Obtención de inhibidores de la Dipeptidil Peptidasa-IV del grano de Amaranto (Amaranthus hypochondriacus L.) y su efecto en la Glucemia de ratones Diabéticos
Amaranth (Amaranthus hypochondriacus L), is a pseudocereal that shows protein content between 16-20%, this protein content is higher than the one found in most common cereals. Because of its remarkable protein content, amaranth has been subjected to several studies to find encrypted peptide sequence...
| Autor: | |
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| Tipo de recurso: | tesis de maestría |
| Estado: | Versión publicada |
| Fecha de publicación: | 2017 |
| País: | México |
| Institución: | Universidad Autónoma Metropolitana |
| Repositorio: | Repositorio Institucional de la UAM Iztapalapa |
| Idioma: | español |
| OAI Identifier: | oai:bindani.izt.uam.mx:fj2362114 |
| Acceso en línea: | https://doi.org/10.24275/uami.fj2362114 |
| Access Level: | acceso abierto |
| Palabra clave: | info:eu-repo/classification/LEM/Amaranto -- Investigación info:eu-repo/classification/LEM/Glicemia info:eu-repo/classification/LEM/Amaranths -- Research info:eu-repo/classification/LEM/Diabetes -- Tratamiento alternativo info:eu-repo/classification/LEM/Diabetes -- Alternative treatment info:eu-repo/classification/LEM/Blood sugar info:eu-repo/classification/cti/6 |
| Sumario: | Amaranth (Amaranthus hypochondriacus L), is a pseudocereal that shows protein content between 16-20%, this protein content is higher than the one found in most common cereals. Because of its remarkable protein content, amaranth has been subjected to several studies to find encrypted peptide sequences that may possess biological activities. However, just a few bioactive amaranth peptides have been identified and characterized. Some in silico and in vitro experiments have shown that these encrypted peptide sequences in the amaranth protein fraction are bioactive molecules with activities such as: antithrombotic, antihypertensive, anticarcinogenic, antioxidant, antiglycemic, among others. The antiglycemic property of amaranth peptides is related with the ability of these bioactive peptides to act as an inhibitor of the dipeptidyl peptidase IV (DPP-IV) enzyme. This enzyme is heavily involved in the postprandial glycemic regulation in humans. Considering the pharmacological potential of hydrolyzed amaranth peptides with biological activity, the objective of this work was: (a) to test, in vitro and in vivo, the ability of different amaranth peptides, derived from the hydrolysis of amaranth, to inhibit the enzymatic activity of DPP-IV; (b) to clarify the mechanism of inhibition of DPP-IV by these amaranth peptides; (c) to characterize and partially purify these hydrolyzed peptides by gel filtration chromatography; (d) to identify the relationship between amaranth peptides inhibitory effect and their relative molecular mass; and finally (e) to test the biological activity of hydrolyzed amaranth peptides in an in vivo system, by administration of the hydrolysed amaranth protein extracts to STZ-induced diabetic mice, and evaluation of glucose tolerance, insulin and glucagon concentrations. Four protein fractions were found in the amaranth seeds. The three main fractions were: Alb1 46%, Glu 22% and Glo 12%. An additional minor fraction was identified as Pro (<1%). These protein fractions made up to 83% of the total protein found in the amaranth seeds analyzed. Enzymatic hydrolysis of amaranth protein by using alcalase was extensive, however, the Glu fraction was easily hydrolysed, this fraction showed a degree of hydrolysis of 75 ± 0.92% at 24 h. On the other hand, Alb1 was highly resistant to the alcalase activity. This fraction required a E/S= 0.8 AU/g and showed a degree of hydrolysis of 43 ± 0.34% at 48 h. SDSprotein profiles indicated that the hydrolysis of the different amaranth protein fractions was faster and more effective on the proteins having a higher molecular mass when the enzymatic hydrolysis by alcalase was used. Conversely, proteins showing a molecular mass between 2024 kDa were more resistant to the alcalase activity. After 24 h of hydrolysis, the fraction with the best capacity to inhibit DPP-IV was Glu (85 0.2%), meanwhile, Alb1 and Glo showed an inhibitory effect of 45 0.2% and 63 0.5%, respectively, after 48 h of hydrolysis. The hydrolyzed amaranth protein extracts that showed the highest inhibition capacities were partially purified by molecular exclusion chromatography. It was found that peptides having a molecular mass of 0.86, 0.65 and 0.45 kDa, isolated from the fractions Alb1, Glo and Glu, respectively, were more effective to inhibit DPP-IV in the in vitro experiments. The IC50 for the Alb1, Glo and Glu peptides were 1.98 0.01, 0.25 0.04 and 0.12 0.006 mg/mL, respectively. Based on these results it was found that there was an inverse relationship between molecular mass and the inhibitory effect of the isolated peptides on the activity of DPP-IV. Moreover, these three peptides inhibit DPP-IV by a competitive mechanism. The most potent peptide, tested in the in vitro experiments, was Glu III (0.45 kDa). This peptide showed a catalytic efficiency of 5.6 x 10-6 min-1, Km´= 0.75 mM and a Ki= 0.11 mg/mL. Results obtained from the acute administration of amaranth protein fractions in the in vivo experiments, showed that the protein fraction having the most positive effect on reducing postprandial glycaemia (around 26% higher compared to the control group) was the Glu fraction. When 300 mg Glu/kg were administer to the mice units (having a glucose oral charge of 2g/kg), the insulin released increased three times (1.2 ng/mL) and glucagon concentration fell 30% compared to the control units (p <0.05). Sub-acute administration (30 day period) of amaranth protein fractions showed that the Glu fraction was the most effective to decrease postprandial glucose up to 17%. The protein fraction also increased the insulin content to 2.25 ng/L in the in vivo units tested, however, no significant changes were found on plasma glucacon levels. Hydrolyzed amaranth protein extracts obtained from the Glu fraction by alcalase hydrolysis and under the conditions described in the present work, could be used to develop functional foods having an inhibitory effect on DPP-IV. These functional food ingredients could help to treat diabetes type 2 and improve the quality of life of patients suffering from this type of diabetes. Nowadays, amaranth is not considered as a high value-added crop. However, the identification and characterization of biomolecules with biological activity, like the ones reported in this work will help to make a better use of amaranth nutritional properties and to spread the cultivation of this plant. |
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