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Assessment of the lupin seed glucose-lowering protein intestinal absorption by using in vitro and ex vivo models
Abstract
A lupin seed glycoprotein, termed γ-conglutin, has previously been found to display insulin-mimetic activity in myocyte models and reduce plasma glucose concentration when orally administered to both rats and humans. To envisage the possible metabolic fate of this bioactive protein, we used in vitro cell and ex vivo tissue models to monitor its transit through the intestinal barrier. Caco-2 cell monolayers and rat intestinal everted sacs were treated with purified γ-conglutin and the protein was immuno-assayed by chemi-luminescence-enhanced Western blotting. The in vitro approach showed that the intact protein can transit from the apical to the basolateral side of the cell monolayers. The unmodified lupin protein was also detected inside the intestinal everted sacs. Proper controls of cell monolayer and sac integrity ruled out the possibility of protein passive leakage.
Supplementary resource (1)
Data
June 2013
... Over the last two decades, lupins (Lupinus albus, L. angustifolius, and L. mutabilis) have attracted particular attention for their hypoglycaemic, hypocholesterolemic, and anti-inflammatory properties, especially regarding protein fractions [7][8][9][10]. Among them, γ-conglutin (γ-C) has been extensively studied for its postprandial glycaemic regulating activity in vitro [11,12], in vivo [13][14][15][16][17][18][19][20], ex vivo [21], and in humans [11,[22][23][24]. The consumption of foods containing bioactive proteins and/or peptides that can help in managing different chronic diseases (e.g., diabetes) is nowadays gaining attention. ...
... Under the adopted experimental conditions, no new peptides were detectable in the basolateral chamber, likely indicating that no proteolysis of γ-C occurred during the transport. Alternatively, γ-C once internalised might be degraded inside the cells and the residual intermediate products could not be transported to the basolateral compartment [21,35]. Furthermore, a study was conducted to assess the transport of γ-C from apical to basolateral chambers of transwell inserts. ...
... On the apical chamber, proteins were applied at two different concentrations (0.50 and 1.00 mg/mL) compared with the control without any protein. The peak at 37 min is the γ-C and is present in both chambers, essentially confirming the ability of Caco-2 cells to transport the protein through a model of the intestinal barrier, as previously described by Capraro et al. [21]. Under the adopted experimental conditions, no new peptides were detectable in the basolateral chamber, likely indicating that no proteolysis of γ-C occurred during the transport. ...
γ-Conglutin (γ-C) is the glycoprotein from the edible seed L. albus, studied for long time
for its postprandial glycaemic regulating action. It still lacks clear information on what could happen
at the meeting point between the protein and the organism: the intestinal barrier. We compared
an in vitro system involving Caco-2 and IPEC-J2 cells with an ex vivo system using pig ileum and
jejunum segments to study γ-C transport from the apical to the basolateral compartment, and its
effects on the D-glucose uptake and glucose transporters protein expression. Finally, we studied its
potential in modulating glucose metabolism by assessing the possible inhibition of α-amylase and
α-glucosidase. RP-HPLC analyses showed that γ-C may be transported to the basolateral side in the
in vitro system but not in the pig intestines. γ-C was also able to promote a decrease in glucose uptake
in both cells and jejunum independently from the expression of the SGLT1 and GLUT2 transporter
... The mechanism of action of γ-conglutin has been investigated using different experimental models. In particular, it has been shown that the intact protein is absorbed by the human intestinal epithelium (Capraro et al., 2011) using two different models. Caco2 were treated with a purified sample of γ-conglutin and the protein was measured by chemiluminescence-enhanced Western blotting, demonstrating that the intact protein is transferred from the apical to the basolateral side of the intestinal cell monolayer (Capraro et al., 2011). ...
... In particular, it has been shown that the intact protein is absorbed by the human intestinal epithelium (Capraro et al., 2011) using two different models. Caco2 were treated with a purified sample of γ-conglutin and the protein was measured by chemiluminescence-enhanced Western blotting, demonstrating that the intact protein is transferred from the apical to the basolateral side of the intestinal cell monolayer (Capraro et al., 2011). The paper, however, does not clarify the modality of γ-conglutin transit, i.e. receptor-mediated versus endocytic mechanism. ...
... The paper, however, does not clarify the modality of γ-conglutin transit, i.e. receptor-mediated versus endocytic mechanism. In parallel, in an ex vivo model, the unmodified lupin protein was detected inside the intestinal everted sacs of rat ileum again by using chemiluminescence-enhanced Western blotting for the quantification (Capraro et al., 2011). As a consequence, most molecular investigations have been performed on intact γ-conglutin. ...
... As stated before, many aspects of the possible bioactivities of γC on model human cells have yet to be further elucidated. We focused on the effects on the Caco-2 cell, since previous works on the subject have been carried out using this cell model [11]. Caco-2 is a cell line derived from human colon adenocarcinoma used as a common model of the intestinal barrier for in vitro toxicology studies and to test the biological activities of food-derived molecules [32]. ...
... Indeed, when Caco-2 cells were treated with the peptides obtained by the in vitro-simulated gastro-intestinal digestion of γC, no negative effects were observed ( Figure 1B). subject have been carried out using this cell model [11]. Caco-2 is a cell line derived from human colon adenocarcinoma used as a common model of the intestinal barrier for in vitro toxicology studies and to test the biological activities of food-derived molecules [32]. ...
The search for bioactivities influencing the human wellbeing of food proteins and peptides is a topic of broad and current interest. γ-Conglutin (γC) is a lupin seed protein drawing remarkable pharmacological and/or nutraceutical interest, as it is able to reduce hyperglycemia in humans and animal models. The present work deepens our investigations to understand the molecular basis of the in vitro effects of γC by testing the possible metabolic effects on cultivated Caco-2 cells. γC and its derived peptides (obtained via simulated gastrointestinal digestion) did not influence the cell viability at incubation times up to 24 h. The incubation of cells with native or digested γC caused no detectable inflammation processes mediated by Nuclear Factor kappa B (NFκB). We checked if treatment with γC or its derived peptides can elicit the expression of two peptide transporters (Pept-1 and Htp-1) by using an RT-qPCR approach. Native γC caused the halving of Pept-1 expression compared to untreated cells, but this effect disappeared when γC was digested. Either native γC or γC peptides reduced the expression levels of Hpt-1. Finally, this work also sheds light on the possible structural modifications of γC that may occur in the gastrointestinal tract, using an in vitro simulated dispersed system with polystyrene nanoparticles (NPs).
... The way γ-conglutin reaches the blood torrent from the intestine had been somewhat elusive up to then. However, Capraro et al. [74] proposed a putative metabolic pathway for γ-conglutin protein (L. albus), and tested it in an in vitro and ex vivo model of rat intestine. ...
... (B) Suggested pathway for γ-conglutin to exert its normoglycemic effects. Drawn from the results of Capraro et al.[74], Terruzzi et al.[73], Capraro et al.[75] and Vargas-Guerrero et al.[71]. IR = Insulin receptor. ...
Biological significance:
Some important topics concerning storage proteins from lupin seeds are presented and analyzed in an integrated way in this review. Proteomic approaches have been essential in characterizing lupin seed protein diversity, which goes far beyond gene diversity since the protein level adds to the latter differential proteolytic cleavage of conglutin pro-proteins and a diverse array of glycosylation forms and sites. Proteomics has also proved helpful for screening and studying Lupinus germplasm with the future aim of exploiting and improving food production, quality, and nutritional values.
... No literature could be found on intestinal protein absorption using the everted sac technique with human tissue. However, the everted sac technique has been applied for investigating the absorption of human ␥-globulin and ovalbumin across the ileum of rabbits and guinea pigs [83], as well as ␥-conglutin absorption in rats [69]. A disadvantage of everted sac method against the Ussing chamber technique is that with everted sac the muscle layer is still present and this may lead to the underestimation of the protein transport value. ...
... A positive result in an ELISA or RIA could be caused by only a specific part of the protein, so even if the protein is degraded intra-cellularly it may still be recognised by the antibody used in these assays. Immunoblotting [69,71,87,92] and high-performance liquid (gel permeation) chromatography [16,30,79,99] are good alternatives, since these techniques separate the proteins by their molecular weight, thus proving the presence of intact protein. ...
