Developing Antihypertensive Peptides from Spent Laying Hen Muscle Proteins

  • Author / Creator
    Fan, Hongbing
  • Hypertension is the leading cause of global morbidity and mortality, afflicting > 20% of adults worldwide (~1 in 4 in Canada). Pharmaceutical drugs are the primary treatment for hypertension, but are generally associated with side effects. Recently, food-derived antihypertensive peptides are emerging alternatives. Central to the pathophysiology of hypertension is the renin-angiotensin system, which is regulated by two enzymes, angiotensin-converting enzyme (ACE) and ACE2. ACE converts angiotensin (Ang I) into Ang II that elevates blood pressure (BP) via Ang II type 1 receptor (AT1R), whereas ACE2 reverts this process by degrading Ang II into Ang (1–7) followed by binding with Mas receptor (MasR).
    Spent hens are laying hens reaching the end of egg-laying cycle, with an average yearly production of > 30 million in Canada over the last decade. Despite being a byproduct, spent hen is a rich source of muscle proteins that can be biotransformed into bioactive peptides with antihypertensive properties. The overall objective of this thesis is to purify and characterize novel antihypertensive peptides from spent hen muscle proteins targeting both the ACE-Ang II-AT1R (by inhibiting ACE) and the ACE2-Ang (1-7)-MasR (by upregulating ACE2) axes.
    In the first study, eighteen spent hen muscle protein hydrolysates (SPHs) were prepared and were screened for the ACE inhibitory (ACEi), ACE2 upregulating (ACE2u), antioxidant, and anti-inflammatory activities, as well as their fates during gastrointestinal digestion and transepithelial transport. Three SPHs were screened and fed to spontaneously hypertensive rat (SHR), while only SPH prepared by thermoase PC10F (SPH-T) reduced BP. The second study further investigated the antihypertensive effect of SPH-T at two doses (high dose, 1,000 mg/kg body weight [BW], and low dose, 250 mg/kg BW). Its BP reduction was associated with increased circulating ACE2 and Ang (1-7) but lowered Ang II levels, upregulated vascular expression of ACE2, as well as ameliorated vascular inflammation, oxidative stress, and fibrosis.
    The third study aims to purify ACEi peptides and ACE2u peptides from SPH-T. Finally, five potent ACEi peptides, VRP, LKY, VRY, KYKA, and LKYKA (IC50 values of 0.034–5.77 μg/mL) and four ACE2u peptides, VKW, VHPKESF, VVHPKESF (V-F), and VAQWRTKYETDAIQR-TEELEEAKKK (upregulated ACE2 expression by 0.52–0.84 folds) were identified. Among them, four with the highest ACEi and/or ACE2u activities from the major muscle proteins were selected for animal study; they were categorized into 3 groups: VRP (ACEi activity), V-F (ACE2u activity), and LKY and VRY (both ACEi and ACE2u activities).
    Prior to assessing the in vivo efficacies of VRP, LKY, VRY, V-F, their antioxidant and anti-inflammatory effects were evaluated in vascular smooth muscle A7r5 cells (VSMCs) and endothelial EA.hy926 cells (ECs), upon stimulation by Ang II and tumor necrosis factor alpha (TNF), respectively. All four peptides showed antioxidant activity in VSMCs, whereas only V-F attenuated inflammation, manifested by inhibiting expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) in VSMCs and VCAM-1 expression in ECs. VRP, LKY, and VRY exhibited antioxidant activity by acting as direct free radical scavengers whereas V-F also activated endogenous antioxidant enzymes. The anti-inflammatory effect of V-F was likely through modulation of the nuclear factor kappa B p65 and p38 mitogen-activated protein kinase pathways and was partially dependent on MasR.
    VRP, LKY, VRY, and V-F were orally administrated to SHRs at a dose of 15 mg/kg BW; only V-F reduced BP significantly over an 18-day treatment period. Associated with BP reduction were the increased circulating ACE2 and Ang (1-7) but reduced Ang II levels, upregulated vascular ACE2 and MasR expressions, as well as attenuated vascular inflammation and oxidative stress. Notably, V-F was not gastrointestinal stable and its fragment, VVHPK, was also an ACE2u peptide.
    The present study demonstrated the presence of antihypertensive peptides in spent hen muscle proteins, supporting the use of spent hens as antihypertensive functional food ingredients. Furthermore, this study reported for the first time the antihypertensive effect of an ACE2u peptide (V-F) identified using in vitro method. The discovery and characterization of ACE2u peptides in this work manifested their feasibilities of being isolated from proteins and uses for hypertension management.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.