Efeito vasodilatador de um hidrolisado de soro de leite (Whey protein) após digestão in vitro: importância do endotélio e do óxido nítrico

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia, Florianópolis, 2019.A hipertensão arterial sistêmica ocorre quando mecanismos fisiológicos de controle da pressão se encontram ineficientes. Essa condição afeta 32,5% de adultos no Brasil. Devido a disfunção arterial contribuir para a hipertensão, a procura por novos agentes que possuam atividade nos vasos se mantém foco de muitos estudos. Dessa maneira, os efeitos vasculares de peptídeos derivados de alimentos têm sido amplamente investigados. Dentre eles, peptídeos derivados do soro de leite são descritos como inibidores da enzima conversora de angiotensina (ECA). O presente estudo teve como objetivo investigar a hipótese de que metodologias de digestão in vitro poderiam potenciar o efeito vasodilatador de peptídeos derivados de soro de leite. Além do mais, o mecanismo de ação envolvido nesta atividade vasodilatadora foi explorado em vasos de condutância e resistência, em ratos. Nossos resultados revelaram que as frações de hidrolisado de soro de leite submetidas à digestão in vitro (denominado HDE) exerceram relaxamento concentração-dependente com maior potência em aorta e artérias mesentéricas, quando comparado à hidrolisados não digeridos. Ademais, o relaxamento máximo induzido por HDE foi reduzido de 94 ± 7% em anéis com endotélio intacto, para 10 ± 9% em anéis sem endotélio funcional, indicando que esse relaxamento é dependente de endotélio. Notavelmente, a incubação de anéis de aorta com 50 ?g/mL de HDE não interferiu na resposta contrátil à angiotensina II após adição de angiotensina I, sugerindo que não há atividade inibitória sobre a ECA. Por outro lado, o relaxamento vascular induzido por HDE em aorta foi abolido por L-NAME (inibidor das sintases de óxido nítrico), c-PTIO (sequestrador de NO) e ODQ (inibidor da guanilato ciclase solúvel, bem como pelo TEA (bloqueador de canais de potássio ativados por cálcio). Por outro lado, apesar do ODQ bloquear o relaxamento induzido por HDE em artérias de resistência, apenas uma inibição parcial foi obtida após incubação com L-NAME. Além disso, em artérias mesentéricas, o TEA não foi capaz de reduzir a atividade de HDE. Esses achados confirmam a hipótese de que produtos obtidos após digestão de hidrolisados de soro de leite induzem vasorrelaxamento com maior potência comparado com hidrolisados não digeridos. Esse efeito não parece ser relacionado à inibição da ECA e ocorre de maneira dependente de endotélio. Apesar da diferença encontrada, o relaxamento induzido por HDE é ao menos em parte relacionado à ativação da via da via NO/guanilato ciclase em aorta e artérias de resistência. São necessários estudos adicionais para explorar a eficácia desses produtos em modelos in vivo.Abstract: Systemic arterial hypertension occurs when physiological mechanisms of blood pressure control are ineffective. This condition affects about 32,5% of adults in Brazil. Because arterial dysfunction contributes to hypertension, the search for new agents with activity in vessels remains the focus of several studies. In this way, the vascular effects of food-derived peptides have been widely investigated in the last decades. Among them, peptides derived from whey protein are described as inhibitors of angiotensin-converting enzyme (ACE). The present study was designed to investigate the hypothesis that in vitro digestion approaches could potentiate the vasodilatory effects of whey protein-derived peptides. Besides, the mechanism of action involved in such vasodilatory action was further explored in both conductance (aorta) and resistance (small mesenteric) arteries from rats. Our results revealed that the whey protein hydrolysed fractions subjected to in vitro digestion (here named HDE) present enhanced concentration-dependent vasodilatory effect in both aorta and small mesenteric arteries, compared with non-digested hydrolysates. Besides, the maximal relaxation induced by HDE was reduced from 94 ± 7% in endothelium-intact to 10 ± 9% in endothelium-denuded aortic rings, indicating that this relaxation was highly dependent on endothelial function. Notably, the previous exposure of aortic rings to 50 ?g/mL HDE did not prevent angiotensin II-mediated contractile responses obtained after addition of angiotensin I, suggesting the lack of any inhibitory effect on ACE activity. On the other hand, HDE-induced vascular relaxation in aortic rings was abolished by L-NAME (a nitric oxidase synthase inhibitor), c-PTIO (a nitric oxide scavenger), and ODQ (a soluble guanylate cyclase inhibitor), as well as by TEA (a non-selective calcium-activated potassium channel blocker). Differently, although ODQ did block the relaxation induced by HDE in small mesenteric arteries, only partial inhibition was found after incubation with L-NAME. Moreover, in these resistance arteries, TEA failed to reduce the activity of HDE. These findings confirm the hypothesis that products obtained after digestion of whey protein hydrolysates can induce vascular relaxation with enhanced potency, compared with non-digested products. This effect does not appear to be associated with ACE inhibition and occurs in an endothelium-dependent manner. Moreover, despite the differences found, HDE-induced relaxation is at least in part associated with the activation of the nitric oxide/guanylate cyclase pathway in both conductance and resistance arteries. Additional experiments must be carried out to explore the efficacy of these products in in vivo models

