RESUMEN
Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse Brassica napus genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (pI) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of pI 8.65 and 9.9, and one acidic of pI 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC50 values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.
Asunto(s)
Antihipertensivos/farmacología , Brassica napus/química , Dipeptidil Peptidasa 4/química , Inhibidores Enzimáticos/farmacología , Hipoglucemiantes/farmacología , Peptidil-Dipeptidasa A/química , Secuencia de Aminoácidos , Antihipertensivos/química , Antihipertensivos/aislamiento & purificación , Brassica napus/genética , Brassica napus/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Genotipo , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/aislamiento & purificación , Focalización Isoeléctrica , Cinética , Extracción Líquido-Líquido/métodos , Peptidil-Dipeptidasa A/metabolismo , Extractos Vegetales/químicaRESUMEN
The extraction of phenolic compounds from canola meal produces functional health products and renders the canola meal a more digestible animal feed. The extracted phenolics may have novel bioactivity worth investigation. In this study, several solvents were evaluated for their ability to extract phenolic compounds from canola meal: water (WE) and various 80% organic solvent/water mixtures of methanol (ME), acetone (AE), ethanol (EE), butanol (BE), chloroform (CE) and hexane (HE). The in vitro antioxidant and anti-obesity properties of various extracts were investigated. Anti-obesity properties were studied using adipogenic differentiation inhibition of a murine mesenchymal stem cell line (C3H10T1/2) and a pancreatic lipase inhibition assay. AE, ME, and BE showed significant (p < 0.05) adipogenesis and pancreatic lipase inhibitory activities and may have more pharmacological properties. AE down-regulated the gene expression of the major adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), correlating to phenolic content in a dose-dependent manner. The chemical characterization of AE revealed the presence of sinapic acid, ferulic acid, and kaempferol derivatives as main bioactive phenols.
Asunto(s)
Adipogénesis/efectos de los fármacos , Antioxidantes/farmacología , Brassica napus/química , Fenoles/farmacología , Extractos Vegetales/farmacología , Animales , Antioxidantes/química , Línea Celular , Cromatografía Líquida de Alta Presión , Regulación de la Expresión Génica/efectos de los fármacos , Espacio Intracelular/metabolismo , Lipasa/antagonistas & inhibidores , Lipasa/química , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , PPAR gamma/genética , Fenoles/química , Extractos Vegetales/química , Solventes , Espectrometría de Masas en TándemRESUMEN
A normal phase high performance liquid chromatography (HPLC) method was developed to simultaneously quantify several prominent bioactive compounds in canola oil vis. α-tocopherol, γ-tocopherol, δ-tocopherol, ß-carotene, lutein, ß-sitosterol, campesterol and brassicasterol. The use of sequential diode array detection (DAD) and tandem mass spectrometry (MS/MS) allowed direct injection of oils, diluted in hexane without derivatisation or saponification, greatly reducing sample preparation time, and permitting the quantification of both free sterols and intact sterol esters. Further advantages over existing methods included increased analytical selectivity, and a chromatographic run time substantially less than other reported normal phase methods. The HPLC-DAD-MS/MS method was applied to freshly extracted canola oil samples as well as commercially available canola, palm fruit, sunflower and olive oils.