In view of the imminent deficiency of protein sources for human consumption in the near future, new protein sources need to be identified. However, safety issues such as the risk of allergenicity are often a bottleneck, due to the absence of predictive, validated and accepted methods for risk assessment. The current strategy to assess the allergenic potential of proteins focuses mainly on homology, stability and cross-reactivity, although other factors such as intestinal transport might be of added value too. In this review, we present an overview of the knowledge of protein transport across the intestinal wall and the methods currently being used to measure this. A literature study reveals that protein transport in sensitised persons occurs para-cellularly with the involvement of mast cells, and trans-cellularly via enterocytes, while in non-sensitised persons micro-fold cells and enterocytes are considered most important. However, there is a lack of comparable systematic studies on transport of allergenic proteins. Knowledge of the multiple protein transport pathways and which model system can be useful to study these processes may be of added value in the risk assessment of food allergenicity.
... Its oral administration is facilitated by this characteristic. Additionally, Capraro and colleagues [37] revealed that Cγ could cross the Caco-2 cells' basolateral membranes, an in vitro model that resembled the human intestinal epithelium, in an undamaged state. We proposed that, following absorption, Cγ may induce pancreatic insulin secretion similarly to glucagon-like peptide-1 (GLP-1), which binds to its pancreatic receptor and stimulates insulin exocytosis [38]. ...
Early insulin resistance and a progressive loss of pancreatic β cell function combine to cause type 2 diabetes (T2D), which leads to insufficient insulin production followed by hyperglycemia. Purified from Lupinus albus seed, conglutin gamma (Cγ) is a protein that lowers blood sugar. The primary function of adipocytokines, hormones released by adipose tissue, is to alert important organs to maintain metabolic balance. This study aimed to identify and compare the role of Cγ and glimepiride in controlling hyperglycemia, insulin secretion, insulin resistance, and hyperlipidemia in high-fat diet (HFD) and low-dose streptozotocin (STZ) induced diabetes in experimental rats. Male Sprague-Dawley rat groups were divided into seven groups; normal, Cγ control, T2D control, and four T2D groups which received Cγ (30, 60, and 120 mg/kg) and Glimepiride (0.1 mg/kg) treatments. Administration of Cγ successfully eliminated hyperglycemia and increased insulin secretion and sensitivity. In addition, when compared to (STZ+HFD) control rats, treatment with Cγ improved the expression of leptin, adiponectin, and their blood concentrations, as well as the activity of the enzyme chitotriosidase. It also significantly decreased the expression of apelin, nicotinamide phosphoribosyltransferase and RBP4. The present data suggests that Cγ has an effective role in controlling hyperglycemia induced by diabetes through amelioration of leptin, adiponectin, lipid profile, and metabolic syndrome.
... Western blot with Concanavalin A (ConA) detection was carried out to evaluate the presence of mannose residues. Proteins were loaded on a 12% SDS-PAGE and electroblotted onto nitrocellulose [24]. Membranes were washed three times for 5 min with Tris Buffered Saline (TBS), pH 7.4, saturated with 1% fish gelatin in TBS for 1.5 h and incubated with ConA (50 mg/mL in TBS, Merck) for 1 h. ...
γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have 'escaped' the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein.
... When different dosages of γ-conglutin (630, 315, and 157.5 mg) were eaten 30 min before the carbohydrate supply for 7 weeks, a relevant hypoglycemic effect could be observed in human subjects [54]. Lupin seed protein could be transported by Caco-2 cell monolayers and everted intestinal sacs, suggesting it has the potential to be developed as an oral agent to manage T2D [84]. ...
Diabetes mellitus is a complex disorder characterized by insufficient insulin production or insulin resistance, which results in a lifelong dependence on glucose-lowering drugs for almost all patients. During the fight with diabetes, researchers are always thinking about what characteristics the ideal hypoglycemic drugs should have. From the point of view of the drugs, they should maintain effective control of blood sugar, have a very low risk of hypoglycemia, not increase or decrease body weight, improve β-cell function, and delay disease progression. Recently, the advent of oral peptide drugs, such as semaglutide, brings exciting hope to patients with chronic diabetes. Legumes, as an excellent source of protein, peptides, and phytochemicals, have played significant roles in human health throughout human history. Some legume-derived peptides with encouraging anti-diabetic potential have been gradually reported over the last two decades. Their hypoglycemic mechanisms have also been clarified at some classic diabetes treatment targets, such as the insulin receptor signaling pathway or other related pathways involved in the progress of diabetes, and key enzymes including α-amylase, α-glucosidase, and dipeptidyl peptidase-IV (DPP-4). This review summarizes the anti-diabetic activities and mechanisms of peptides from legumes and discusses the prospects of these peptide-based drugs in type 2 diabetes (T2D) management.
... The method that was described previously by [20] for the preparation of everted gut sacs of rat ileum was followed for this study also. Pentobarbital sodium 40 mg/kg was used to anesthetize the rats, and the small intestine was removed [21]. The intestinal digesta was removed and cleaned with ice-cold saline, and the distal ileum (about 15 cm each) was extracted and everted using a glass rod. ...
Background
Metoprolol is a substrate of CYP3A4, 2B6, CYP2D6, CYP2C9, and p -glycoprotein ( p -gp). Hesperetin was reported as an inhibitor of cytochrome P-450 (CYP) enzymes and p -gp. The objective of this study was to investigate the effect of hesperetin on the pharmacokinetics of metoprolol in rats and in vitro models. In in vivo studies, male Wistar rats were treated with metoprolol (30 mg/kg) once a day for 15 consecutive days alone and in combination with hesperetin (25, 50, and 100 mg/kg). Blood samples were withdrawn from the tail vein on the 1st day in the single-dose pharmacokinetic study and on the 15th day in the repeated-dose pharmacokinetic study. In in vitro studies, metoprolol was incubated in the presence or absence of hesperetin and traditional p -gp inhibitors using rat-everted gut sacs. Reverse phase-high-performance liquid chromatography (RP-HPLC) was used to determine the amounts of metoprolol in the plasma and incubated samples (RP-HPLC).
Results
The C max , AUC, and half-life ( t 1/2 ) of metoprolol significantly increased by twofold compared to the metoprolol group in rats pre-treated with hesperetin. The clearance and volume of distribution both decreased significantly. Metoprolol transport was dramatically increased in the presence of hesperetin and quinidine (standard p -gp inhibitor) in in vitro study.
Conclusion
The present study results revealed that hesperetin significantly increased the absorption of metoprolol in rats and everted gut sacs in vitro might be due to the inhibition of CYP and p -gp.
... 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. ...
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.
... Many different in vivo models have been used to test the positive health effects of lupin seed γ-conglutin. Capraro et al. (2011), using Caco2 cell line, showed that the human intestinal epithelium absorbs the intact form of this protein. The molecular basis of antidiabetic activity was investigated in a myocyte model of C2C12 cells (Terruzzi et al., 2011). ...
A number of scientific data indicate that γ-conglutin can be internalised by different human cells and undergoes secretion from the seed in response to high temperature. In both of these cases, the protein must interact in some manner with biological membranes, however, the mechanisms underlying this phenomenon remain unknown. Herein, we found that the remarkable change of total surface hydrophobicity after appropriate heat treatment of γ-conglutin monomer led to its interaction with model membranes (liposomes). Before the interaction, the protein undergoes an intriguing thermal unfolding pattern which was studied based on a spectroscopic approach. Insight into the interaction mechanism with liposomes was possible thanks to applying two molecular probes that were differentially localised in the lipid bilayer. The results show that the thermal rearranged γ-coglutin monomer affects hydrocarbon chains in membranes leading to their morphology change and disruption. The main driving force of this phenomenon is based on hydrophobic interaction.
... The evaluation of their bioavailabilities and metabolic fate was outside the scope of this work. However, previous studies have shown that some plant proteins introduced with the diet arrive in the intestinal tract in intact form and may be absorbed by intestinal cells [79]. The susceptibility of proteins and peptides to digestion and their bioavailability is influenced by diverse conditions, including extreme pH values and plasma membranes phospholipids interaction [80]. ...
Food proteins and peptides are able to exert a variety of well-known bioactivities, some of which are related to well-being and disease prevention in humans and animals. Currently, an active trend in research focuses on chronic inflammation and oxidative stress, delineating their major pathogenetic role in age-related diseases and in some forms of cancer. The present study aims to investigate the potential effects of pseudocereal proteins and their derived peptides on chronic inflammation and oxidative stress. After purification and attribution to protein classes according to classic Osborne’s classification, the immune-modulating, antioxidant, and trypsin inhibitor activities of proteins from quinoa (Chenopodium quinoa Willd.), amaranth (Amaranthus retroflexus L.), and buckwheat (Fagopyrum esculentum Moench) seeds have been assessed in vitro. The peptides generated by simulated gastro-intestinal digestion of each fraction have been also investigated for the selected bioactivities. None of the proteins or peptides elicited inflammation in Caco-2 cells; furthermore, all protein fractions showed different degrees of protection of cells from IL-1β-induced inflammation. Immune-modulating and antioxidant activities were, in general, higher for the albumin fraction. Overall, seed proteins can express these bioactivities mainly after hydrolysis. On the contrary, higher trypsin inhibitor activity was expressed by globulins in their intact form. These findings lay the foundations for the exploitation of these pseudocereal seeds as source of anti-inflammatory molecules.
