoting properties of PP. The PP in conjugates and complexes tend to be more unfolded than in their native state, which often improves their functional attributes. PP–PC conjugates and complexes often exhibit improved in vitro digestibility, antioxidant activity, and potential allergy‐reducing activities. Consequently, they may be used as antioxidant emulsifiers, edible film additives, nanoparticles, and hydrogels in the food industry. However, studies focusing on the application of PP–PC conjugates and complexes in real foods are still scarce. Further research is therefore required to determine the structure–function relationships of PP–PC conjugates and complexes that may influence their application as functional ingredients in the food industry.
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A multidimensional analysis aimed to determine the thermal impact on γ-conglutin at the two oligomeric states was carried out. A wide range of biophysical and bioinformatic methods allowed to get insight into a thermal unfolding process mechanism. The determined midpoint transition temperature (Tm) values were remarkably different, being 56.5°C and 71.1°C for γ-conglutin monomer and hexamer, respectively. The unfolding pattern for hexamer molecules included aggregation/precipitation, while monomers tended to form soluble aggregates after heat exposure. Interestingly, differences in the aromatic amino acid residues movements indicate that during thermal treatment of γ-conglutin hexamer red-shift occurred contrary to the monomer in the case of which blue-shift was noted. The obtained results provide an essential contribution to expand our knowledge about the molecular characterization of this intriguing lupin seed protein.
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Digestion is the key step for delivering nutrients and bioactive substances to the body. The way different food components interact with each other and with digestive enzymes can modify the digestion process and affect human health. Understanding how food components interact during digestion is essential for the rational design of functional food products. Plant polyphenols have gained much attention for the bioactive roles they play in the human body. However, their strong beneficial effects on human health have also been associated with a negative impact on the digestion process. Due to the generally low absorption of phenolic compounds after food intake, most of the consumed polyphenols remain in the gastrointestinal tract, where they then can exert inhibitory effects on enzymes involved in the degradation of saccharides, lipids, and proteins. While the inhibitory effects of phenolics on the digestion of energy-rich food components (saccharides and lipids) may be regarded as beneficial, primarily in weight-control diets, their inhibitory effects on the digestion of proteins are not desirable for the reason of reduced utilization of amino acids. The effect of polyphenols on protein digestion is reviewed in this article, with an emphasis on food processing methods to improve the antinutritive properties of polyphenols.
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Lupin seeds are already widely used as an ingredient in different food products. Their attractiveness is related mainly to their high protein content that is characterised by a favourable amino acid composition, as well as the desired technological properties. However, with the increase of lupin seeds usage in food manufacture, their potential allergic properties have been demonstrated. The aim of this work was to study the immunoreactivity changes taking place during the enzymatic hydrolysis of the major seed proteins of narrow-leafed (Lupinus angustifolius, varieties Zeus and Bojar) and yellow (L. luteus, var. Lord and Parys) lupin species. Two digestion systems were used, namely the in vitro model simulating digestion taking place in digestive track, and specific hydrolysis carried out by trypsin. The obtained hydrolysates were analysed by means of one-dimensional electrophoresis, and their immunoreactivity was assessed with the use of a sera pool from patients with lupin-specific IgE. An important reduction in allergenicity of lupin seed proteins was observed when trypsin digestion was applied. The digestion in the in vitro model revealed the possibility of formation of neoallergens which were identified on the basis of mass spectrometry results as β-conglutin fraction.
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Background: Proteins enzymatic digestion is a very complex process, during which some components are degraded, while others remain in an unchanged form. Moreover, the enzymatic hydrolysis is one of the most popular methods used to reduce the allergenicity of food proteins. In this study, the efficiency of enzymatic hydrolysis of lupin seeds proteins was assessed by proteomic analysis performed by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry identification. Two digestion systems were used, namely, oriented digestion carried out by trypsin and the model in vitro digestion mimicking conditions present in the gastrointestinal tract. Results: The comparisons of 2-DE maps of proteins isolated form different lupin seeds species revealed that the differences in proteins expression were observed mainly in the central parts of gels, i.e., in the molecular weight range from 20 to 70 kDa, and the pH range 5-7. A total number of 27 differentially expressed proteins spots were successfully identified by mass spectrometry analysis. An important reduction in the number of proteins spots on 2-DE maps was observed when trypsin, and the in vitro digestion model were applied. The protein spot insensitive to digestion in both hydrolysis systems was identified as β-conglutin. Conclusions: The results provide insight into the nature of the digestion process that may take place after lupin seed proteins intake and highlight the important fact that some of the proteins are insensitive to digestive enzymes activity. Moreover, evaluation of digestion activity of trypsin towards lupin seed proteins may be used for the development of specific processes of hypoallergenic food production.
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Background: Interest in protein-phenol interactions in biological systems has grown substantially in recent decades. Methods: The interest has focused largely on food systems in response to reports on the prominent roles of phenolic compounds in nutrition and health. Results: Phenolic compounds can have both favourable and adverse nutritional effects. Polyphenols are widely known for their antioxidant, anti-inflammatory, anticancer and antiaging properties; however, they have also been ascribed anti-nutritional effects resulting from interactions with some proteins and enzymes. Interactions between proteins and polyphenols can additionally influence food quality by altering some physical-chemical and sensory properties of foods. These effects may be useful to develop new products in food science and technology provided the nature of physical-chemical interactions between proteins and phenols is accurately elucidated. In this paper, we review the different possible modes of interaction between selected food proteins and phenolic compounds. Conclusion: Existing knowledge on the mechanisms behind polyphenol-protein reactions, the structures of the resulting products and their potential uses is reviewed.
