Effect of digested total protein, protein fractions, and pure peptides from chia seeds (Salvia hispanica L.) on infl ammation, atherosclerosis and adipogenesis in silico and in vitro

• Ano de defesa: 2020
• Autor(a) principal: Grancieri, Mariana
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Federal de Viçosa
• Palavras-chave em Português: Hidrolisados de Proteína; Proteínas; Peptídeos; Inflamação; Adipósitos; Macrófagos; Bioquímica da Nutrição
• Link de acesso: https://locus.ufv.br//handle/123456789/27781

Cardiovascular diseases (CVD) are the leading cause of death in the world. Some pathologies are associated with the development of CVD, such as low-grade inflammation, atherosclerosis and obesity. Chia seed (Salvia hispanica L.) is a food that has shown promising results in experimental and clinical studies. These seeds are a rich source of total proteins and storage proteins that, after gastrointestinal digestion, can generate bioactive peptides with benefits in specific physiological functions. The general objective of this study was to investigate the effects of total digested protein (DTP), digested protein fractions (DPF) and pure chia seed peptides (Salvia hispânica L.) on inflammation, atherosclerosis and adipogenesis in silico and in vitro. Manuscript 1 was a review study that aimed to identify the composition and beneficial effects of chia seeds (Salvia hispanica L.), their proteins, peptides and their potential impact on health. The Scopus and PubMed databases were used to identified studies that investigated the effects of chia seed consumption on humans. In addition, chia proteins were identified using the Uniprot database and their bioactive peptides were evaluated for their potential biological effects through the BIOPEP® database. As a result, it was observed that the chia seed has proteins with promising bioactive peptides, especially with antioxidant, hypoglycemic and hypotensive effects, which can be correlated to the beneficial effects resulting from the consumption of this seed in studies with humans. However, there was a lack of studies that investigate these effects of chia protein in chronic diseases. Therefore, in Manuscript 2, it aimed to identify and characterize the DTP and DPF peptides of chia seeds (Salvia hispanica L.) and to determine their potential antioxidant, anti-inflammatory and anti- atherosclerotic effect. Chia seed grown in Brazil was used, which was ground to obtain the flour and, from this, the mucilage and lipids were removed. Such mucilage and lipid-free flour was used to extract total protein by alkaline precipitation, as well as protein fractions by solvent solubility. Then, the proteins, total and fractions, underwent gastrointestinal digestion simulated with pepsin and pancreatin and their bioactive peptides were identified by Ultra Efficiency Liquid Chromatography, coupled to the mass spectrometer (UPLC-ESI-MS). The biological potential, parental proteins and physicochemical properties were characterized using the databases BIOPEP®, BLAST tool® and PedDraw®, respectively. In addition, the antioxidant and anti-inflammatory effects were evaluated using biochemical and in silico analyzes. DTP and digested albumin, globulin and glutelin showed the ability to eliminate superoxide, hydrogen peroxide, nitric oxide and DPPH and inhibition of the enzymes 5-LOX, COX-1-2 and iNOS in biochemical analyzes. In addition, the peptides from the samples mentioned above had interaction with the NF-κB, LOX-1, TLR4 and COX-1 markers in in silico analyzes. In Manuscript 3, the objective was to determine the effect of DTP and DPF (albumin, globulin and glutelin) from chia seeds (Salvia hispanica L.) on inflammation and atherosclerosis in macrophages and the mechanisms of action. Macrophages RAW.264.7 had inflammation and atherosclerosis induced by the addition of lipopolysaccharide (1μM) and oxidized low density lipoprotein (80 μM), respectively. It was observed that in the inflammatory process, DTP and DPF reduced the expression and translocation of NF-κB to the nucleus and, consequently, the expression of p-NF-κB, iNOS, p-JNK and AP-1 was also reduced. Digested glutelin reduced the secretion of nitric oxide (-65.1%), reactive oxygen species (-19.7%), prostaglandins (-34.6%), TNF-α (-24.1%), among others. As an anti- atherosclerotic, DTP and digested glutelin reduced the expression of iCAM (-86.4%, -80.8%), LOX-1 (-37.3%, -35.7%), iNOS (-67, 0%, -42.2%) and NF-κB (-57.5%, -71.1%). DTP was effective in reducing nitric oxide secretion (-43.4%), accumulation of lipids in macrophages (- 41.9%) and prostaglandin secretion (-41.9%), TNF-α (-43, 3%), MCP-1 (-47.6%) and IL-6 (- 50.5%). In addition, in Manuscript 4, the objective was to evaluate the in vitro effect of DTP and DPF (albumin and glutelin) and pure peptides from chia seed (Salvia hispanica L.) on adipogenesis, as well as on the prevention and inhibition of induced inflammation in 3T3-L1 adipocytes. The DTP, DPF samples, and two pure peptides NSPGPHDVALDQ (Pep1) and RMVLPEYELLYE (Pep2) were tested for the effects of preventing adipogenesis. In addition, 3T3-L1 cells were induced to mature adipocytes and were exposed to conditioned media (CM) of inflamed macrophages and the efficacy of DTP and DPF in preventing inflammation was tested (samples were added together with CM) inhibition (samples were added after adding CM). All treatments prevented adipogenesis, reducing the expression of PPAR by more than 50% and, to a lesser extent, LPL, FAS, SREBP1, lipase and triglyceride activity. Inflammation induced by conditioned means was reduced mainly during prevention. The expression of NF-κB, iNOS and COX-β and the secretions of nitric oxide, PGEβ and TNFα were reduced in all treatments. In general, the DPF fractions and digested proteins: albumin, globulin and glutelin showed promising effects on inflammation, atherosclerosis and adipogenesis, reducing expression and markers related to these pathways. In addition, DTP, as a set of proteins, presented more potent effect compsred to the isolated proteins. Thus, chia seed proteins can be a tool in reducing the risk of developing cardiovascular disease markers. Keywords: Adipocyte. Hydrolyzates. Macrophages. Peptides. Protein. Inflammation.