... On the other hand, though, other events or factors cannot be ruled out. One possibility is that LcC and HcC may be internalized in the cytoplasm, as it occurs for other seed proteins [71], exerting their activity in intracellular pathways such as in IkB translocation [72], for example. ...
Several food-derived molecules, including proteins and peptides, can show bioactivities toward the promotion of well-being and disease prevention in humans. There is still a lack of information about the potential effects on immune and inflammatory responses in mammalian cells following the ingestion of seed storage proteins. This study, for the first time, describes the potential immunomodulation capacity of chenopodin, the major protein component of quinoa seeds. After characterizing the molecular features of the purified protein, we were able to separate two different forms of chenopodin, indicated as LcC (Low charge Chenopodin, 30% of total chenopodin) and HcC (High charge Chenopodin, 70% of total chenopodin). The biological effects of LcC and HcC were investigated by measuring NF-κB activation and IL-8 expression studies in undifferentiated Caco-2 cells. Inflammation was elicited using IL-1β. The results indicate that LcC and HcC show potential anti-inflammatory activities in an intestinal cell model, and that the proteins can act differently, depending on their structural features. Furthermore, the molecular mechanisms of action and the structural/functional relationships of the protein at the basis of the observed bioactivity were investigated using in silico analyses and structural predictions.
... Lupine seed is mentioned in the ancient and traditional pharmacopoeia books as an ant diabetic product (Bertoglio et al., 2011) they demonstrated that the active protein responsible for the claimed anti-diabetic effect of the Lupine seed is effective in man, in addition to animal models. Also another authors report on the glucose lowering effect of Lupine -gamma-conglutin in human subjects (Capraro et al., 2011). Fontanari et al., (2012) demonstrated that protein isolate from Lupine has a metabolic effect on endogenous cholesterol metabolism and a protector effect on development of hepatic statuses. ...
... It displayed glucose-controlling properties when b-conglutin was orally administered to both rats and humans. These findings represent the first molecular evidence of the possible use of a legume protein in the control of glycemia ( Capraro et al., 2010). ...
... It displayed glucose-controlling properties when b-conglutin was orally administered to both rats and humans. These findings represent the first molecular evidence of the possible use of a legume protein in the control of glycemia ( Capraro et al., 2010). ...
... The intact protein can transit from the apical to the basolateral side of Caco-2 cell monolayers. Unmodified protein was also detected inside intestinal everted sacs [7]. These findings imply that the protein may be translocated across the intestinal barrier. ...
Interaction with model phospholipid membranes of lupin seed γ-conglutin, a glycaemia-lowering protein from Lupinus albus seeds, has been studied by means of Fourier-Transform infrared spectroscopy at p²H 7.0 and at p²H 4.5. The protein maintains the same secondary structure both at p²H 7.0 and at p²H 4.5, but at p²H 7.0 a higher ¹H/²H exchange was observed, indicating a greater solvent accessibility. The difference in Tm and TD1/2 of the protein at the abovementioned p²H's has been calculated around 20 °C. Infrared measurements have been then performed in the presence of DMPG and DOPA at p²H 4.5. DMPG showed a little destabilizing effect while DOPA exerted a great stabilizing effect, increasing the Tm of γ-conglutin at p²H 4.5 of more than 20 °C. Since γ-conglutin at p²H 4.5 is in the monomeric form, the interaction with DOPA likely promotes the oligomerization even at p²H 4.5. Interaction between DMPG or DOPA and γ-conglutin has been confirmed by turbidity experiments with DMPC:DMPG or DOPC:DOPA SUVs. Turbidity data also showed high-affinity binding of γ-conglutin to anionic SUVs made up with DOPA. The molecular features outlined in this study are relevant to address the applicative exploitation and to delineate a deeper comprehension of the natural functional role of γ-conglutin.
... These findings may help explain the beneficial effects observed in vivo in animal and human studies Duranti et al., 2008). It is, however, useful to add that a recent literature has shown that γ-conglutin, a specific hypoglycaemic lupin protein, is absorbed intact in Caco-2 cells (Capraro et al., 2011). ...
... Diabetes mellitus is a serious health problem with continuously increasing rates of incidence and mortality. Many studies have confirmed the benefits of medicinal plants with hypoglycemic effects in the management of diabetes mellitus [1][2]. The literature review noted that very few isolations and pharmacological activities were reported on Uraria lagopoides (L.) and the whole plant was not yet used in isolation. ...
... Lupin ingredients have been included in a range of highly palatable breads and other baked goods, meat products and beverages [45]. Studies have also indicated the substantial health attributes of lupin such as diets supplemented with lupin grain may play an important role in treating type 2 diabetes, particularly in overweight and obese people, beneficially influence satiety (appetite suppression) and energy balance, improve blood lipids, lower hypertension and improve bowel health [45,46,47,48]. ...
Crops, livestock and trees are major components of farming systems in Ethiopia. Crop production is dominant in Ethiopian agriculture as well as in the farming system. Legumes are among the various crops produced in all regions of the country in different volumes after cereals. More than twelve legume species are grown in the country. Pulses production by volume has been increased by 71.92% for the duration of nearly 20 years and with a growth rate of 3.78% per annum. Area coverage by pulse crops for the same period grown by 53% with a growth rate of 3% per year. Total pulses grain yield, which is volume of production per unit area, showed good increment from 8.79 quintal s per hectare in the cropping year 1994/1995 to 14.76 quintals per hectare in
... This property facilitates its oral administration. Furthermore, Capraro and colleagues [21] demonstrated that Cγ could transit, in an intact form, from the apical to the basolateral membranes of Caco-2 cells, an in vitro model that mimicked the human intestinal epithelium. Similar to glucagon-like peptide-1 (GLP-1), which binds to its pancreatic receptor and stimulates insulin exocytosis [22], we hypothesized that, upon absorption, Cγ may stimulate pancreatic insulin secretion in a similar manner. ...
Several studies support the health-promoting benefits of lupins, particularly lupin proteins. It has been demonstrated that Lupinus albus gamma conglutin (Cγ) protein lowered blood glucose levels; thus, Cγ showed promise as a new anti-diabetic compound for type 2 diabetes (T2D) treatment. The aim of this study was to evaluate the effect of Cγ on Ins-1 gene expression and on pancreatic insulin content in streptozotocin-mediated diabetic rats. Cγ was isolated from Lupinus albus seeds. Its identification was confirmed with polyacrylamide gel electrophoresis under native and denaturing conditions. We used streptozotocin (STZ) to induce T2D on the 5th day of life of newborn male Wistar rats (n5-STZ). After 20 weeks post-induction, these animals (glycemia > 200 mg/dL) were randomly assigned to three groups that received the following one-week treatments: vehicle, 0.90 % w/v NaCl (n5 STZ-Ctrl); glibenclamide, 10 mg/kg (n5 STZ-Glib); or Cγ, 120 mg/kg (n5 STZ-Cγ). Glucose and insulin levels were measured before and after treatment. Ins-1 gene expression was quantified using real time polymerase chain reaction and the pancreatic insulin content was evaluated with immunohistochemistry. Post-treatment, the n5 STZ-Cγ and n5 STZ-Glib groups showed reductions in glucose, increments in serum insulin, and increases in Ins-1 gene expression and beta cell insulin content compared to the n5 STZ-Ctrl group. The results showed that Cγ had beneficial effects on Ins-1 gene expression and pancreatic insulin content. These biological effects of Cγ strengthen its promising potential as a nutraceutical and/or new agent for controlling hyperglycemia.
... Male Wistar rats weighing about 180-220 g were deprived of food for 1 d and provided with only double-distilled water before the experiments. The rats were anesthetized with pentobarbital sodium 40 mg/kg, and the small intestine of rats was dissected out 23 . The intestines were rinsed with ice cold saline (0.9%) in order to discard the intestinal digesta, and the distal ileums of the rat intestines (approximately 15 cm each) were taken. ...
