Considering this, in vivo assays are always required to demonstrate in vivo bioactivity of these protein hydrolysates able to inhibit ACE in vitro. doses and Captopril. Initial SBP values of 55 and 85 mg/kg were recovered 24 or 8 h post-administration, respectively, 55 mg/kg being the most effective dose. At this dose, a reduction in the plasmatic ACE activity in the SHR was found. However, Hpp11 did not unwind the aortic ring preparations. Therefore, ACE inhibition could be the mechanism underlying Hpp11 antihypertensive effect. Remarkably, Hpp11 did not change SBP in WKY rats, showing that this decreased SBP effect is usually specific to the hypertensive state. were provided by a local farm (Granja Gai, La Riera de Gai, Spain). Protamex? (Novozymes, Bagsv?rd, Denmark) (EC 3.4.21.62 and 3.4.24.28, 1.5 AU/g from and for 20 min at 4 C, and the supernatant was filtered through a 0.45 m membrane, collected and lyophilized. Hpp11 was reconstituted in water to carry out the following experiments. Hpp11 was characterized before its administration to SHR. Hpp11 protein C7280948 content was estimated by the determination of total nitrogen compounds content of Hpp11 by the Kjeldahl method, multiplying the decided nitrogen content by 6.25 and the humidity determination was carried out following the AOAC official methods [29]. The degree of hydrolysis was determined by the TNBS method according to Adler-Nissen (1979) [30], in which free -amino groups were decided. The Hpp11 ACEI activity was decided according to Quirs et al. [31]. The fluorescence measurements were performed after 30 min in a multi-scan microplate fluorimeter (FLUOstar optima, BMG Labtech, Offeuburg, Germany). The excitation and emission C7280948 wavelengths were 360 and 400 nm, respectively. The software used to process the data was FLUOstar control (version 1.32 R2, BMG Labtech, Offeuburg, Germany). The inhibition pattern of Hpp11 around the ACE substrate o-Abz-Gly-p-Phe(NO2)-Pro-OH was assayed at the following concentrations: 7.2, 3.6, 1.8, 0.9, 0.45, 0.23 and 0 mM. The inhibition kinetics of ACE in the presence of Hpp11 was determined by LineweaverCBurk plots [30]. All the analyses were performed in triplicate. 2.3. Experimental Process in the SHR and WKY Rats Male SHR and WKY rats (17C20-week-old, weighing 300C350 g) were obtained from Charles River Laboratories Espa?a S.A. (Barcelona, Spain). The animals were housed at a heat of 23 C with 12 h light/dark cycles and consumed tap water and a standard diet (A04 Panlab, Barcelona, Spain) ad libitum during the experiments. Different doses of the hydrolysate (25, 55 and 85 mg/kg bw) or a single dose of Hpp11 (55 mg/kg bw) were administered by gastric intubation to SHR or WKY rats, respectively, between 9 and 10 am. Tap water was used as a negative control for the SHR and WKY rats, and 50 mg/kg Captopril dissolved in tap water was given as a positive control to the SHR. The total volume of water, Captopril or Hpp11 orally administered to the rats was between 1.5 and 2 mL. The systolic blood pressure (SBP) was recorded in the rats by the tail-cuff method [32] before and 2, 4, 6, 8, 24 and 48 h post-administration. Before the measurement, the animals were kept at 38 C for 10 min in order to detect the pulsations of the tail artery. Changes in the SBP were expressed as the differences between C7280948 the mean values of these variables before and after the administration of the treatment. To minimize stress-induced variations in BP, all measurements were taken by the same person, in the same peaceful environment. Moreover, before starting the experiments, we established C7280948 a 2-week training period for the rats to become accustomed to the procedure. Data are expressed as the mean values standard error of the means (SEM) for a minimum of six experiments. Rabbit Polyclonal to RAB2B Additionally, twelve 20C23-week-old SHR weighing 350C380 g were administered Hpp11 at 55 mg/kg bw or water to determine the plasmatic ACE activity. The Hpp11 and water were orally administered by gastric intubation between 9 and 10 am. Blood samples were collected at 6 h post-administration via the saphenous vein using heparin vials. The samples were centrifuged at 2000 for 15 min at 4 C to obtain plasma. The procedure that was used to determine the plasmatic ACE activity is usually explained below. 2.4. Determination of the Plasmatic ACE Activity The plasmatic ACE activity was performed by a fluorometric method reported by Miguel et al. [28]. The measurements were performed in a multi-scan microplate fluorimeter (FLUOstar optima, BMG Labtech) at 37 C and 350 nm excitation with 520 nm emission filters. ACE at different concentrations was added to each plate to obtain a calibration curve. ACE activity was expressed as the mean SEM mU ACE/mL.