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Vitexin and isovitexin are active components of many traditional Chinese medicines, and were found in various medicinal plants. Vitexin (5, 7, 4-trihydroxyflavone-8-glucoside) has recently received increased attention due to its wide range of pharmacological effects, including but not limited to anti-oxidant, anti-cancer, anti-inflammatory, anti-hyperalgesic, and neuroprotective effects. Isovitexin (apigenin-6-C-glucoside), an isomer of vitexin, generally purified together with vitexin, also exhibits diverse biological activities. Latest research has suggested that vitexin and isovitexin could be potential substitute medicines for diversity diseases, and may be adjuvants for stubborn diseases or health products. This review summarized recent findings on various pharmacological activities and associative signalling pathways of vitexin and isovitexin to provide a reference for future research and clinical applications.
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The aim of this work was to study the processes taking place during the enzymatic hydrolysis of seed globulins isolated from narrow-leaf (var. Zeus and Bojar) and yellow (var. Lord and Parys) lupin seeds species cultivated in Poland. In lupin seed globulins hydrolysis studies, there were used enzymes typical for the human gastrointestinal tract, such as: pepsin, pancreatin, trypsin and chymotrypsin. The obtained hydrolysates were assessed based on the results of electrophoretic, immunoblotting, as well as chromatographic separations. The evaluation of lupin seed globulins digestion susceptibility was supported by the bioinformatics analyses. Analysis of hydrolysates showed that the proteins present in globulins are completely hydrolysed by: pepsin, double digestion model (pepsin followed by pancreatin) or chymotrypsin. The high specificity of pancreatin and trypsin action results in limited lupin globulins hydrolysis. Protein fraction resistant to the action of these enzymes was γ-conglutin which also retains its antigenic properties. Its insensitivity to the hydrolysis might be associated with the formation of complexes with flavonoids which were released from other protein connections during digestion, as well as with relatively low number of cleavage sites for trypsin. The analysis of the three-dimensional structure of γ-conglutin enabled a very accurate description of the amino acid residues localisation, at which trypsin hydrolysis should occur.
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γ-Conglutin from lupin seeds is an unusual 7S basic globulin protein. It is capable of reducing glycaemia in mammals, but the structural basis of this activity is not known. γ-Conglutin shares a high level of structural homology with glycoside hydrolase inhibitor proteins, although it lacks any kind of inhibitory activity against plant cell-wall degradation enzymes. In addition, γ-conglutin displays a less pronounced structural similarity to pepsin-like aspartic proteases, but it is proteo­lytically dysfunctional. Only one structural study of a legume 7S basic globulin, that isolated from soybean, has been reported to date. The quaternary assembly of soybean 7S basic globulin (Bg7S) is arranged as a cruciform-shaped tetramer comprised of two superposed dimers. Here, the crystal structure of γ-conglutin isolated from Lupinus angustifolius seeds (LangC) is presented. The polypeptide chain of LangC is post-translationally cleaved into α and β subunits but retains its covalent integrity owing to a disulfide bridge. The protomers of LangC undergo an intricate quaternary assembly, resulting in a ring-like hexamer with noncrystallographic D3 symmetry. The twofold-related dimers are similar to those in Bg7S but their assembly is different as a consequence of mutations in a β-strand that is involved in intermolecular β-sheet formation in γ-conglutin. Structural elucidation of γ-conglutin will help to explain its physiological role, especially in the evolutionary context, and will guide further research into the hypoglycaemic activity of this protein in humans, with potential consequences for novel antidiabetic therapies.
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Many of the potential health benefits of flavonoids have been associated with their specific chemical and biological properties including their ability to interact and bind non-covalently to macronutrients in foods. While flavonoid-protein interactions and binding have been the subject of intensive study, significantly less is understood about non-covalent interactions with carbohydrates and lipids. These interactions with macronutrients are likely to impact both the flavonoid properties in foods, such as their radical scavenging activity, and the food or beverage matrix itself, including their taste, texture and other sensorial properties. Overall, non-covalent binding of flavonoids with macronutrients is primarily driven by van der Waals interactions. From the flavonoid perspective, these interactions are modulated by characteristics such as degree of polymerization, molecular flexibility, number of external hydroxyl groups, or number of terminal galloyl groups. From the macronutrient standpoint, electrostatic and ionic interactions are generally predominant with carbohydrates, while hydrophobic interactions are generally predominant with lipids and mainly limited to interactions with flavonols. All of these interactions are involved in flavonoid-protein interactions. While primarily associated with undesirable characteristics in foods and beverages, such as astringency, negative impact on macronutrient digestibility and hazing, more recent efforts have attempted to leverage these interactions to develop controlled delivery systems or strategies to enhance flavonoids bioavailability. This paper aims at reviewing the fundamental bases for non-covalent interactions, their occurrence in food and beverage systems and their impact on the physico-chemical, organoleptic and some nutritional properties of food.
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This short paper presents an overview of the most important characteristics of lupin, a legume that is regaining much interest in human nutrition, after having been neglected for many years. Particular emphasis is placed on the nutraceutical properties of lupin protein that are able to impact positively on hypercholesterolemia, hypertension and hyperglycaemia.