Abstract The aim of this study was to investigate the effect of naringenin on the pharmacokinetics (PK) of felodipine in rats and membrane permeability across rat everted gut sacs in vitro. Rats were simultaneously co-administered with felodipine 10 mg/kg, p.o. and naringenin (25, 50 and 100 mg/kg, p.o.) for 15 consecutive days. Rats of the control groups received the corresponding volume of vehicle. Blood samples were withdrawn from retro-orbital plexus on first day in single dose PK study (SDS) and on 15th day in multiple dosing PK study (MDS). The PK parameters were calculated using Thermo kinetica. The co-administration of naringenin significantly elevated the Cmax and increased the AUCtotal of felodipine in dose-dependent manner. The Cmax of felodipine was increased from 173.25 ± 14.65 to 275.61 ± 44.62 and 223.26 ± 26.35 to 561.32 ± 62.53 ng/mL in SDS and MDS, respectively, at the dose of naringenin 100 mg/kg. The AUCtotal of felodipine was significantly (p < 0.001) increased from 2050.48 ± 60.57 to 3650.22 ± 78.61 and 3276.51 ± 325.61 to 7265.25 ± 536.11 (ng/mL/h) in SDS and MDS, respectively. The permeability of felodipine was increased in presence of naringenin and ritonavir (standard P-glycoprotein (P-gp) and Cytochrome P450 (CYP)3A4 inhibitor). Felodipine is a substrate of CYP3A4, and naringenin was reported to be a modulator of P-gp and CYP3A4. These results suggest that naringenin significantly increased the Cmax and AUC of felodipine is due to P-gp and CYP3A4 inhibition.
... Further studies on the oral administration of c-Conglutin to animal models and healthy humans confirmed its remarkable capacity of decreasing glycaemia [5]. In a study aimed at identifying the metabolic fate of the protein, in vitro and ex vivo approaches showed that the protein can be transcytosed through a CaCo2 cell monolayer and cross the intestinal barrier in an intact form [10]. This set of findings is in line with the traditional medicine claims which include lupin seeds and flours amongst the natural anti-diabetic food products [11]. ...
... This has led to the use of the commercial term 'Australian sweet lupins' for L. angustifolius seed exported from Australia, highlighting that it is safe for both food and feed purposes. There is good evidence of the human health benefits of lupins, including: increasing satiety and appetite reduction for weight loss (Lee et al. 2006), reducing blood pressure (Lee et al. 2009), glucose and insulin response (Capraro et al. 2011), and improving bowel (Johnson et al. 2006) and eye health (Fryirs et al. 2008). Allergenicity is unlikely to impede the use of lupins in human food because a relatively low proportion of the human population is allergic to lupin protein (Peeters et al. 2007;Goggin et al. 2008). ...
The narrow-leafed lupin is a legume with much to offer to agriculture and human wellbeing through its adaptation to N and P deficient, acid, sandy soils, and production of nutritious, very low glycemic index grain with manifold health benefits. However, the industry has exploited only a small fraction of the genetic and adaptive diversity residing in the species, reflecting a short and fragmented domestication history. Given declining global production, unlocking the potential residing in untapped sources of genetic diversity to maximize yield and value is critical for the future of the crop.
To this end a wide range of genetic resources are under evaluation. The Australian Lupin Collection (ALC) comprises almost 4,600 diverse, mostly wild accessions, many of which have been genotyped using DArT markers, and collection sites characterized to facilitate ecophysiology of contrasting material. Additional exotic genetic resources include recombinant inbred line and mutant populations, as well as inter-specific crosses. These resources are being used to investigate specific adaptation, genetic and molecular control of key traits, all of which will be expedited by current efforts to provide a reference genome sequence for L. angustifolius. Genetic base broadening is the current breeding focus, combining distantly related wild and domestic material with elite cultivars in double back- or topcrosses with dramatic effects on yield. In future this will be complemented by marker-based targeted trait introgression to improve narrow-leafed lupin adaptation, quality/value and fit into the farming system.
... Lupine is increasingly recognized as a human health food as the grain is high in protein and dietary fi ber, gluten-free and low in fat, with virtually no starch and thus has a very low glycemic index ( Sipsas, 2008 ). Potential benefi ts to human health for lupines include: satiety and reducing appetite for weight loss ( Lee et al., 2006 ), bowel health ( Johnson et al., 2006 ), hypocholesterolemic activity ( Sirtori et al., 2004 ;Hall et al., 2005 ;Martins et al., 2005 ;Sirtori et al., 2011 ), reduced blood glucose and insulin response ( Hall et al., 2005 ;Capraro et al., 2011 ), reduced blood pressure ( Lee et al., 2009 ), and eye health ( Fryirs et al., 2008 ). Lupines have a number of advantages over the market-dominant protein grain, soybean, with respect to seed quality and nutrition for food end uses ( Wolko et al., 2011 ). ...
Lupines (Lupinus species; Fabaceae) are an ancient crop with great potential to be developed further for high-protein feed and food, cover crops, and phytoremediation. Being legumes, they are capable of symbiotically fixing atmospheric nitrogen. However, Lupinus species appear to be nonmycorrhizal or weakly mycorrhizal at most; instead some produce cluster roots, which release vast amounts of phosphate-mobilizing carboxylates (inorganic anions). Other lupines produce cluster-like roots, which function in a similar manner, and some release large amounts of carboxylates without specialized roots. These traits associated with nutrient acquisition make lupines ideally suited for either impoverished soils or soils with large amounts of phosphorus that is poorly available for most plants, e.g., acidic or alkaline soils. Here we explore how common the nonmycorrhizal phosphorus-acquisition strategy based on exudation of carboxylates is in the genus Lupinus, concluding it is very likely more widespread than generally acknowledged. This trait may partly account for the role of lupines as pioneers or invasive species, but also makes them suitable crop plants while we reach "peak phosphorus".
Soybean seed basic 7S globulin (Bg7S)-like proteins are found in many plant species. Bg7S was originally thought to be a major seed storage protein but was later found to be multifunctional, with stress response, antibacterial activity, hormone receptor-like activity. Moreover, functional differences between Bg7S proteins from legumes and other plants have been revealed. In non-leguminous plants, Bg7S molecules inhibit the invasion of pathogenic microorganisms. However, although leguminous plants have a peptide called leginsulin that can bind to Bg7S, non-leguminous plants do not have leginsulin. Bg7S in leguminous plants and other plants may have evolved in functionally different directions. Several homologs of Bg7S in plants are reported, but there is no homolog of this protein in peas, suggesting that the pea evolution might have followed a different route when compared to other leguminous plants. Although the functions of Bg7S are well documented in plants, recent studies suggest that this protein is also important in controlling blood glucose level, blood pressure and plasma cholesterol level, and cancer cell antiproliferative actions.
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.
Fabaceae is the third largest flowering plant family, commonly known as legumes that are the major source of dietary protein; some are source of oil, fodder, timber, and medicine, and some are ornamental plants. Legume enriches soil health-adding atmospheric nitrogen. This potential crop is often under threat in terms of growth, physiology, developmental processes, and yield potential due to high temperature induced by climate change. There are various approaches to combat high temperature stress. Agronomic approaches and exogenous application of phytoprotectants including osmoprotectants, nutrients, antioxidants, polyamines, trace elements, phytohormone, and signaling molecule are potential ways to mitigate high temperature damages of Fabaceae plants. Few research findings also developed transgenic plants showing improved tolerance to high temperature stress. This chapter accommodates information regarding high temperature effects on Fabaceae plants and different approaches mitigating high temperature induced-damages which will draw the attention of the scientists and researchers and will help them in converting the Fabaceae plants to be more prominent against the threat of climate change.
BACKGROUND
Lupin seeds are rich in proteins, which are utilized in food industry. There is an increased interest in lupin research due to its association with health related benefits, such as reduction of hypertension and hyperglycemia. However, the study on the peptides derived from lupin proteins is rare.
RESULTS
Lupin protein hydrolysates (LPHs) were prepared by proteolysis using alcalase, trypsin and pepsin, respectively. All the hydrolysates demonstrated higher antioxidant and ACE inhibitory activities compared to lupin proteins. The hydrolystes were fractionated into three fractions based on molecular weight (MW), and the peptides with MW < 3 kDa (LPH3) had the highest antioxidant and ACE inhibitory activities compared to other fractions. Cell model study revealed LPH3 fraction had the highest protection against the generation of reactive oxygen species in HepG2 cells, which was associated with increased activities of super oxide dismutase and glutathione peroxidase through up‐regulation of SOD1, GPX1, GCLM, SLC7A11 and SRXN1 expressions.
CONCLUSION
The analysis of amino acids composition indicated that the peptides were characterized with high content of hydrophobic amino acids, which may be responsible for the greatest antioxidant activity. This study highlights the promising potential of lupin peptides as functional ingredient in healthy foods.
