Data set on effect of amaranth proteins on the RAS system. In vitro, in vivo and ex vivo assays

Data set presented in this article is related to the research paper entitled “Effect of amaranth proteins on the RAS system. In vitro, in vivo and ex vivo assays”, available in Food Chemistry [1]. In this article, we evaluated the effect on systolic blood pressure of spontaneously hypertensive rats (SHR) of different samples with amaranth proteins/peptides. The effect of these samples on RAS system was evaluated using in vitro and ex vivo assays. The concentration of renin and angiotensin converting enzyme (ACE) was evaluated using two commercial ELISA kits. Renin concentration was estimated through a direct immunoassay and ACE concentration with an immunoassay based on a competitive inhibition. In addition, the ACE inhibitory activity in plasma was evaluated using a spectrophotometric assay according to [2]. Ex vivo experiments were done with thoracic aorta extracted during the surgical procedure employed to obtain blood samples according to [3]. Data presented in this article recollect a very extensive work on how can be affect the RAS system in SHR model using amaranth protein/peptides as potential antihypertensive samples. These data could be useful to design novel functional foods for hypertensive individuals.


Data description
Data describes the effect of amaranth protein/peptides on RAS system inquiring into the mechanism of action of these samples using in vitro, in vivo and ex vivo approaches [1]. Treatment groups were: 1. G W : Negative control group. Animals treated with water, which did not receive amaranth proteins.
In order to compare mean values, a one way analysis of variance (ANOVA) multiple comparisons was applied. The critical significance level was set at p < 0.05. All samples were compared to G W (negative control group). Table 1 shows the reduction in SBP values exerted in each experimental group. Data were expressed as the decrease of SBP in mmHg of animals 3 h after the administration of each sample with respect to the SBP measured at the beginning of the experiment (SBP 3h -SBP 0hi ). DP values are presented as mean ± SEM. Animals belonging to the G E and G EþVIKP groups showed the most significant reduction in the SBP reaching reduction values of 42 ± 2 mmHg and 35 ± 2 mmHg respectively. The administration of API, AH or VIKP in water as vehicle (G API , G AH y G VIKP groups) caused a reduction in SBP values that was significantly lower than those observed in the groups mentioned above (25 ± 14 mmHg, 26 ± 3 mmHg and 21 ± 3 mmHg respectively.) Table 2 shows ACE plasma concentration of different groups assayed. This ELISA immunoassay is based on a competitive inhibition. Calibration curve was calculated according to the manufacturer's directions and the values are as follows: y ¼ 0.7089 e (À0.001405.X) þ 0.107 where "y" means OD at 450nm and "x" is ACE sample concentration in mg/ml. It can be observed that G W group presented extremely low values of ACE levels (0.17 ± 0.02 mg/ml), whereas ACE concentration in G C G A G E y G EþVIKP groups were 13.6e25.8 times higher than control group. API, AH and VIKP (G API ,G AH ,G VIKP groups respectively) induced an increase in the ACE levels that was 7.6 to 5.3 times higher than control group (1.3 ± 0.2 mg/ml, 0.90 ± 0.3 mg/ml and 1.1 ± 0.3 mg/ml respectively. p < 0.05). The same trend has been observed in studies evaluating synthetic drugs in hypertension treatments [4,5]. Table 3 shows renin plasma concentration in the different samples assayed. Calibration curve obtained was y ¼ 0.005x þ 0.008 where "y" means OD at 450 nm and "x" represented renin concentration in pg/ml. Only the G A , G API and G AH groups presented differences in plasma renin levels, as compared to G W group. No differences were found in the levels of this enzyme between G C , G E , G VIKP and G EþVIKP groups and the control group G W . Table 4 shows % ACE activity/mg ACE in plasma collected after treatments. Data are expressed as relative to 100% ACE activity to water control group (G W ). The lowest activity values (4e7% active ACE/ mg ACE) corresponded to groups G C , G A , G E and G EþVIKP , whereas the highest activity values were found in G VIKP , G AH and G API groups, which presented 20e13% active ACE/mg ACE. The administration of different samples decreases the enzymatic activity of ACE, together with an increase in plasma levels ( Table 2), probably to counter balance the inhibitory effect exerted by the hypotensive peptides. Table 5 shows the contractile activity determined in presence of potassium ions. Contractile force was higher in G W , G C and G A groups (roughly 0.45 g/mg), whereas this activity was significantly lower in the animals belonging to groups G API , G VIKP , G EþVIKP , G AH and G E (0.34e0.29 g/mg). Upon treating aorta rings with physiological concentrations of norepinephrine, statistically differences were observed in G VIKP and G EþVIKP groups.
-Commercial ACE and renin inhibitors (captopril and aliskiren, respectively) were employed as positive controls.