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Apigenin (4',5,7-trihydroxyflavone, 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a flavonoid found in many fruits, vegetables, and herbs, the most abundant sources being the leafy herb parsley and dried flowers of chamomile. Present in dietary sources as a glycoside, it is cleaved in the gastrointestinal lumen to be absorbed and distributed as apigenin itself. For this reason, the epithelium of the gastrointestinal tract is exposed to higher concentrations of apigenin than tissues at other locations. This would also be true for epithelial cancers of the gastrointestinal tract. We consider the evidence for actions of apigenin that might hinder the ability of gastrointestinal cancers to progress and spread. Apigenin has been shown to inhibit cell growth, sensitize cancer cells to elimination by apoptosis, and hinder the development of blood vessels to serve the growing tumor. It also has actions that alter the relationship of the cancer cells with their microenvironment. Apigenin is able to reduce cancer cell glucose uptake, inhibit remodeling of the extracellular matrix, inhibit cell adhesion molecules that participate in cancer progression, and oppose chemokine signaling pathways that direct the course of metastasis into other locations. As such, apigenin may provide some additional benefit beyond existing drugs in slowing the emergence of metastatic disease.
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Flavonoids are structurally diverse and the most ubiquitous groups of dietary polyphenols distributed in various fruits and vegetables. In this study, the interaction between five flavonoids, namely formononetin-7-O-β-D-glucoside, calycosin- 7-O-β-D-glucoside, calycosin, rutin, and quercetin, and bovine serum albumin (BSA) was investigated by fluorescence and UV-vis absorbance spectroscopy. In the discussion, it was proved that the fluorescence quenching of BSA by flavonoids was a result of the formation of a flavonoid-BSA complex. Fluorescence quenching constants were determined using the Stern-Volmer and Lineweaver-Burk equations to provide a measure of the binding affinity between the flavonoids and BSA. The binding constants ranked in the order quercetin>rutin>calycosin>calycosin-7-O-β-D-glucoside ≈ formononetin-7-O-β-D-glucoside. The results of thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures indicated that the hydrophobic interaction played a major role in flavonoid-BSA association. The distance r between BSA and acceptor flavonoids was also obtained according to Förster's theory of non-radiative energy transfer.
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Solvent polarity and the local environment have profound effects on the emission spectral properties of fluorophores. The effects of solvent polarity are one origin of the Stokes shift, which is one of the earliest observations in fluorescence. Emission spectra are easily measured, resulting in numerous publications on emission spectra of fluorophores in different solvents, and when bound to proteins, membranes, and nucleic acids. One common use of solvent effects is to determine the polarity of the probe binding site on the macromolecule. This is accomplished by comparison of the emission spectra and/or quantum yields when the flu-orophore is bound to the macromolecule or dissolved in solvents of different polarity. However, there are many additional instances where solvent effects are used. Suppose a fluorescent ligand binds to a protein or membrane. Binding is usually accompanied by spectral shift or change in quantum yield due to the different environment for the bound ligand. Alternatively, the ligand may induce a spectral shift in the intrinsic or extrinsic protein fluorescence. In either case the spectral changes can be used to measure the extent of binding. The effects of solvent and environment on fluorescence spectra are complex, and are due to several factors in addition to solvent polarity. The factors that affect fluorescence emission spectra and quantum yields include:
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Available data suggest that substitution of refined carbohydrate in the diet with protein and fiber may benefit blood pressure. Lupin kernel flour is high in protein and fiber and low in carbohydrate. Our objective was to determine the effects on blood pressure of a diet moderately higher in dietary protein and fiber achieved by substituting lupin kernel flour for wheat flour in bread. Overweight and obese men and women (n = 88) were recruited to a 16-wk parallel-design study. Participants were randomly assigned to replace 15-20% of their usual daily energy intake with white bread (control) or lupin kernel flour-enriched bread (lupin). Measurements, including 24-h ambulatory blood pressure, were taken at baseline and 16 wk. Seventy-four participants (37 per group) completed the intervention. Baseline mean (+/-SD) systolic/diastolic blood pressures were 122.1 +/- 9.6/70.8 +/- 7.2 mm Hg (control) and 120.1 +/- 9.5/71.2 +/- 5.9 mm Hg (lupin). For lupin relative to control, the estimated mean (95% CI) net differences in protein, fiber, and carbohydrate intakes during the intervention were 13.7 g/d (95% CI: 2.3, 25.0 g/d), 12.5 g/d (95% CI: 8.8, 16.2 g/d), and -19.9 g/d (95% CI: -45.2, 5.5 g/d), respectively. Differences in systolic blood pressure, diastolic blood pressure, pulse pressure, and heart rate were -3.0 mm Hg (95% CI: -5.6, -0.3 mm Hg; P = 0.03), 0.6 mm Hg (95% CI: -1.0, 2.2 mm Hg; P = 0.47), -3.5 mm Hg (95% CI: -5.3, -1.8 mm Hg; P < 0.001), and 0.0 beats/min (95% CI: -1.7, 1.7 beats/min; P = 0.99), respectively. Increasing protein and fiber in bread with lupin kernel flour may be a simple dietary approach to help reduce blood pressure and cardiovascular risk. This trial was registered at the Australian New Zealand Clinical Trials Registry at http://www.anzctr.org.au/trial_view.aspx?ID=1014 as ACTRN12606000034538 on 25 January 2006.
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Determination of influence of lupin natural phenolic compounds on antibacterial properties of its seeds was carried out. Raw material were seeds of Lupinus albus, L. luteus, and L. angustifolius. The methods included the determination of the content of proteins, total phenolic compounds, free phenolic acids, and tannins as well as antibacterial properties with ethanol extracts. The content of total phenolic compounds was smaller in testas than in cotyledons and the highest levels are observed in bitter cultivars of Lupinus albus cv. Bac and L. angustifolius cv. Mirela. Lupin tannins mainly occurred in cotyledons of the white lupin, predominantly in the bitter cultivar Bac. Free phenolic acids were mainly found in testas. Only extracts from the testas displayed antibacterial properties, which excludes the possibility of alkaloid influence on the results. The results suggest that inhibition of test bacteria growth depended mainly upon the content of the total phenolic compounds.