Background
Lupin, the largest legume crop in Australia, is gaining global attention because of its unique protein γ-conglutin, which has shown promise as a nutraceutical for controlling blood glucose level and thus reducing the risk of type II diabetes development. Type II diabetes is a chronic condition affecting millions of individuals worldwide, which urgently requires natural side effect free therapies as alternatives to currently used drugs. Purification of γ-conglutin opens up a new avenue for high-value products from lupin seeds as nutraceuticals, the market for which is predicted to reach US$ 250 billion by 2018 (Dutta, Mahabir, & Pathak, 2013).
Scope and approach
Previously, several research groups have reported trials on extracting and purifying proteins from lupin seed. However, most of these methods have focussed on protein isolates as food ingredients. Very few reports have aimed to purify γ-conglutin from the total proteins, but the methods reported are time-consuming and unsuitable for commercial scale production of high purity γ-conglutin due to the involvement of many processing steps for nutraceutical application. Hence there is a need to fully understand all reported γ-conglutin extraction and purification processes in terms of their advantages and limitations, so that an effective scalable purification process for nutraceutical grade γ-conglutin may be designed in the future.
Key findings and conclusions
This article reviews reported extraction and purification methods for γ-conglutin, to provide a basis for the development of novel purification technique/process for this potentially highly valuable protein.
Scope: We have investigated the potential use of β-conglutin protein isoforms from narrow-leafed lupin (Lupinus angustifolius L.) as a diabetes treatment.
Methods and results: We produced purified recombinant β1-, β2-, β3-, β4-, and β6-conglutin proteins and showed that β1, β3 and β6 could bind to insulin. To assess β-conglutin proteins modulatory effect on insulin-activation meditated kinases, whole blood and peripheral blood mononuclear cell (PBMC) cultures from Type 2 diabetes (T2D) and healthy control subjects (C) were incubated with conglutin proteins. Treatment of PBMCs from T2D patients with β1, β3, and β6 proteins increased up to 3-folds mRNA and protein levels of genes important in insulin signalling pathways, namely IRS-1/p85 /AKT/GLUT-4. This was accompanied by a comparable fold-change decrease in the mRNA expression level of pro-inflammatory genes (iNOS and IL-1β) and proteins compared to healthy controls. The β2 and β4 isoforms had no effect on the insulin signalling pathway. However, these β-conglutin proteins elicited pro-inflammatory effects since levels of mRNA and proteins of iNOS and IL-1β were increased.
Conclusion: Our results raise the possibility of using these particular β-conglutin proteins in the prevention and treatment of diabetes, as well as their potential as anti-inflammatory molecules.
Blue lupin is native to southern Europe, Middle East and northern Africa. It has naturalized in parts of Australia, South Africa and North America. It is widely distributed in all Mediterranean countries, from Portugal, south-west France and the Maghreb countries to the Levante and Turkey.
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.
The effects of hesperetin on the pharmacokinetics and the role of P-glycoprotein (P-gp) in the transport of felodipine were investigated in rats and in vitro. Felodipine was administered orally (10 mg/kg) without or with hesperetin (25, 50 and 100 mg/kg) to rats for 15 consecutive days. Blood samples were collected at different time intervals on 1(st) day in single dose pharmacokinetic study (SDS) and on 15(th) day in multiple dose pharmacokinetic study (MDS). The area under the plasma concentration-time curve (AUC0-∞ ) and the peak plasma concentration (Cmax ) of felodipine were dose-dependently increased in SDS and MDS with hesperetin compared to control ( p < 0.001). The half-life (t1/2 ) and mean residence time was longer than the control group in both studies. The role of P-gp determined using everted rat gut sacs in vitro by incubating felodipine with or without hesperetin and verapamil (typical P-gp and CYP3A4 inhibitor). The in vitro experiments revealed that the verapamil and hesperetin increased the intestinal absorption of felodipine (p < 0.01). Concurrent use of hesperetin dramatically altered the pharmacokinetics of felodipine leading to an increase in systemic exposure. The likely mechanism is inhibition of CYP3A4-mediated first-pass metabolism and P-gp in the intestine and the liver. Copyright © 2013 John Wiley & Sons, Ltd.
The present work is to study for the first time the anti-diabetic properties of aqueous extract of roots of Anacyclus pyrethrum L. in normal and streptozotocin (STZ)-induced diabetic rats and to achieve a primary pharmacological screening contained in the aqueous extract. A total of 20 rats including 10 diabetics and 10 normal rats were used for this study. The anti-diabetic activity of aqueous extract of roots was evaluated by using normal and STZ induced diabetic rats at a dose of 250 mg/kg p.o daily for 21 days. Blood glucose levels were measured using GOD-POD. Screening for major classes of phytochemical was done using standard chemical tests. Per oral administration of the aqueous extract of the roots (250 mg/kg body weight) to streptozotocin-induced diabetic rats exhibited a significant antihyperglycemic activity in STZ-induced diabetic rats, whereas in normal rats no hypoglycemic activity was observed. Phytochemical screening showed a wealth in compounds: Tannins, saponins, alkaloids, amino acids, steroids and terpenoids. Aqueous extract of roots exhibit attractive properties and can therefore, be considered a promising candidate for future application as alternative therapeutic agents, particularly in the development of anti-diabetic drugs.
A lupin seed γ-conglutin-enriched preparation was tested in a glucose overload trial with both murine models and adult healthy volunteers. The results with rats showed a dose-dependent significant decrease of blood glucose concentration, which confirmed previous findings obtained with the purified protein. Moreover, three test-product doses equivalent to 630, 315, and 157.5 mg γ-conglutin, orally administered 30 min before the carbohydrate supply, showed a relevant hypoglycemic effect in human trials. Insulin concentrations were not significantly affected. The general hematic parameters did not change at all. This is the first report on the glucose-lowering effect of lupin γ-conglutin in human subjects.
The transport of [14C]-radiolabelled β-lactoglobulin and α-lactalbumin through Caco-2 cell monolayers grown on permeable filters was studied in order to evaluate the different protein pathways through the intestinal epithelium. β-Lactoglobulin or α-lactalbumin (0.25-3 mg/ml) was introduced on the apical side of the monolayer and both the transport and the release of labelled material from the cells were measured following different incubation times. The labelled material was analysed by either trichloroacetic acid precipitation or by high pressure liquid chromatography. Despite some differences between the 2 proteins, the overall mechanism followed approximately the same pattern. Part of the intact internalized protein was either recycled (10-15%) or transportedvia transcytosis (about 5%). Another pathway corresponded to the intracellular degradation of the protein. The calculation of the different routes followed by the proteins indicated that the main part of the degraded fraction (about 70%) was recycled whereas approximately 30% was transported to the other side. Moreover, 5-10% of the endocytosed material was retained intracellularly.
The amino acid absorption from legume protein isolates (from chickpeas, CPI; and lupins, LPI) was studied in in vivo and in vitro experiments in comparison to animal proteins (casein and lactalbumin). In the in vivo experiment on rats, the diets were isoenergetic and isonitrogen (per kg diet 15.5 MJ digestible energy and 150 g protein, respectively). At 150 min after feeding, the concentration of free amino acids in arterial and portal blood plasma of rats fed legume proteins was significantly different (p < 0.05) from rats fed animal protein (lactalbumin). In arterial plasma the concentration for Met, Leu, Trp and Lys in rats fed legume proteins was lower than lactalbumin controls and for Val and Cys higher; in portal plasma, the concentration of free Met, Leu, Trp and Lys was lower, and the concentration of Cys was higher in rats fed legume proteins than in rats fed lactalbumin. The cumulative (total mM at 195 min after ingestion) and net absorption (% of ingested amounts) of Met, Leu, Trp and Lys were higher (p < 0.01) for rats fed lactalbumin as compared to those fed legume protein isolates or casein. In the in vitro study (Caco-2 cell monolayers), after 2 h incubation the transport values of all the individual amino acids in CPI and LPI, except for Glu, Val and Ile, were lower (p < 0.01) than for casein or lactalbumin. The results indicate that amino acids from chickpea and lupin protein isolates are absorbed at slower rates than those from animal proteins, which might explain the lower nutritional utilisation of legume storage proteins as compared with lactalbumin or casein.
Lupin seed is referred to as an antidiabetic product in traditional medicine. Conglutin-γ, a lupin seed glycoprotein, was found to cause a significant plasma glucose reduction when orally administered to rats in glucose overload trials. Conglutin-γ was identified as being responsible for the claimed biological activity, and the aim of this work was to envisage its hypothetical insulin-mimetic cellular mechanism of action. Insulin is responsible for proteosynthesis control through IRS/AKT/P70S6k/PHAS1 pathways modulation, glucose homeostasis through PKC/Flotillin-2/caveolin-3/Cbl activation and muscle differentiation/hypertrophy via muscle-specific MHC gene transcription control.