Gastrointestinal digestion improving the antihypertensive activity of whey peptides

Whey proteins are byproducts of dairy industry, known for presenting bioactivity in several biological systems, including the cardiovascular. Its peptides are able to exert antihypertensive activity directly via the intestinal lumen and also after absorption. Despite all knowledge concerning whey peptides, there are only few products commercially available with this proposal, emphasizing the need of more studies regarding this theme. Aiming to make a better use of this agroindustrial byproduct, together with the need to meet the demands of this market, the present study proposed to evaluate the in vitro vasorelaxant activity of a whey hydrolysate during gastrointestinal digestion. A whey protein concentrate solution (1.25%w/w) was hydrolysed by a low-cost commercial pepsin (1.91%w/w, 37°C, 3h). The hydrolysate obtained was then spray-dried and submitted to in vitro static digestion, including oral, gastric and enteric phases. Samples were collected to chemical analysis and in vitro biological assays, using rat aortic rings. Non digested samples showed varied peptide profiles, with complete α-Lactalbumin hydrolysis and partial β-Lactoglobulin hydrolysis. During gastric digestion, more than 70 peptides were formed, ranging from high to low polarity and a β-Lactoglobulin hydrolysis was also observed. After the addition of pancreatin, peptides were extensively hydrolysed and 2 intense peaks were formed in 2.87 and 9.75min of a RP-HPLC run. The biological assays corroborate with the chromatographic analysis, as the vasorelaxation increased during the digestive process. Non digested samples showed a vasorelaxant activity of 80.45% ±7.12 (10mg.mL-1). This biofunctionality was maintained during in vitro gastric digestion (86.19% ±10.87; 10mg.mL-1, p>0.05), however an increase of approximately 100 times-fold was observed after the enteric phase, achieving 91.59% ± 9.90 (100μg.mL-1) of vasorelaxation. The results showed that the whey hydrolysate with reduced cost of production presented a high vasorelaxant activity after gastrointestinal digestion, which may be of great interest for food and nutraceutical industries

Optimizing the development of an antihypertensive whey hydrolysate in semi-pilot scale

Whey proteins and peptides are byproducts of dairy industry recognized for reducing risk factors of numerous diseases, including hypertension Despite the need of better use of this agroindustrial residue and the high prevalence of hypertension worldwide, whey based-products that are able to modulate blood pressure are very scarce in food and nutraceutical markets, emphasizing the need of more research regarding this theme. The development of new products requires several stages in which scaling-up the production is comprised. In this manner, the present study proposed to optimize the process of development of a new antihypertensive whey hydrolysate in semi-pilot scale, assessing the influence of drying technologies and enzyme inactivation conditions on its biofunctionality. A commercial pepsin (1.91% w/w; 0.28μU.mL-1) was used to catalyse the hydrolysis of a 88% whey protein concentrate solution (1.25%; pH2, 1M HCl; 20L). Reactions occurred for 3h at 37°C, then the enzyme was inactivated either by increasing the temperature to 80°C/5min or the pH to 7 (5M NaOH).The drying method (freeze- and spray-drying) was also assessed concerning the vasorelaxant activity, which was evaluated in rat aortic rings. Peptide profiles of the samples inactivated by temperature were more varied than the chemically inactivated ones. In all samples α-Lactalbumin was completely hydrolyzed while β-Lactoglobulin remained partially resistant. Corroborating with the chemical analyses, hydrolysates thermally inactivated showed vascular relaxations of 72.06% ± 11.36 (freeze-dried) and 81.00% ± 14.27 (spray-dried) (p>0.05), while the samples inactivated by pH reached 20.6% ± 5.4 (freeze-dried) and -9.25% ± 10.18 (spray-dried) of vascular relaxation (p>0.05), which may be possibly related to the formation of NaCl. The results showed that it was possible to develop a whey protein hydrolysate in semi-pilot scale with high antihypertensive activity using a more affordable technology to food industrie