Article Cite This: J. Agric. Food Chem. 2019, 67, 7810−7820
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Discovery and Study of Novel Antihypertensive Peptides Derived from Cassia obtusifolia Seeds Ya-Hui Shih,†,§ Fu-An Chen,‡,§ Li-Fei Wang,∥ and Jue-Liang Hsu*,†,⊥,#,∇
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Department of Biological Science and Technology, ⊥Research Center for Tropic Agriculture, #Research Center for Animal Biologics, and ∇Research Center for Austronesian Medicine and Agriculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan ‡ Department of Pharmacy and Master Program, Tajen University, Pingtung, Taiwan ∥ Hospitality and Tourism Research Center, National Kaohsiung University of Hospitality and Tourism, Kaohsiung City 81271, Taiwan S Supporting Information *
ABSTRACT: Antihypertensive peptides were screened from thermolysin hydrolysate of Cassia obtusifolia seeds (Jue Ming Zi) using two independent bioassay-guided fractionations, reversed-phase high-performance liquid chromatography (RP-HPLC), and strong cation-exchange (SCX) liquid chromatography coupled with angiotensin I-converting enzyme (ACE) inhibitory assay. The identical peptide in the most active RP-HPLC and SCX fractions was simultaneously de novo sequenced as FHAPWK with high-resolution mass spectrometry. FHAPWK (IC50 = 16.83 ± 0.90 μM) was further identified as a competitive inhibitor and a true inhibitor on ACE by a Lineweaver−Burk plot and preincubation experiment, respectively. The molecular docking simulation indicated that FHAPWK could interact with several key residues of the ACE active site, which is consistent with the result of the inhibitory kinetics study. Moreover, its antihypertensive effect was demonstrated using the animal model of spontaneously hypertensive rats. It is concluded that FHAPWK is the first reported antihypertensive peptide derived from thermolysin hydrolysate of C. obtusifolia seeds. KEYWORDS: Cassia obtusifolia, antihypertensive peptide, Angiotensin-I converting enzyme (ACE), de novo sequencing
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INTRODUCTION Hypertension is one of the key risks for cardiovascular diseases, and it is correlated to 45% of deaths caused by heart disease and 51% of deaths caused by stroke (WHO, 2013).1 For clinical treatment or daily blood-pressure control, several types of antihypertensive drugs have been developed based on different therapeutic targets, such as diuretics, calcium channel blockers, beta blockers, angiotensin-II receptor blockers, and angiotensin-I converting enzyme (ACE) inhibitors.2 Among them, ACE inhibitors are the most frequently prescribed drugs due to less adverse effects according to the guidelines on management of hypertension, heart failure, myocardial infarction, and coronary artery disease.3,4 In the reninangiotensin-aldosterone system (RAAS), ACE plays dual roles in regulating blood pressure by catalyzing the formation of strong vasoconstrictor octapeptide (angiotensin II) from a decapeptide (angiotensin I) as well as triggering the inactivation of bradykinin, an important vasodilator. Therefore, inhibition or inactivation of ACE is recognized as a direct approach for reduction of blood pressure.5 Due to inspiration of the first ACE inhibitory (ACEI) drug captopril which was derived from snake venom peptide and successfully introduced to hypertension treatment in 1980s,6 ACEI peptides originated from natural proteins have attracted scientists’ attention. Edible proteins from either animal or plant sources are especially concerned for exploitation of novel ACEI peptides.7−9 Plant seeds are also deemed as important targets for bioactive peptide production owing to their rich protein © 2019 American Chemical Society
contents. In recent years, ACEI peptides derived from various plant seeds, such as adzuki bean seeds,10 Parkia speciosa seeds,11 cherry seeds,12 bitter melon seeds,13 hemp seeds,14 amaranth seeds,15 walnuts,16 soybeans,17 peanuts,18 and rice brans,19 have been extensively reported. Cassia obtusifolia L. (Leguminosae) is a plant prevalent across America, Asia, Africa, and Oceania.20 Jue Ming Zi, the Chinese name of C. obtusifolia seeds (COS), has long been used as a traditional medicine for the treatment of eye inflammation, photophobia, or other vision problems in China, Korea, and Japan.21 Several modern pharmacological studies have also confirmed its potential roles as complementary medicine for some liver and eye diseases as well as inflammatory disorders.22−25 Most of these biological activities of COS were contributed by the constituents of anthraquinones, flavonoids, and phytosterols. To explore more health benefits of COS, this study was aimed to investigate the antihypertensive peptides from enzymatic hydrolysate of COS. Typically, bioactive peptides are the short-chain protein fragments generated by enzymatic hydrolysis of proteins.26 In this study, four proteases, including trypsin, chymotrypsin, pepsin, and thermolysin, were used to hydrolyze COS proteins, and the ACEI activities of these hydrolysates were tested by an Received: Revised: Accepted: Published: 7810
