Blood pressure-lowering peptides from neo-fermented buckwheat sprouts: a new approach to estimating ACE-inhibitory activity

Abstract

Neo-fermented buckwheat sprouts (neo-FBS) contain angiotensin-converting enzyme (ACE) inhibitors and vasodilators with blood pressure-lowering (BPL) properties in spontaneously hypertensive rats (SHRs). In this study, we investigated antihypertensive mechanisms of six BPL peptides isolated from neo-FBS (FBPs) by a vasorelaxation assay and conventional in vitro, in vivo, and a new ex vivo ACE inhibitory assays. Some FBPs demonstrated moderate endothelium-dependent vasorelaxation in SHR thoracic aorta and all FBPs mildly inhibited ACE in vitro. Orally administered FBPs strongly inhibited ACE in SHR tissues. To investigate detailed ACE-inhibitory mechanism of FBPs in living body tissues, we performed the ex vivo assay by using endothelium-denuded thoracic aorta rings isolated from SHRs, which demonstrated that FBPs at low concentration effectively inhibited ACE in thoracic aorta tissue and suppressed angiotensin II-mediated vasoconstriction directly associated with BPL. These results indicate that the main BPL mechanism of FBP was ACE inhibition in living body tissues, suggesting that high FBP's bioavailability including absorption, tissue affinity, and tissue accumulation was responsible for the superior ACE inhibition in vivo. We propose that our ex vivo assay is an efficient and reliable method for evaluating ACE-inhibitory mechanism responsible for BPL activity in vivo.

Figure 1. FBP concentration-vasorelaxation curves of phenylephrine-preconstricted thoracic aorta rings from SHRs.

Dose dependent response to cumulatively increasing concentration of DVWY (A), FDART (B), VVG (C), FQ (D), VAE (E), and WTFR (F). Vasorelaxant rate is expressed as the percentage reduction of evoked contraction. Each data point and bar represent the mean ± S.E. (n = 3). *p<0.05, **p<0.01, versus pre-treatment tension, Student's t-test.

Figure 2. Tissue and plasma ACE activities in SHRs after treatment with FBPs.

Animals were treated with FBPs at a dose of 10 mg/kg BW. The levels of ACE activity in thoracic aorta (A), heart (B), liver (C), kidney (D), lung (E), and plasma (F) were determined 6 hours after administration. Data represent the mean ± S.E. (n = 6). *p<0.05, **p<0.01 versus the control group (purified water administration), Student's t-test.

Figure 3. Angiotensin II production after incubation with angiotensin I and DVWY, FDART, FQ, WTFR, and captopril.

Angiotensin II production was measured in SHR aorta rings incubated in 0.2 µM angiotensin I with DVWY, FDART, FQ, WTFR, and captopril (positive control). Data represent the mean ± S.E. (n = 3). *p<0.05, **p<0.01 versus the negative control group (Krebs solution treatment), Student's t-test.

Figure 4. Vascular tension changes after incubation with angiotensin I and DVWY, FDART, FQ, WTFR, and captopril.

Tension changes were measured using SHR thoracic aorta rings incubated in 0.2 µM angiotensin I with DVWY, FDART, FQ, WTFR, and captopril (positive control). Data represent the mean ± S.E. (n = 3). *p<0.05, **p<0.01 versus the negative control group (Krebs solution treatment) by Student's t-test.

Figure 5. Correlation between suppression of Ang II production and vasoconstriction by DVWY, FDART, FQ, and WTFR.

Correlation analysis of suppressive effects of Ang II production (IC₅₀) and vasoconstriction (EC₅₀). Correlation coefficients (r) and two-tailed P values are given in the inserts.