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White lupin (Lupinus albus, L.), a widely cultivated crop that has been consumed for many years in Western Europe, may provide a useful alternative for individuals wishing to substitute animal with plant proteins for cardiovascular disease prevention. Lupin seeds have a very low content of isoflavones, and lupin protein isolates are essentially isoflavone free. In rats fed a casein-based cholesterol + cholic acid diet, a relatively low daily intake (50 mg/d by gavage for 2 wk) of total lupin protein extract reduced plasma total and VLDL + LDL cholesterol concentrations by 21 and 30%, respectively (both P<0.001). In an attempt to elucidate the lipid-lowering mechanism, LDL receptor activity was evaluated in a human hepatoma cell line (HepG2). In this model, the lupin total protein extract was essentially inactive, whereas one purified minor protein component, conglutin gamma, had a remarkable upregulatory effect, with maximal increases of 53 and 21% (both P<0.05) for LDL uptake and degradation, respectively. This initial study indicates that lupin, although isoflavone free, has hypocholesterolemic activity similar to that of other leguminous proteins in an established animal model. Further, the cholesterol reduction appears to be associated with stimulation of LDL receptors by a well-defined protein component of the lupin seeds as demonstrated by in vitro studies.
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The beneficial cardiovascular effects of soy protein have been studied intensively in recent years. Another protein-rich legume is lupin, which has been shown to have similar effects to those of soy in lowering serum cholesterol levels. In this study we compared the effects of lupin and soy protein on hypertension and vascular functions in spontaneously diabetic Goto-Kakizaki (GK) rat, which develop hypertension when fed a high-salt diet. The rats were fed with a 6% NaCl diet containing either lupin or soy protein isolate (20% weight/weight) for two weeks. In the end of the study the SBP was 18.6 mmHg lower (p<0.001) in the lupin group, and 12.0 mmHg lower (p<0.01) in the soy group than in the control group. Lupin and soy treatments normalised the decreased vasocontraction observed in the NaCl-fed control group, but only lupin treatment improved the impaired endothelium-dependent vasorelaxation. The attenuation of hypertension is likely to be mediated by the corrected vascular dysfunction, whose precise mechanism and the possible clinical relevance remains to be studied further.
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The effects of embedding up to 60 mol% of alpha-tocopherol (alpha-Toc) on the morphology and structure of the egg phosphatidylcholine (PC) membrane were studied using spectroscopic techniques. The resulting vesicles were subjected to turbidometric and dynamic light scattering measurements to evaluate their size distribution. The alpha-Toc intrinsic fluorescence and its quenching was used to estimate the tocopherol position in the membrane. Optical microscopy was used to visualize morphological changes in the vesicles during the inclusion of tocopherol into the 2 mg/ml PC membrane. The incorporation of up to 15 mol% of tocopherol molecules into PC vesicles is accompanied by a linear increase in the fluorescence intensity and the simultaneous formation of larger, multilamellar vesicles. Increasing the tocopherol concentration above 20 mol% induced structural and morphological changes leading to the disappearance of micrometer-sized vesicles and the formation of small unilamellar vesicles of size ranging from 30 to 120 nm, mixed micelles and non-lamellar structures.
Chapter
Peptide synthesis has undergone a major transformation in the last three decades, building on the solid-phase synthesis methodology of Bruce Merrifield first published in 1963 (1). During the 1970s, the first automation of peptide synthesis was undertaken using Boc chemistry. In the 1980s, improvements were made in the Boc chemistry automated process and, consequently, the synthesis of more-difficult sequences, as well as longer polypeptides became possible (2). Solid-phase Fmoc synthesis was developed in the early 1980s (3,4) and was also applied to automated systems (5). The 1990s saw improvements in both Boc and Fmoc chemistry together with novel modes of activation of the amino acids in both chemistries (6,7). The result was faster cycle times and, hence, reduced synthesis times. The range of protecting groups and resins available today means that sophisticated syntheses utilizing a combination of Boc and Fmoc chemistry are possible (8).
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The three globulins of the seeds of L. angustifolius cv. Uniwhite may be satisfactorily resolved in 10 min by electrophoresis on cellulose acetate strips. These globulins, conglutins α, β and γ, vary markedly in their amino acid compositions, with conglutin Ω differing from conglutins α and β and most other legume storage proteins in its relatively high content of cystine and methionine and lower content of arginine and glutamic acid. When examined on sodium dodecyl sulphate-polyacrylamide gels, both in the absence and presence of β-mercaptoethanol, the three globulins were found to differ completely in the type of subunit proteins they contain and in the significance of intrachain disulphide bonding. Conglutin α was found to contain three or four types of non-covalently linked subunits with apparent molecular weights in the range 55 000-80 000, each of which may contain a disulphide-bonded moiety with a molecular weight near 20 000. Conglutin γ was found to contain disulphide-bonded chains of molecular weights 17 000 and 30 000, whereas the four major subunits of conglutin β, whose molecular weights lie in the range 20 000-60 000, were not covalently linked together. The latter globulin does not appear to be homogeneous, for it can be separated by fractional precipitation with ammonium sulphate into a series of fractions which differ markedly in the proportion of subunit types they contain.
