Naturally Occurring Angiotensin I‑Converting Enzyme Inhibitory Peptide from a Fertilized Egg and Its Inhibitory Mechanism Xiang Duan, †,‡ Fengfeng Wu, †,‡ Mei Li, †,‡ Na Yang, †,‡ Chunsen Wu, †,‡ Yamei Jin, †,‡ Jingjing Yang, † Zhengyu Jin, †,‡ and Xueming Xu* ,†,‡ † State Key Laboratory of Food Science and Technology, and ‡ School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi Jiangsu 214122, People’s Republic of China ABSTRACT: This study was performed to investigate the angiotensin I-converting enzyme (ACE) inhibitory activity of peptides derived from fertilized eggs and elucidate the inhibition mechanism of these peptides. During incubation, ACE inhibitory activity of the peptides remained stable before day 12 and then increased markedly on day 15. Two ACE inhibitory peptides, VGVIKAVDKKAGGAGKVT and HLFGPPGKKDPV, were purified from peptides on day 15 by consecutive chromatography. Because HLFGPPGKKDPV possessed a higher ACE inhibitory activity (IC 50 = 125 μM), an antihypertensive effect of this peptide was further evaluated in vivo. The result showed that this peptide had an antihypertensive effect in spontaneously hypertensive rats (SHRs) at a dosage of 10 mg/kg. Furthermore, Lineweaver-Burk plots suggested that HLFGPPGKKDPV played as a non-competitive inhibitor against ACE, as supported by docking simulation. These data indicated that a fertilized egg has potential as antihypertensive components in functional foods and nutraceuticals. KEYWORDS: ACE inhibitory peptide, fertilized egg, inhibitory pattern, molecular docking, spontaneously hypertensive rat ■ INTRODUCTION Hypertension is one of the major risk factors for the development of cardiovascular diseases, stroke, and the end stage of renal disease. 1 Treatment of hypertension is an effective way to reduce the risk of these diseases. 2 The renin- angiotensin system (RAS) plays a key role in maintaining blood pressure homeostasis. 3 Angiotensin I-converting enzyme (ACE) is a key enzyme in RAS; it raises blood pressure by converting the inactive decapeptide angiotensin I to the potent vasoconstrictor octapeptide angiotensin II as well as inactivat- ing the vasodilating nonapeptide bradykinin. 4 Therefore, inhibition of ACE is considered as an effective approach to controlling hypertension. 1 Since the discovery of ACE inhibitors in snake venom, 5 many chemical ACE inhibitors, such as captopril, enalapril, alacepril, and lisinopril, were designed. 6 However, these chemical ACE inhibitors are known to have various side effects, such as coughing, skin rashes, and angioedema. 7,8 Therefore, many studies have been initiated to develop novel, safe, and natural ACE inhibitors from natural sources. 9,10 In recent years, several ACE inhibitory peptides have been isolated from a wide range of food sources, such as gelatin, 11 casein, 12 tuna, 13 corn, 14 soy beans, 15 egg white, 16 and dried bonito. 17 For a better understanding of inhibitory properties of peptides, the structure-activity relationship should be clarified. Terashima et al. 18 investigated a series of peptides derived from an ACE inhibitory peptide (VTVNPYKWLP) and concluded that the Pro residue in the C-terminal end strongly enhanced ACE inhibition activity. In addition, the length of peptides also played a key role in ACE inhibitory ability. 19 Recently, Wu et al. 20 have contructed a database consisting of 168 dipeptides and 140 tripeptides to study the quantitative structure-activity relationships of ACE inhibitory peptides. It showed that amino acid residues with bulky side chains as well as hydrophobic side chains were preferred for dipeptides, while for tripeptides, the most favorable residues for C terminus, middle position, and N terminus were aromatic amino acids, positively charged amino acids, and hydrophobic amino acids, respectively. However, these structure-activity correlation studies were mainly based on amino acid sequence analysis. In recent years, the crystal structures of ACE and its inhibitors have been analyzed to elucidate the relationship between the inhibition mechanism and the molecular structure of inhibitors. 21-23 These studies provided a new method to analyze the structure-activity relationship of ACE inhibitory peptides. Traditionally, fertilized eggs are widely considered as natural dietary supplements in many Asian countries. 24 During chick embryonic development, RAS plays a principal role in regulation of blood pressure. 25 Savary et al. 25 reported that ACE was produced at a very early stage and its activity increased during incubation. Whether naturally occurring peptides derived from fertilized eggs have ACE inhibitory effect is still unknown. To our knowledge, this is the first research to investigate ACE inhibitory activity and the structure-activity relationship of the peptides from fertilized eggs. Because the fertilized eggs that are eaten by Asian consumers are commonly 12-day-old eggs, 26 the fertilized eggs before day 15 of incubation were chosen for investigation in this paper. The objectives of the present study were to (1) determine ACE inhibitory activity of peptides derived from fertilized eggs and (2) investigate the inhibition mechanism of a purified ACE inhibitory peptide. Received: November 20, 2013 Revised: May 19, 2014 Accepted: May 27, 2014 Published: May 27, 2014 Article pubs.acs.org/JAFC © 2014 American Chemical Society 5500 dx.doi.org/10.1021/jf501368a | J. Agric. Food Chem. 2014, 62, 5500-5506