April 1, 2019 June 19, 2019 June 21, 2019 June 21, 2019 DOI: 10.1021/acs.jafc.9b01922 J. Agric. Food Chem. 2019, 67, 7810−7820
Article
Journal of Agricultural and Food Chemistry
After 16 h of incubation, all of the hydrolysis reactions were quenched by heating at 95 °C for 10 min. The hydrolysates were centrifuged at 14000 rpm/4 °C for 15 min, and the supernatent was lyophilized as the whole COS hydrolysates, which were redissolved in PBS buffer and filtered through the 3 kDa MWCO ultrafiltration membrane to acquire the short-chain peptides ( 285, and the collision-induced dissociation (CID) energy was set at 19 V. The content of FHAPWK in the COS protein hydrolysate could be revealed by comparing the peak area of FHAPWK in the SRM chromatogram of the thermolysin hydrolysate of COS protein to the calibration curve generated from the synthetic FHAPWK of various concentrations. Molecular Docking Simulation. To simulate the molecular-level interaction between the ACEI peptide FHAPWK and ACE, a molecular docking simulation was performed in the medium of the Discovery Studio Visualizer (Accelrys Software, UK) coupled with the CHARMm (Chemistry at HARvard Macromolecular Mechanics) program for dynamic energy calculation. The three-dimensional structure of human testicular ACE−lisinopril complex (1O8A.pdb) imported from the Protein Data Bank (with a resolution of 2.0 Å) was used as the target structure for molecular docking.13 The ligand structure of FHAPWK was constructed, and its binding energy was minimized by CHARMm. Before docking simulation, lisinopril and all water molecules in the ACE−lisinopril complex were removed but zinc and chloride ions were retained.31 The simulation was performed using cavity detection with a radius of 8 Å centered at the coordinates (x, y, z = 37.53, 35.24, 45.22). The molecular docking results were reviewed according to the docking score of each pose, and the pose 7812
DOI: 10.1021/acs.jafc.9b01922 J. Agric. Food Chem. 2019, 67, 7810−7820
Article
Journal of Agricultural and Food Chemistry
Figure 2. (A) RP-HPLC chromatogram of thermolysin hydrolysate of COS protein. (B) ACE inhibitory activities of the 12 RP-HPLC fractions. (C) ACE inhibitory activities of the 10 SCX fractions. with the best score was regarded as the most suitable docking for the ligand of FHAPWK to interact with ACE. In Vivo Study on the Antihypertensive Effect of ACEI Peptide FHAPWK. Eighteen 10-week-old male spontaneously hypertensive rats (SHRs), purchased from the National Laboratory Animal Center (Taipei, Taiwan), were acclimated for 1 week individually in steel cages under a controlled environment condition (25 ± 1 °C and 12 h light/dark cycle) and given ad libitum access to tap water and common laboratory diet. During this period, the rats became accustomed to the procedure of blood pressure measurement. For the experiment, all SHRs were divided into three groups to be orally administered with a single dose of distilled water, captopril (2 mg/kg bw) and FHAPWK (2 mg/kg bw), respectively. Their systolic blood pressure (SBP) was measured using a tail-cuff sphygmomanometer (NP-NIBP model MK-2000ST, Three Dian Technology Co., Ltd.) at 0, 2, 4, 6, 8, and 24 h after administration. The SHRs were treated in accordance with the international guidelines approved by the Institutional Animal Care and Use Committee (IACUC) in National Pingtung University of Science and Technology (NPUST) with Protocol Number: NPUST-103-065. Statistical Analysis. The experiments were performed in triplicate, and the data were expressed as the mean ± standard deviation. SBP data measured from six SHRs was statistically analyzed using SPSS 16.0 software (SPSS, Inc., Chicago, IL). Significant differences between means were determined by analysis of variance (ANOVA) and Dunnett’s multiple comparison at the resulting p value lower than 0.05 (p < 0.05).
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thermolysin, and trypsin, were used to hydrolyze COS protein. Generally, smaller peptides are better absorbed by intestines and more accessible toward the active sites of ACE.32 Therefore, the COS protein hydrolysates were filtered through 3 kDa MWCO ultrafiltration membranes to gather the shorter peptides (