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The seed globulins of Lupinus albus were extracted and 12 ditterent proteins were separated: four of them correspond to vicilins and two to legumin
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Secondary plant metabolites are important native food components, which are becoming more and more interesting due to their physiological effects on human beings. One of the largest groups of these compounds is represented by plant phenols. This review summarizes the structure, classification and distribution of the phenolic compounds in plant foods, their chemistry and signification with regard to food processing and -storage as well as their physiological effects. This work focuses mainly on such reactions of the phenolic substances with proteins and enzymes that lead to covalent bonds. The derivatives formed have been characterized in terms of changes in their physicochemical and structural properties. The effect on the proteolytic in vitro digestion has been also illustrated. Further aspects reported include the influence on enzyme activity and -kinetic parameters. The different aspects of the nutritional-physiological consequences of such reactions in food and body, especially considering their significance to food science and technology are discussed.
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There is currently much interest in phytochemicals as bioactive components of food. The roles of fruit, vegetables and red wine in disease prevention have been attributed, in part, to the antioxidant properties of their constituent polyphenols (vitamins E and C, and the carotenoids). Recent studies have shown that many dietary polyphenolic constituents derived from plants are more effective antioxidants in vitro than vitamins E or C, and thus might contribute significantly to the protective effects in vivo. It is now possible to establish the antioxidant activities of plant-derived flavonoids in the aqueous and lipophilic phases, and to assess the extent to which the total antioxidant potentials of wine and tea can be accounted for by the activities of individual polyphenols.
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The seed globulins separated from Lupinus albus are all oligomeric proteins. Vicilins, i.e. globulins 4,5,6 and 7, and to a minor extent legumins,
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The phenolic constituents in the defatted flours and hulls of 10 legume species were fractionated into free acid, soluble ester, and residue components, followed by alkaline hydrolysis and quantitation by capillary gas-liquid chromatography. The flours contained only soluble esters, and hydrolyses revealed the presence of trans-ferulic, trans-p-coumaric, and syringic acids in nearly all species. Mung bean, field pea, lentil, fababean, and pigeon pea contained only 2-3 mg of phenolic acids/100 g of flour, but higher levels were obtained in navy bean, lupine, lima bean, chickpea, and, especially, cowpea. The hulls contained p-hydroxybenzoic, protocatechuic, syringic, gallic, trans-p-coumaric, and trans-ferulic acids in the soluble ester fraction and, to a lesser extent, in the insoluble residue. Dehulling would reduce the phenolic composition of the flour substantially for pigeon pea, fababean, mung bean, and lentil but would have little effect on the phenolic composition of field pea, navy bean, lima bean, or chickpea products.
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In this study a method was designed to assess non-destructively the type of UV-screening compounds present in the leaf epidermis. The method is based on the recording and calculation of the ratio of UV-excitation spectra of chlorophyll fluorescence (FER) from the adaxial and abaxial sides of bifacial leaves, or from older and younger segments of monocotyledonous leaves. The logarithm of this ratio (logFER) matched the absorption spectrum of the UV-absorbers present in the leaf, as confirmed by its overlap with the absorption spectrum of the methanolic extract of the leaf or of the isolated epidermis. By using the logFER approach, it was possible to demonstrate that the concentration but not the classes of compounds present in the epidermis that are responsible for UV-screening is affected by the side and the age of the leaves. In contrast, measurements from the leaves of seven dicots and one monocot indicated large difference in the classes of these compounds between species. Finally, it was shown that the logFER in the UV is independent of the emission wavelength, and that the method can be used for quantitative measurements. This method expands to the spectral domain the use of ChlF for the estimation of the leaf epidermal transmittance.
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Studies on lupin seed storage proteins have been performed using purification techniques that are often time-consuming and lead to poor resolution between the individual globulins. In this paper we have utilized anion exchange chromatography on the MONO Q column of the FPLC as a high-resolution, easy and rapid method to fractionate the three main globulins from two cultivars of Lupinus albus, namely the sweet cv Multolupa and the bitter cv Torres Vedras. The structure of the purified globulins, designated by γ-, β- and α-conglutins, was studied by electrophoresis performed under non-denaturing conditions, under denaturing, non-reducing conditions and denaturing, reducing conditions, and by two-dimensional electrophoresis. We have also developed a rapid, easy and sensitive method to determine the polypeptide chain composition of proteins or protein subunits which is based on the lateral diffusion of 2-mercaptoethanol during electrophoresis. We propose a nomenclature for the polypeptide chains and individual subunits of L. albus globulins that can be extended to other seed storage proteins.
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In this work, the effect of germination of lupin seeds (Lupinus angustifolius L., c.v. Zapatón) on bioactive phenolic compounds as well as on the antioxidant activity was studied. Phenolic compounds were analysed by HPLC-PAD-ESI/MS. The antioxidant activity was determined by spectrophotometry, evaluating the free radical scavenging activity of the samples. Germination produced significant changes in flavonoids and non-flavonoid phenolic compounds. In the analysed samples, isoflavones, flavones and dihydroflavonols in free and conjugated forms were identified. The results obtained indicate that germination modifies the quantitative and qualitative polyphenolic composition of lupin (Lupinus angustifolius L.) seeds during the different days of the process, with a significant increase of flavonoids. An increase in the antioxidant activity was also observed as a consequence of the process. Germination was shown to be a good process to increase the phenolic content of lupin seeds as well as their antioxidant activity.
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Bovine serum albumin (BSA) was modified by covalent attachment of chlorogenic acid using different concentrations at pH 9. The derivatization was accompanied by a reduction of lysine, cysteine and tryptophan residues. The isoelectric points were shifted to lower pH values and formation of high molecular weight fractions was noted. The structural changes were studied using circular dichroism, differential scanning calorimetry (DSC), intrinsic fluorescence, and binding of anilinonaphthalenesulfonic acid. The results showed that the content of α-helix decreased with a parallel increase in unordered structures with higher degrees of derivatization. DSC revealed a decrease in both denaturation temperature and enthalpy. Surface hydrophobicity declined, indicating that hydrophilic regions were exposed on the molecular surface. Proteolytic digestion showed that, at a lower degree of derivatization, the tryptic degradation was most adversely effected, whereas the peptic digestion declined with increasing modification. A trypsin inhibitory effect of the breakdown products released from derivatized BSA was also observed.