2.2.
In vivo assays 2.2.1. Indirect measurement of blood pressure The systolic blood pressure was measured according to [3]. In order to determine baseline values, blood pressure values were recorded at least three times on different days for each rat. After recording the last baseline blood pressure value, an aqueous suspension of each sample was administered to each animal. Three hours after the administration, blood pressure values were recorded with a tail cuff and a pulse sensor (NarcoBiosystems, Houston, TX).

Determination of plasma ACE and renin concentrations
A commercial ELISA kit (Rat Angiotensin converting enzyme MBS703086, MyBioSource, CA, USA) was employed to determine ACE concentration according to manufacturer's directions. This immunoassay is based on a competitive inhibition. Briefly, microtitre plates are coated with ACE. Samples and standards are incubated together with an anti-ACE HRP-labeled conjugate to generate the competition. Plasma renin concentration was determined with a commercial ELISA kit (Rat renin ELISA kit MBS041519 MyBioSource) following the manufacturer's directions. This immunoassay is a direct ELISA, which has an analytical measurement range of 6.25e200 pg/ml. The final colour reaction was read in a microtiter plate reader (Biotek Synergy HT, Winooski, VT, USA) at 450 nm.

Determination of plasma ACE activities
ACE-inhibitory activity was assayed according to [2]. Briefly, to determine the enzymatic activity, 50 ml of buffer [0.2M sodium borate pH 8.3; 2M NaCl), 25 ml of milli Q water, 25 ml of the commercial enzyme (maximum activity control)], or plasma samples were incubated with 100 ml of synthetic substrate (HHL) at 37 C for 30 min. The reaction was stopped by heating the mixture over a water bath at 90 C for 15 min. After cooling, 600 ml of 0.2M potassium pH 8.2 and 515 ml of colour reagent, which reacts with the hippuric acid generated during the enzymatic reaction, were added and stirred vigorously with a vortex and then centrifuged for 10 min at 20 C and 3000Âg. The absorbance was measured at 382 nm in a spectrophotometer (Beckman DU 650). The reaction blank was obtained by incubating the synthetic substrate with neither the plasma samples nor the enzyme, completing the reaction volume with milli Q water. Reaction blanks without the substrate (HHL was replaced by 100 ml of borate buffer) and containing plasma samples were also included. Controls containing plasma samples and captopril were also assayed.

Ex vivo experiments
During the surgical procedure employed to obtain blood samples, the thoracic aorta was resected and placed in saline solution bubbled with 5% CO 2 and 95% O 2 . The adjacent connective tissue was carefully removed avoiding distention of the vessel and damage to the endothelium. The aorta was  then cut into 2 mm long rings. Assay was performed according to [3]. The rings were gently suspended between two stainless steel wires in a water-jacketed organ baths kept at 37 C and filled with saline solution, bubbled with a mixture of 5% CO 2 and 95% O 2 , giving a pH of 7.40. The lower wire was fixed to a vertical plastic rod immersed in the organ bath, while the upper one was rigidly connected to a force transducer (Grass FT.03D, Grass Telefactor, West Warwick, CT, USA). Preparations were then stretched to obtain a passive force of 2 g and stabilized during 1 h, changing the solution in the chamber every 20 min. Tissue rings were then exposed to a solution containing 80mM potassium or norepinephrine 10 À6 M. For each condition, the contractile response was recorded. At the end of the experiment, tissue rings were dried on filter paper and weighed on a precision scale. The contraction intensity was calculated as the quotient between strength and the weight of the ring (mgF/mg).