To assess whether conglutin-γ modulates the same insulin-activated kinases, myoblastic C2C12 cells were incubated after 72 h of differentiation with 100 nM insulin or 0.5 mg/mL (∼10 μM) conglutin-γ. Metformin-stimulated cells were used as a positive control. The effect on the above mentioned pathways was evaluated after 5, 10, 20 and 30 min. In the control cells medium insulin, conglutin-γ and metformin were not added. We demonstrated that insulin or conglutin-γ cell stimulation resulted in the persistent activation of protein synthetic pathway kinases and increased glucose transport, glut4 translocation and muscle-specific gene transcription regulation.
Our results indicate that conglutin-γ may regulate muscle energy metabolism, protein synthesis and MHC gene transcription through the modulation of the same insulin signalling pathway, suggesting the potential therapeutic use of this natural legume protein in the treatment of diabetes and other insulin-resistant conditions, as well as the potential conglutin-γ influence on muscle cells differentiation and regulation of muscle growth.
Bowman-Birk inhibitor (BBI) from soyabeans is a naturally occurring protease inhibitor with potential anti-inflammatory and chemopreventive properties within the gastrointestinal tract (GIT). In a previous paper, we reported that significant amounts of BBI-related proteins reach the terminal ileum functionally and biologically active. We have now investigated: (a) if soyabean BBI is biotransformed by faecal microbiota which would reduce its potential colorectal chemopreventive properties and (b) the potential influence of this protease inhibitor on the modulation of faecal microbiota. In vitro incubation studies of native soyabean BBI at a physiological level (93 microM) with mixed faecal samples of pigs for 24 h at 37 degrees C demonstrated that BBI remains active and its intrinsic trypsin and chymotrypsin inhibitory activities were not significantly influenced by the enzymic or metabolic activity of faecal microbiota. Soyabean BBI did not affect the growth of the different bacterial groups studied (lactobacilli, bifidobacteria, bacteroides, coliforms, enterobacteria, clostridia and total anaerobes). It was concluded that protease inhibitory activities, intrinsically linked to the chemopreventive properties of soyabean BBI, were largely unaffected by faecal microbiota in vitro. BBI retains significance, therefore, as a bioactive compound in the human GIT.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
Certain protease inhibitors are effective at preventing or suppressing carcinogen-induced transformation in vitro and carcinogenesis in animal model systems. One protease inhibitor, the soybean-derived Bowman-Birk inhibitor (BBI) is particularly effective in suppressing carcinogenesis. BBI is a protein of a molecular weight of 8000 with a well-characterized ability to inhibit trypsin and chymotrypsin. BBI has been extensively studied, both as purified BBI and as an extract of soybeans enriched in BBI called BBI concentrate (BBIC). Purified BBI and BBIC have comparable suppressive effects on the carcinogenic process in a variety of in vivo and in vitro systems. BBI appears to be a universal cancer preventive agent. Purified BBI and BBIC suppress carcinogenesis as follows: in 3 different species (mice, rats, and hamsters); in several organ systems and tissue types [eg, colon, liver, lung, esophagus, cheek pouch (oral epithelium), and cells of hematopoietic origin]; and in cells of epithelial and connective tissue origin when given to animals by several different routes of administration, including the diet, leading to different types of cancer (eg, squamous cell carcinomas, adenocarcinomas, and angiosarcomas), and induced by various chemical and physical carcinogens. About half of an oral dose of BBI is taken up into the bloodstream and distributed throughout the body, with excretion via the urine. Pharmacokinetic studies of BBI have been performed in animals with radioactively labeled BBI, whereas antibodies that react with reduced BBI are being used in pharmacokinetic studies in humans. The calculated serum half-life is 10 h in both rats and hamsters. BBIC achieved Investigational New Drug status from the FDA in April 1992 (IND no. 34671; sponsor, Ann R Kennedy), and studies to evaluate BBIC as an anticarcinogenic agent in human populations began. Both BBI and BBIC prevent and suppress malignant transformation in vitro and carcinogenesis in vivo without toxicity.
This work describes the in vitro interaction between a lupin seed protein, namely, conglutin gamma, and insulin. The binding to an insulin-immobilized matrix occurs in the pH range from 7.5 to 4.2 and is strongly affected by ionic strength, suggesting that it is driven primarily by electrostatic interactions. The quantitative parameters of the binding were determined by surface plasmon resonance. On the basis of the conditions required for the interaction to take place and the quantitative binding parameters, it appeared that the interaction is specific, despite the fact that the origin of the two protein molecules is completely different. The effect of the oral administration of conglutin gamma on the glycemic levels of rats subjected to glucose overloading was a statistically significant reduction in glycemia comparable to that of metformin, a well-known glucose lowering drug. These findings represent the first molecular evidence of the possible use of a legume protein in the control of glycemia.
The AIN-93 rodent diets were formulated to substitute for the previous version (AIN-76A) and to improve the performance of animals that consume them. They are called AIN-93G, formulated for growth, and AIN-93M, for maintenance. Major changes included substituting cornstarch for sucrose and soybean oil for corn oil and increasing the amount in order to supply both essential fatty acids (linoleic and linolenic). L-Cystine was substituted for DL-methionine to supplement the casein component. The mineral mix was reformulated to lower the amounts of phosphorus, manganese and chromium, to increase the amount of selenium, and to add molybdenum, silicon, fluoride, nickel, boron, lithium and vanadium. The amounts of vitamins E, K-1 and B-12 were increased over those in the AIN-76A vitamin mix. The AIN-93G diet contains 200 g of casein and 70 g of soybean oil/kg diet. The maintenance diet (AIN-93M) contains 140 g of casein and 40 g of soybean oil/kg diet. The 1993 diets have a better balance of essential nutrients than the 1976 diet and are better choices for studies with laboratory rodents.
The seed globulins of Lupinus albus were extracted and 12 ditterent proteins were separated: four of them correspond to vicilins and two to legumin
Basic 7S globulin in soybean seeds is a cysteine-rich glycoprotein with insulin-binding activity, and has 27-and 16-kDa subunits linked by disulfide bonds. After TV-glycanase treatment, the 27-kDa subunit did not react with insulin, but the 16-kDa subunit still did. Soybean seeds release much basic 7S globulin when immersed in hot water. Protein kinase activity was detected in the proteins released. The protein kinase activity of the globulin was measured by the phosphorylation method in a gel. The activity was conferred by the 16-kDa subunit. The 16-kDa subunit and the sugar chain of the 27-kDa subunit could bind with insulin, whereas protein kinase activity seemed to be associated with the 16-kDa subunit. © 1994, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.
This review examines the use of Caco-2 monolayers in the prediction of intestinal drug absorption. First, the different routes of drug transport in Caco-2 monolayers are compared with those seen in vivo. Second, the prediction of drug absorption in vivo from transport experiments in cell monolayers is discussed for different classes of drugs. Finally, the use of Caco-2 monolayers as a reference model in physico-chemical and theoretical predictions of drug absorption is discussed. We conclude that Caco-2 monolayers can be used to identify drugs with potential absorption problems, and possibly also to select drugs with optimal passive absorption characteristics from series of pharmacologically active molecules generated in drug discovery programs.
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,
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.
Leguminous seeds are a valuable source of food proteins. A reappraisal of the beneficial effects of legume seed dietary intake, which are the basis for various health claims, is currently taking place. Proteins and peptides concur in the observed biological/functional activities of legume seeds. However, the molecular determinants of these beneficial activities in most cases are far from being understood. A multidisciplinary research work, including the characterisation at molecular level of these molecules and the evaluation of their biological activities, is crucial to unveil food protein nutri-functional properties and optimise their exploitation. These are the approaches of the molecular nutraceutics.
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.
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.
In the present study, the effect of free amino acid (FAA) diets on the intestinal absorption rate of methionine and leucine was studied 'ex vivo' with rats adapted for different periods of time to the diets, using the everted sac method. The adaptation period to the 21% FAA diet with an amino acid content based on casein was either, 0 (no adaptation, N-ADA), 5 (short-term adaptation, ST-ADA), or 26-33 days (long-term adaptation, LT-ADA). Within the ST-ADA and the LT-ADA groups, three different levels of methionine were included: 50%, 100% and 200% of the level normally present in casein. All diets were iso-nitrogenous and iso-caloric. After the adaptation period (0, 5, or 26-33 days), intestinal everted sacs were prepared. Methionine or leucine was added to the medium as transport substrate. The methionine absorption rate of the rats of the LT-ADA groups was higher than that of the N-ADA groups. Furthermore, adaptation to 200% dietary methionine levels caused a significantly slower leucine absorption compared to the 100%, and 50% group. Methionine absorption was similar in the 100% and 200% groups, but the absorption of methionine in the 50% group was enhanced in the distal part of the intestines. We concluded that in response diets with 21% FAAs as only amino acid source, amino acid absorption is decreased to avoid toxic effects of high levels of methionine in the circulation.
