RESUMO
The pH shift method was utilised for the recovery of proteins from salmon trimmings (ST), yielding 93% (w/w) protein. ST protein (STP) hydrolysates were generated with different enzyme preparations. STP incubated with Corolase PP for 1h (STP-C1) had the most potent angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory and oxygen radical absorbance capacity (ORAC) activities. Analysis of fractions of STP-C1 using UPLC-MS/MS identified sixteen peptides/amino acids. Tyr-Pro had the highest ACE inhibitory activity (ACE IC50=5.21±0.94µM). The highest DPP-IV inhibitory activity was found with the amino acid Tyr (DPP-IV IC50=75.15±0.84µM). Val-Pro had the highest ORAC activity (19.45±2.15µmol of TEg-1). To our knowledge, the peptides Gly-Pro-Ala-Val, Val-Cys, and Phe-Phe have not been previously identified to have the activities tested in this study. These results indicate that STP hydrolysates are potential sources of bioactive peptides.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Inibidores da Dipeptidil Peptidase IV/isolamento & purificação , Proteínas de Peixes/isolamento & purificação , Peptídeos/isolamento & purificação , Salmo salar/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Inibidores da Dipeptidil Peptidase IV/farmacologia , Proteínas de Peixes/farmacologia , Peptídeos/farmacologia , Espectrometria de Massas em TandemRESUMO
Salmon gelatin (Salmo salar, SG) enzymatic hydrolysates were generated using Alcalase 2.4L, Alcalase 2.4L in combination with Flavourzyme 500L, Corolase PP, Promod 144MG and Brewer's Clarex. The hydrolysate generated with Corolase PP for 1h (SG-C1) had the highest angiotensin converting enzyme (ACE, IC50=0.13±0.05mgmL-1) and dipeptidyl peptidase IV (DPP-IV, IC50=0.08±0.01mgmL-1) inhibitory activities, and oxygen radical absorbance capacity (ORAC, 540.94±9.57µmolTEg-1d.w.). The in vitro bioactivities of SG-C1 were retained following simulated gastrointestinal digestion. Administration of SG and SG-C1 (50mgkg-1 body weight) to spontaneously hypertensive rats (SHR) lowered heart rate along with systolic, diastolic and mean arterial blood pressure. The SG-C1 hydrolysate was fractionated using semi-preparative RP-HPLC and the fraction with highest overall in vitro bioactivity (fraction 25) was analysed by UPLC-MS/MS. Four peptide sequences (Gly-Gly-Pro-Ala-Gly-Pro-Ala-Val, Gly-Pro-Val-Ala, Pro-Pro and Gly-Phe) and two free amino acids (Arg and Tyr) were identified in this fraction. These peptides and free amino acids had potent ACE and DPP-IV inhibitory, and ORAC activities. The results show that SG hydrolysates have potential as multifunctional food ingredients particularly for the management of hypertension.
Assuntos
Anti-Hipertensivos , Antioxidantes , Dipeptidil Peptidase 4 , Proteínas de Peixes , Peptídeos , Salmo salar , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/farmacologia , Proteínas de Peixes/química , Proteínas de Peixes/farmacologia , Gelatina , Masculino , Peptídeos/química , Peptídeos/farmacologia , Hidrolisados de Proteína/química , Ratos , Ratos Endogâmicos SHR , Espectrometria de Massas em TandemRESUMO
The aim of this study was to standardize a method of DNA extraction from formalin-fixed and paraffin-embedded tissues (PETs) using a salt solution to precipitate protein and isopropanol to precipitate DNA. The samples were submitted to a DNA extraction method in which two different concentrations of ammonium acetate (2 and 4M) were compared with a phenol-chloroform extraction method and with a commercial DNA isolation kit. DNA was qualified and quantified by spectrophotometer analysis, electrophoresis, and amplification by PCR. The 167 and 268bp fragments of APC and beta-globin genes, respectively, were amplified equally from DNA extracted by all tested methods and in all cases. However, the 536bp fragment of beta-globin gene was not amplified in all cases. According to our results, the extraction method using ammonium acetate proved to be simple and suitable for obtaining DNA of good quality, which can be easily amplified by PCR.