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This review deals with the main proteins of white lupin seed (Lupinus albus, L.) and reports on the current knowledge of the structural and functional properties of these proteins with the aim of providing the first comprehensive, accurate and up-to-date survey on this topic. Lupin seed's four main protein families of globulins, termed α-, β-, γ- and δ-conglutins, are reviewed with specific regard to their molecular and biological features. Their nutritional, technological, nutraceutical and allergenic potentials are also considered. The review is intended to provide nutritionists, food technologists and various stakeholders with the molecular background for a better exploitation of this valuable and accessible protein-rich source.
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Dietary polyphenols have received attention for their biologically significant functions as antioxidants, anticarcinogens or antimutagens, which have led to their recognition as potential nutraceuticals. Polyphenols also characteristically possess a significant binding affinity for proteins, which can lead to the formation of soluble and insoluble protein–polyphenol complexes. Questions remain concerning whether and to what extent the protein–polyphenol interaction influences functionality. For example, is the formation of protein–polyphenol complexes an obstacle to the nutritional bioavailability of either species? This article discusses the development of suitable methodologies to investigate the physicochemical basis of protein–polyphenol interactions and the influence of structure–activity relationships on binding affinities.
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Chlorination can significantly enhance the antioxidant and antitumor activity of genistein. In this paper, genistein, 8-chlorogenistein, and 3',8-dichlorogenistein were selected to investigate the binding to bovine serum albumin (BSA) using fluorescence spectroscopy and circular dichroism (CD). The results showed that chlorination, especially at position 3', had significant effects on the binding constant value of chlorinated genistein derivatives to BSA; however, the binding site and the binding number were slightly affected. The thermodynamic parameters indicated that hydrophobic and electrostatic forces played important roles in the binding process and the enhanced binding affinity mainly associated with the increase of the hydrophobicity caused by the chlorine atom substitution. Furthermore, the CD data demonstrated that the conformation of BSA was slightly altered in the presence of genistein, 8-chlorogenistein, and 3',8-dichlorogenistein, with different reduced α-helix contents. The results obtained herein will be of biological significance in toxicology investigation and genistein derivative drug design.
Article
γ-Conglutin is a blood glucose-lowering protein purified from lupin (Lupinus albus, L.) seed. Despite various features of this protein have already been studied, no function in the seed nor any mechanism of action as a hypoglycemic nutraceutical compound have been identified so far. The lupin protein was shown to exist both in monomeric and multimeric forms as a function of pH. However, a detailed description of the pH-dependent structural dynamics of this protein, as the basis to investigate the reason/s of its functional behaviour, is not available yet. In this study, multiple and independent spectroscopic approaches, including light scattering associated to size exclusion chromatography of both untreated and covalently cross-linked protein, near and far UV circular dichroism, intrinsic and extrinsic fluorescence measurements, have been used to monitor oligomeric and conformational modifications caused by pH changes. Altogether, the results revealed a tetramer-dimer-monomer transition between neutral to slightly acidic pH and a dramatic and abrupt conformational change below pH 3.5. According to these findings, a model depicting γ-conglutin structural dynamics was drawn. This model highlights the primary role of amino acid side group electrostatic interactions in the oligomer association/dissociation equilibria and in the pH-driven collapse of the native conformation.
Article
gamma-Conglutin, a glycoprotein from Lupinus albus seed, has been characterized at molecular level but its physiological function is still unknown. gamma-Conglutin shares a high structural similarity with xyloglucan-specific endo-beta-1,4-glucanase inhibitor proteins (XEGIPs) and Triticum aestivum xylanase inhibitor (TAXI-I), which act specifically against fungal glycosyl hydrolase belonging to families 12 and 11, respectively. To assess the possible involvement of gamma-conglutin in plant defense, germinating lupin seeds were incubated with chitosan. The relative quantification of gamma-conglutin mRNA extracted from cotyledons was then carried out by RT-qPCR and indicated that chitosan strongly elicited the expression of gamma-conglutin. Moreover, biochemical trials aimed to test the inhibitory capacity of the protein have been also carried out. gamma-Conglutin failed to inhibit representative fungal endo-glucanases and other cell wall-degrading enzymes. To explain the lack of inhibitory capacity we investigated the possible structural differences between gamma-conglutin and XEGIPs and TAXI-I, including the construction of a predictive 3D model of the protein. Bioinformatic analysis suggests that the lack of inhibitory activity of gamma-conglutin can be attributed to sequence differences in the inhibitor interaction domains, and in particular to a sequence deletion in one of the functional loops.
Article
Lupin seed gamma-conglutin, orally administered to animal models, has been shown to display glucose-controlling properties. Therefore, we have addressed the study of gamma-conglutin susceptibility to proteolytic enzymes in vitro as the basis to unveil its metabolic fate in the body. Pepsin treatment at pH 2.0 and 3.0 caused extensive proteolytic breakdown, while at pH 4.0, where pepsin is minimally active, gamma-conglutin was unaffected. Aliquots of the pepsin-treated protein were further incubated with pancreatin at neutral pH. If the protein backbone was already cleaved by pepsin action, then the breakdown by pancreatin was almost complete; alternatively, pancreatin did not affect at all gamma-conglutin polypeptide chain. This was not due to an inhibitory activity of gamma-conglutin, because co-incubation with casein showed complete breakdown of the milk protein. Furthermore, gamma-conglutin was incubated with bromelain, a proteinase effective between pH 4.0 and 7.0. A sharp transition from the uncleavable to the fully cleavable form of gamma-conglutin was observed below pH 4.25. Therefore, it was concluded that (i) gamma-conglutin is resistant to proteolysis at pH greater than 4.0, likely because of a compact native conformation, (ii) an acidic pH renders the protein susceptible to proteases, suggesting the occurrence of a trans conformation, which has also been observed by circular dichroism spectral analysis, and (iii) the protein undergoes an "all or none" degradation pathway, regardless of the enzyme used.