There is now no reasonable doubt that small quantities of intact proteins do cross the gastrointestinal tract in animals and adult humans, and that this is a physiologically normal process required for antigen sampling by subepithelial immune tissue in the gut. It is too small to be nutritionally significant in terms of gross acquisition of amino-nitrogen, but since it has important implications relating to dietary composition it must receive consideration from nutritionists. The process of intact protein absorption occurs without eliciting harmful consequences for most individuals, but it appears likely that a small number of people absorbing these "normal" amounts may react idiosyncratically; also, some individuals may absorb excessive amounts, and they may suffer clinically significant consequences. Likewise, individuals with diminished absorption of intact protein may be at risk. Normal absorption probably occurs predominantly by transcellular endocytosis with some possible contribution by a route between cells; increased net entry of protein to the circulation may reflect (a) increased paracellular (intercellular) passage, (b) increased transcellular passage, and/or (c) decreased lysosomal proteolysis. Tests to distinguish among these possibilities are strongly desirable. Intact protein absorption may be involved in the pathogenesis of inflammatory bowel disease, "food allergies," and other diseases, including even major psychiatric disorders, but the current evidence is mainly indirect and suggestive. Great caution and careful objective studies are needed to establish whether such relationships with disease do exist and to unravel the underlying basic physiological mechanisms. Now that interest has developed in the assessment of intestinal permeability to small- and medium-sized molecules, it is hoped that equally simple methods for studying macromolecular permeability will be developed and applied. Therapeutic methods for enhancing intact polypeptide absorption would be valuable for vaccine and peptide drug administration by the oral route. Therapeutic reduction of the process may be relevant in food-sensitive patients.
Intact, biological active insulin and pancreatic RNase can be absorbed from the intestinal lumen into the blood circulation. The absorption is dependent on the addition of bile acid (sodium cholate) and proteinase inhibitor. The quantitative absorption of insulin and pancreatic RNase has been demonstrated in an in situ model. The amount of insulin absorbed after 30 min from the ileum to the mesenteric vein was 0.025% of the initial amount. Sodium cholate (10 mg/ml) and 3000 KIU/ml aprotinin enhanced this absorption by 30 times. The amount of pancreatic RNase which was absorbed from the ileum to the blood was 0.002% of the initial amount during 30 min. Sodium cholate (10 mg/ml) and 3000 KIU/ml aprotinin increased the absorption by a factor of 200. No damage occurred to the intestine during the experimental procedures. The sieving characteristics of the intestinal wall were not altered by the presence of sodium cholate and proteinase inhibitor in the intestinal lumen. These results suggest that sodium cholate and proteinase inhibitors can facilitate the absorption of intact, biologically active proteins across the intestinal wall.
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.
Native glycosylated and enzymically deglycosylated conglutin gamma (a lupin seed oligomeric protein) both showed an unusual resistance to tryptic degradation. The result of this treatment was that a single 40-residue peptide was cleaved from the N-terminus of conglutin gamma light subunit. Acid treatment of the two protein forms led to their substantial unfolding, as indicated by CD spectra. After this treatment, both polypeptides were completely degraded by trypsin after a few minutes of incubation. Conversely, trypsin pulse experiments run under renaturing conditions demonstrated a different refolding behaviour of the two proteins: the glycosylated form became resistant to trypsin after a 7-h renaturation, while the deglycosylated form required 42 h renaturation. These results were confirmed by CD spectra and reverse-phase HPLC analyses of the glycosylated and deglycosylated conglutin gamma forms. Therefore, it was concluded that the saccharide chain of conglutin gamma increased the rate of formation of a trypsin-resistant conformation upon refolding of the acid-treated protein, without playing any direct role in the protection of the native protein from proteolysis.
The transport of [14C]-radiolabelled beta-lactoglobulin and alpha-lactalbumin through Caco-2 cell monolayers grown on permeable filters was studied in order to evaluate the different protein pathways through the intestinal epithelium. beta-Lactoglobulin or alpha-lactalbumin (0.25-3 mg/ml) was introduced on the apical side of the monolayer and both the transport and the release of labelled material from the cells were measured following different incubation times. The labelled material was analysed by either trichloroacetic acid precipitation or by high pressure liquid chromatography. Despite some differences between the 2 proteins, the overall mechanism followed approximately the same pattern. Part of the intact internalized protein was either recycled (10-15%) or transported via transcytosis (about 5%). Another pathway corresponded to the intracellular degradation of the protein. The calculation of the different routes followed by the proteins indicated that the main part of the degraded fraction (about 70%) was recycled whereas approximately 30% was transported to the other side. Moreover, 5-10% of the endocytosed material was retained intracellularly.
SDS/PAGE, immune blotting with specific antibodies and amino acid sequence analyses revealed that 90% of the protein released from Lupinus albus seeds incubated in water at 60°C for about 3 h was conglutin γ, a putative storage glycoprotein already present in the protein bodies of mature seeds. Incorporation of [14C]leucine into the protein demonstrated that conglutin γ was newly synthesized during the treatment and the use of protein synthesis inhibitors ruled out the secretion of constitutive conglutin γ. Synthesis and secretion took place only over a narrow temperature range, 57.5–62.5°C, and in a short time interval, 135–180 min, of incubation of the seed. The amount of secreted conglutin γ, i.e. 1 mg/seed, was about three times that present inside the treated or untreated seed.
Secreted conglutin γ contained covalently linked carbohydrate as well as the constitutive protein. Inhibition of the glycosylation by tunicamycin did not affect conglutin γ synthesis, but prevented its secretion from the seed, as indicated by quantifying conglutin γ remaining in the seed. An accumulation of the protein outside the protein bodies and at the cotyledonary cell periphery was shown in these samples by immunocytochemistry.
Peptide mapping of the fragments obtained by incubation of constitutive and secreted conglutin γ with trypsin and pepsin revealed no difference between the two proteins.
Lupin seeds were still viable after the treatment. However no similarities between conglutin γ and heat-shock proteins were observed either in the amino acid sequence or other molecular features.
In order to investigate the fate of orally administered proteins, the absorption of ovalbumin (OVA) from the gastrointestinal tract into both the blood and lymph circulation was quantitatively evaluated. After oral administration, a significant amount of intact OVA was detected in both the plasma and the lymph fluid by means of a two-site enzyme immunoassay. The extent of absorption into the plasma, calculated from the area under the plasma concentration versus time curve of OVA after oral and intravenous administration, was only 0.007-0.008% of the dose. This value is extremely low compared to that after nasal administration, showing the stronger barrier function of the gastrointestinal tract against the invasion of macromolecular proteins into the body. The extent of absorption into the lymph was dose-dependent (0.0007-0.002% of dose), and a higher dose leads to a higher fraction of OVA absorbed into the lymph. Moreover, it was demonstrated that not only the small intestine but also the stomach can absorb OVA. OVA absorbed from the stomach was transferred almost exclusively to the blood circulation, which suggests different mechanisms and/or routes of absorption between the stomach and the small intestine. In order to improve the low oral absorption, OVA was incorporated in liposomes and administered orally. Although the effect of liposomes was not significant, it increased OVA absorption into both the plasma and lymph by about 2 to 3-fold. It was considered that the liposomes suppressed the enzymatic degradation of OVA and released it slowly in the gastrointestinal tract.
This review examines the use of Caco-2 monolayers in the prediction of intestinal drug absorption. First, the different routes of drug transport in Caco-2 monolayers are compared with those seen in vivo. Second, the prediction of drug absorption in vivo from transport experiments in cell monolayers is discussed for different classes of drugs. Finally, the use of Caco-2 monolayers as a reference model in physico-chemical and theoretical predictions of drug absorption is discussed. We conclude that Caco-2 monolayers can be used to identify drugs with potential absorption problems, and possibly also to select drugs with optimal passive absorption characteristics from series of pharmacologically active molecules generated in drug discovery programs.
Purified legume storage proteins (chickpea 11S and 7S globulins, faba bean globulins, and lupin globulins) and casein (casein) were subjected to an in vitro enzyme (pepsin + pancreatin) digestion process. Protein digests were then used in a bicameral Caco-2 cell culture system to determine amino acid transport across the cell monolayer. With digests from legume proteins, absolute amounts of aspartate, glycine, and arginine transported were higher than those found in digested casein, whereas amounts of glutamate, proline, tyrosine, valine, and lysine were lower. However, proportions of amino acids in the basolateral chamber as compared with amounts added in the apical chamber were lower than casein controls for all amino acids except cystine. Results confirm previous in vivo observations that amino acids from legume proteins are probably absorbed at rates different from those in other proteins of animal origin such as casein.
