Short communication Antihypertensive effect of an angiotensin I-converting enzyme inhibitory peptide from bullfrog (Rana catesbeiana Shaw) muscle protein in spontaneously hypertensive rats Zhong-Ji Qian a,1 , Won-Kyo Jung b,1 , Sang-Hoon Lee a , Hee-Guk Byun c , Se-Kwon Kim a,d, * a Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea b Department of NOAA Sea Grant Development and Food Science, Louisiana State University, Baton Rouge, LA 70803, United States c Faculty of Marine Bioscience and Technology, Kangnung National University, Kangnung 210-702, Republic of Korea d Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea Received 15 December 2006; received in revised form 25 April 2007; accepted 18 May 2007 Abstract To investigate biomedical and nutraceutical benefits of bullfrog (Rana catesbeiana Shaw) muscle protein, we examined an angiotensin I- converting enzyme (ACE I) inhibitory activity of various enzymatic hydrolystes of R. catesbeiana muscle protein in the present study. Among the enzymatic hydrolysates prepared using various commercial enzymes such as Alcalase, neutrase, pepsin, papain, a-chymotrypsin, and trypsin, Alcalase-proteolytic hydrolysates showed the highest ACE I inhibitory activity. During consecutive purification using a Hiprep 16/10 DEAE FF anion exchange and an octadecylsilane (ODS) C18 reversed phase liquid chromatographic techniques, a potent ACE I inhibitory peptide composed of 12 amino acids, Gly-Ala-Ala-Glu-Leu-Pro-Cys-Ser-Ala-Asp-Trp-Trp (M w : 1.3 kDa) was isolated from R. catesbeiana muscle hydrolysates degraded by Alcalase. The purified peptide from R. catesbeiana muscle (RCMP-alca) has IC 50 value of 0.95 mM, and Lineweaver–Burk plots suggest that RCMP-alca play act as a non-competitive inhibitor against ACE I. Antihypertensive effect in spontaneously hypertensive rats (SHR) also revealed that oral administration of RCMP-alca can decrease systolic blood pressure significantly (P < 0.05). In addition, MTT assay showed no cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5). The result of this study suggests that the ACE inhibitory peptide derived from R. catesbeiana muscle (RCMP-alca) could be potential candidates to develop nutraceuticals and pharmaceuticals. # 2007 Elsevier Ltd. All rights reserved. Keywords: Bullfrog (Rana catesbeiana Shaw) muscle; Alcalase; Hydrolysates; ACE I inhibitory peptide; Antihypertensive effect 1. Introduction Angiotensin I-converting enzyme (ACE I; dipeptidyl carboxy peptidase; EC 3.4.15.1) is a multifunctional zinc- containing enzyme, located in different tissues. This enzyme plays a key physiological role in the control of blood pressure, by virtue of the rennin–angiotensin system [1]. It acts as an exopeptidase that cleaves dipeptides from the C terminus of various oligopeptides [2]. ACE I converts an inactive form of decapeptide (angiotensin I) to octapeptide (angiotensin II), which is a potent vasoconstrictor, and inactivates bradykinin. Many studies have been attempted in the synthesis of ACE inhibitors such as captopril, enalapril, alacepril, and lisinopril, which are currently used in the treatment of essential hypertension and heart failure in humans [3] since the discovery of ACE inhibitors in snake venom [4]. However, these synthetic drugs are believed to have certain side effects such as cough, taste disturbances, and skin rashes. Therefore, the search for natural and non-toxic ACE inhibitors derived from proteins of livestock, grain, fish, etc., as alternatives to synthetic ones is of great interest among researchers for safe and economical use. Bioactive peptides, which are inactive within the sequence of the parent protein, could be released due to enzymatic hydrolysis. Among these, peptides with specific C-terminal sequences exposed by endo- or exo-proteolysis could interact with ACE I catalytic or binding site. The www.elsevier.com/locate/procbio Process Biochemistry 42 (2007) 1443–1448 * Corresponding author at: Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea. Tel.: +82 51 620 6375; fax: +82 51 628 8147. E-mail address: sknkim@pknu.ac.kr (S.-K. Kim). 1 Both authors have contributed equally to this work. 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.05.013