Article
Daidzein, one of major isoflavones found in soybeans, has a wide spectrum of physiological and pharmacological functions. The observed biological effects involve its interactions with lipid bilayers, usually detected by indirect methods. In this study we use the native fluorescence of daidzein to report changes observed during its interactions with organic solvents and in a phosphatidylcholine membrane. We have investigated interactions of daidzein with lipid bilayers of egg phosphatidylcholine (PC) by absorption and fluorescence methods. The data obtained indicate emission arises from the conjugate anion in excited singlet state. The fluorescence is found to increase with the basicity of the solution and the polarity of the solvent. An increase in fluorescence anisotropy in the presence of membranes suggests partial incorporation of daidzein molecules into the bilayer. Two fluorescence lifetime components, 1.5 ns and 3.5 ns, reflects the partition of daidzein between aqueous and membrane environments, respectively. On the basis of the obtained spectroscopic data we conclude that up to 15% of daidzein is located in hydrophilic region of the membrane whereas the rest is distributed in aqueous bulk and aqueous/membrane interface. For studying the antioxidant activity of daidzein against lipid peroxidation initiated by AAPH the molecule of C11-BODIPY581/591 has been used as a fluorescent oxidation indicator. The results show that the presence of daidzein anions in the membrane interface increases the inhibitory effect on lipid peroxidation compared to the neutral form of daidzein.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
The flavonoids are plant polyphenols found frequently in fruits, vegetables, and grains. Divided into several subclasses, they include the anthocyanidins, pigments chiefly responsible for the red and blue colors in fruits, fruit juices, wines, and flowers; the catechins, concentrated in tea; the flavanones and flavanone glycosides, found in citrus and honey; and the flavones, flavonols, and flavonol glycosides, found in tea, fruits, vegetables, and honey. Known for their hydrogen-donating antioxidant activity as well as their ability to complex divalent transition metal cations, flavonoids are propitious to human health. Computer-controlled high-performance liquid chromatography (HPLC) has become the analytical method of choice. Many systems have been developed for the detection and quantification of flavonoids across one, two, or three subclasses. A summary of the various HPLC and sample preparation methods that have been employed to quantify individual flavonoids within a subclass or across several subclasses are tabulated in this review.
Article
Flavonoids are potent antioxidants. It is also known that flavonoids bind to proteins. The effect of the interaction between tea flavonoids and proteins on the antioxidant capacity was examined. Their separate and combined antioxidant capacities were measured with the Trolox equivalent antioxidant capacity (TEAC) assay. It was observed that the antioxidant capacity of several components of green and black tea with alpha-, beta-, and kappa-casein or albumin is not additive; that is, a part of the total antioxidant capacity is masked by the interaction. This masking depends on both the protein and the flavonoid used. Components in green and black tea, which show the highest masking in combination with beta-casein, are epigallocatechin gallate and gallic acid. The results demonstrate that the matrix influences the efficacy of an antioxidant.
Article
Soy glycinin (SG) and soy trypsin inhibitor (STI) were derivatized by chlorogenic- and caffeic acid (cinnamic acids, C(6)-C(3) structure), and by gallic acid representing hydroxybenzoic acids (C(6)-C(1) structure). Further, the flavonoids, flavone, apigenin, kaempferol, quercetin and myricetin (C(6)-C(3)-C(6) structure) were also caused to react with soy proteins to estimate the influence of the number and the position of hydroxy substituents. The derivatization caused a reduction of lysine, cysteine and tryptophan residues in the soy proteins. The isoelectric points of the derivatives were shifted to lower pH values and formation of high molecular fractions was documented. The derivatives were characterized in terms of their solubility at different pH-values to document the influence on the functional properties. The structural changes induced were studied using circular dichroism (CD), differential scanning calorimetry (DSC), intrinsic fluorescence, and binding of anilinonaphthalenesulfonic acid. The influence of derivatization on the in-vitro digestibility with trypsin, chymotrypsin, pepsin and pancreatin was also assessed. The effect on the trypsin inhibitor activity of all the resulting STI derivatives was studied, the latter being reduced.
Article
Quercetin (3,3',4',5,7-pentahydroxyflavone), a ubiquitous, bioactive plant flavonoid, is known to possess anti-cancer, anti-tumor, and other important therapeutic activities of significant potency and low systemic toxicity. In this communication, we report for the first time a study on the interactions of quercetin with the plasma protein human serum albumin (HSA), exploiting the intrinsic fluorescence emission properties of quercetin as a probe. Quercetin is weakly fluorescent in aqueous buffer medium, with an emission maximum at approximately 538 nm. Binding of quercetin with HSA leads to dramatic enhancement in the fluorescence emission intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. The excitation spectrum suggests occurrence of efficient Förster type resonance energy transfer (FRET) from the single tryptophan-214 residue of HSA to the protein bound quercetin. The emission, excitation, and anisotropy (r=0.18 at [HSA]=30 microM) data (using the native protein) along with emission studies of quercetin using partially denatured HSA (by 8M urea) indicate that the quercetin molecules bind at a motionally restricted site near tryptophan-214 in the interdomain cleft region of HSA. Furthermore, the binding constant (K=1.9 x 10(5)M(-1)) and Gibbs free energy change (deltaG(0)=-30.12 kJ/mol)) for quercetin-HSA interaction have been calculated from the relevant anisotropy data. Implications of these results are examined, particularly in relation to prospective applications in biomedical research.