The allergenicity of seed storage proteins, the major components of edible legume seeds, may cause serious reactions in both children and adult population. Updated methodologies for evaluation of the activity of these proteins are needed. In this paper we used two-dimensional (2D) electrophoretic techniques to investigate the immuno-cross-reactivities of anti Ara h 3 basic subunit IgG to the seed proteomes of three legume species, namely, peanut, soybean, and lupin. The seed proteins, extracted with two different procedures, were separated by 2D electrophoresis, and the electrophoretic maps were analyzed by Western blot. In peanut proteome the antibodies strongly reacted with the 23 kDa polypeptides, corresponding to Ara h 3 basic isoforms, the antigen they were raised to, and three unidentified acidic polypeptides near 45 kDa. Remarkable cross-reactivities with lupin and soybean Ara h 3 homologous polypeptides and nonrelated proteins, namely, lupin conglutin gamma and soybean Bg7S, were detected. Therefore, these proteins may be regarded as new putative allergens. The present findings show the potentiality of 2D electrophoresis in the identification of food allergens and open the way to the traceability of the new cross-reacting proteins in the food chain.
The mechanisms by which food allergens are absorbed and sensitized via the gastrointestinal tract have not been well characterized. In this study, the gastrointestinal absorption of a major soybean allergen, Gly m Bd 30K, in young and older mice, and the effects of dietary fat and exogenous emulsifier were investigated. In Expt. 1, Gly m Bd 30K [0, 500 or 2000 mg/kg body weight (BW)] was administered orally to 24-d-old mice, and blood was sampled at various time points over a 120-min period. Plasma Gly m Bd 30K was measured by sandwich ELISA and immunoblotting. Its concentration peaked at 30 min and was dose dependent. Intact Gly m Bd 30K and its 20-kDa fragments were identified in plasma after absorption. In Expt. 2, 24-d-old mice administered soy milk containing 1 mg Gly m Bd 30K showed a steady increase in plasma Gly m Bd 30K from 60 to 120 min that was significantly higher than that in 10-wk-old mice. In Expt. 3, when corn oil (5 or 30%) was coadministered with Gly m Bd 30K (2000 mg/kg BW) to 24-d-old mice, the plasma concentration increased significantly and generally reached a plateau after 30 min. The absorption after the coadministration of 30% corn oil and 3% sucrose fatty acid ester was higher than after the administration of 30% corn oil alone. Intact Gly m Bd 30K and its fragments that were < 20 kDa survived digestion and were absorbed into the blood. We propose that absorption was enhanced by fat carrier-mediated transport.
The Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybean has been described as a potential cancer chemopreventive agent. We have compared the effects of BBI with those of two variant recombinant pea (Pisum sativum L.) seed protease inhibitors, rTI1B and rTI2B, homologous to BBI but differing in inhibitory activity, on the growth of human colorectal adenocarcinoma HT29 cells in vitro. A significant and dose-dependent decrease in the growth of HT29 cells was observed using all protease inhibitors, with rTI1B showing the largest decrease (IC50 = 46 microM). Inclusion of the pan-caspase inhibitor, Boc-D-FMK, did not negate the effects of rTI1B or rTI2B in the cell assays. The relative effectiveness of rTI1B and rTI2B may correlate with a variant amino acid sequence within their respective chymotrypsin inhibitory domain, in agreement with a chymotrypsin-like protease as a potential target.
The well-established murine model of IgE-mediated food allergy, based on oral administration of antigen and cholera toxin (CT), has within the previous years been used to evaluate various food proteins. Nonetheless, little knowledge on the factors that determine the allergenicity of food proteins is available so far. The use of proteins from the legume seed Lupinus albus as food ingredients calls for an evaluation of their allergenic potential, and therefore, we applied the cited model to investigate the putative allergenicity of three lupin protein preparations representing different matrices in which the four types of conglutins are present in varying concentrations.
Weekly, BALB/c A mice were orally immunized with the three lupin protein products together with CT. Total specific antibodies and IgE were determined by ELISA and Western blotting.
A dose-dependent Ig response against the analyzed proteins was observed for all three lupin products, while IgE responses against conglutins beta, gamma and delta, but not against conglutin alpha, were primarily detected after oral administration of lupin flakes. Whereas no differences among the samples for total specific Ig responses were seen, orally administered lupin flake extracts were much more efficient in inducing a conglutin-specific IgE response compared with fractionated lupin protein products.
Although the lupin-specific Ig response induced by coadministration of CT and lupin proteins appears to be dose dependent, the IgE response appears to depend merely on some intrinsic properties of the proteins as well as some factors of the protein matrix.
We have investigated the absorption rates of two purified major allergen 2S albumins, Ber e 1 from Brazil nuts (Bertholletia excelsa Humb. & Bonpl.) and Ses i 1 from white sesame seeds (Sesamum indicum L.), across human intestinal epithelial Caco-2 cell monolayers following gastrointestinal digestion in vitro. The transport from apical to basolateral side in cell monolayers was evaluated by RP-HPLC-UV and indirect competitive ELISA methods, being confirmed by western-blotting analysis. Significant amounts (approximately 15-25 nmol micromol(-1) initial amount/h) of intact Ber e 1 and Ses i 1 were found in the basolateral side. The absorption rates of both plant allergens through the cell monolayer were shown to be constant during the whole incubation period (4 h at 37 degrees C), verifying that the permeability of the membrane was not altered by the allergen digests. Our findings revealed that both purified 2S albumin allergens may be able to survive in immunologically reactive forms to the simulated harsh conditions of the gastrointestinal tract to be transported across the Caco-2 cell monolayers, so that they would be able to sensitize the mucosal immune system and/or elicit an allergic response.
Because intestinal absorption of food protein can trigger an allergic reaction, the effect of wheat proteins on intestinal epithelial cell permeability was evaluated and the abilities of these proteins in native or pepsin-hydrolyzed state to cross the epithelial cell monolayer were compared. Enterocytic monolayers were established by culturing Caco-2 cells, a model of enterocytes, on permeable supports that separate the apical and basal compartments. Proteins were added into the apical compartment, and the transepithelial resistance (TER) was measured; proteins that crossed the cell monolayer were detected in the basal medium by ELISA. Wheat proteins did not alter the cell monolayer. TER and Caco-2 cell viability were conserved, and the passage of dextran was prevented. Native and pepsin-hydrolyzed forms of omega5-gliadin and lipid transfer proteins were detected in the basal medium. The results suggest that these two major allergens in food allergy to wheat were able to cross the cell monolayer by the transcellular route.
The aim of this study was to evaluate two in vitro models, Caco-2 monolayer and rat intestinal mucosa, regarding their linear correlation with in vivo bioavailability data of therapeutic peptide drugs after oral administration in rat and human. Furthermore the impact of molecular mass (Mm) of the according peptides on their permeability was evaluated.
Transport experiments with commercially available water soluble peptide drugs were conducted using Caco-2 cell monolayer grown on transwell filter membranes and with freshly excised rat intestinal mucosa mounted in Using type chambers. Apparent permeability coefficients (P
app) were calculated and compared with in vivo data derived from the literature.
It was shown that, besides a few exceptions, the Mm of peptides linearly correlates with permeability across rat intestinal mucosa (R
2 = 0.86; y = −196.22x + 1354.24), with rat oral bioavailability (R
2 = 0.64; y = −401.90x + 1268.86) as well as with human oral bioavailability (R
2 = 0.91; y = −359.43x + 1103.83). Furthermore it was shown that P
app values of investigated hydrophilic peptides across Caco-2 monolayer displayed lower permeability than across rat intestinal mucosa. A correlation between P
app values across rat intestinal mucosa and in vivo oral bioavailability in human (R
2 = 0.98; y = 2.11x + 0.34) attests the rat in vitro model to be a very useful prediction model for human oral bioavailability of hydrophilic peptide drugs.
Presented correlations encourage the use of the rat in vitro model for the prediction of human oral bioavailabilities of hydrophilic peptide drugs.
Hypoglycaemic effect of c-conglutin, a white lupin (Lupinus albus, L.) seed protein, in healthy volunteers
- J C Bertoglio
- M A Calvo
- J L Hancke
- R A Burgos
- A Riva
- P Morazzoni
Bertoglio, J. C., Calvo, M. A., Hancke, J. L., Burgos, R. A., Riva, A., Morazzoni, P., et al.
(2011). Hypoglycaemic effect of c-conglutin, a white lupin (Lupinus albus, L.)
seed protein, in healthy volunteers. European Journal of Nutrition, submitted for
publication.