Article
From the aerial parts of Lupinus hartwegii, two new flavone C-glycosides apigenin-7-O-beta-apiofuranosyl-6, 8-di-C-beta-glucopyranoside (1) and apigenin-7-O-beta-apiofuranosyl-6-C-beta-glucopyranosyl-8-C-(6z.qprime;-O-E-feruloyl)- beta-glucopyranoside (2) have been isolated together with two known isoflavonoid glucosides genistein-7-O-beta-glucopyranoside (3) and genistein-7, 4'-di-O-beta-glucopyranoside (4) as well as two known compounds ferulic acid 4-O-beta-glucopyranoside (5) and sparteine (6). The structures of the isolated compounds were verified by means of MS and NMR spectral analyses.
Article
To utilize lupin seeds for food and pharmaceutical applications, lupin seeds were pretreated to remove oil using hexane or carbon dioxide. Two types of lupin protein isolate were prepared. Both types of protein isolate showed good foaming activity, comparable to egg white. Protein isolate extracted under acid conditions showed higher foaming activity than protein isolate extracted at neutral pH. The lipoxygenase activity was much reduced in both of the protein isolates. The protein isolate extracted at neutral pH showed a stronger angiotensin converting enzyme inhibition than the protein isolate extracted under acidic pH. In contrast, the protein isolate extracted under acid conditions had a greater sodium cholate binding capacity, comparable to that of cholestyramine. Lupin samples showed less DPPH radical scavenging activity than deoiled soybean. The deoiling method did not affect the functional properties, lipoxygenase activity, angiotensin converting enzyme inhibition, sodium cholate binding, and radical scavenging activity.
Article
Grain legumes are a valuable source of food proteins. Their exploitation is expected to grow in relation of a growing world's food needs. Moreover, it is currently taking place a reappraisal of the beneficial effects of legume seed dietary intake, which are the basis for various health claims. Proteins and peptides concur to the observed biological activities of legume seeds, but their effect(s) has(ve) not completely been disclosed. Aims of this review are: to report the most relevant putative positive effects of grain legumes on human health and to give an account of the current knowledge on the demonstrated legume seed protein biological activities. Specific effects on the prevention and treatment of various diseases, mostly of which are typical of the affluent countries, are reported. Examples of studies at molecular level aimed at elucidating of the underlying mechanism(s) are given. The prospects on targeted legume protein exploitation in the nutraceutical area, including the biotechnological approaches, are also considered.
Article
The ingestion of dietary products containing sweet lupin (such as Lupinus albus or Lupinus angustifolius) has been reported to cause IgE-mediated allergic reactions. Recent studies have indicated lupin globulins as important IgE binding proteins. The aim of the present study was to generate and characterize monoclonal antibodies (mAbs) against lupin seed proteins. Mice were immunized with a protein isolate from L. albus and mAbs were obtained by hybridoma techniques. Albumins and globulins were extracted, and the globulin fraction was separated further into conglutins by anion exchange chromatography. Specificities, binding patterns and applications of the mAbs were investigated by immunochemical methods. Five mAbs were produced: Lu11 (an IgG2b antibody), Lu8, Lu18, Lu34 and Lu35 (all IgM antibodies). The mAbs reacted strongly with protein isolates from both L. albus and L. angustifolius. All mAbs are directed towards the lupin globulin fraction; Lu11 and Lu18 recognize alpha-conglutin, while Lu8, Lu34 and Lu35 recognize beta-conglutin. In addition, Lu11 inhibited the binding of IgE from patients with positive skin prick tests to lupin proteins in a competitive ELISA by approximately 30%. Furthermore, preliminary results show that Lu11 can be used to develop a sensitive method for the detection of alpha-conglutin in foods. Lupin globulins are immunogenic and alpha-conglutin is a potential allergen. This is the first study describing mAbs against the candidate lupin allergens, emphasizing the importance of additional studies on conglutins in lupin allergy.
Article
Glycosylation at different hydroxyl groups of flavonoids and acylation of sugar moieties are ubiquitous modifications observed in plants. These modifications give rise to simultaneous presence of numerous isomeric and isobaric compounds in tissues and extracts thereof. To develop UPLC-MS method capable for resolution of isomeric malonylated glycoconjugates of flavonoids and recognition of structural differences. Flavonoid glycoconjugates were extracted from leaves of blue lupin (Lupinus angustifolius L.) plants with 80% methanol. Extracts were analysed using ultraperformance liquid chromatography (UPLC) combined with tandem (quadrupole-time of flight, QToF) mass spectrometry. Differentiation of malonylated glycosides of isoflavones and flavones is demonstrated in this paper. The use of UPLC-MS/MS enabled 38 flavonoid conjugates to be distinguished, including the discrimination of five different isomers of a single 3'-O-methylluteolin glycoside. Additionally, pseudo MS(3) experiments (CID spectra registered at high cone voltages) enabled confirmation of the aglycone structures by comparison of their spectra with those obtained from aglycone standards. Application of UPLC-MS/MS allows separation and identification numerous positional isomers of malonylated glycosides of flavonoids and isoflavonoids in plant material. Provided there is strict control of the MS ionisation parameters, this method may be useful for preparation of a flavonoids spectra database, enabling the inter-laboratory comparison of analytical results.
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