Vasorelaxing and antihypertensive activities of synthesized peptides derived from computer-aided simulation of pepsin hydrolysis of yam dioscorin

We reported that yam dioscorin and its peptic hydrolysates exhibited ACE inhibition and antihypertensive effects on SHRs, however, the active peptides are not really isolated until now. Using ACE inhibitory screenings, two penta-peptides, KTCGY and KRIHF, were selected for ex vivo and in vivo experiments. KTCGY, KRIHF, and captopril were shown to have similar vasodilating effects against phenylephrine (PE)-induced tensions in rat endothelium-dependent thoracic aortic rings, however, KTCGYKTCGY (two-repeated KTCGY) and TCGYTCGY (two-repeated TCGY) were showed endothelium-independent vasodilating effects against PE-induced tensions. KTCGY, KRIHF (10 or 20 mg/kg), and captopril (10 mg/kg) were used to evaluate antihypertensive activity during 24-h after a single oral administration to spontaneously hypertensive rats (SHRs). The KTCGY and KRIHF showed significantly different and reduced the systolic blood pressure of SHRs compared to the blank. These results suggest that KTCGY and KRIHF may contribute important roles in yam dioscorin for regulating blood pressure in vivo.


Background
Processed yam tuber is a traditional Chinese medicine used as a juvenescent substance, with no apparent side effects resulting from long-term use, which is ranked the top levels in Chinese Pharmacopoeia. Yams (Dioscorea spp., Dioscoreaceae) are an important tuber crop in Africa, Asia, and Middle and South America and are also a staple food in the Caribbean. Yam dioscorin and its peptic hydrolysates have been reported to exhibit several biological activities in vitro and in vivo (Lu et al. 2012), including antioxidant (Hou et al. 2001;Liu et al. 2006;Han et al. 2013Han et al. , 2014a, antihypertensive (Hsu et al. 2002;Lin et al. 2006;Liu et al. 2009a), immunomodulatory (Liu et al. 2007;Liu et al. 2009b), and enzyme activities (Hou et al. 1999a, b;Hou et al. 2000).
The untreated high blood pressure is considered to be the central factor in stroke which accounts approximately 33% deaths (Mark and Davis 2000). There are several classes of pharmacological agents which have been used in the treatment of hypertension, and one class of antihypertensive drugs known as angiotensin I converting enzyme (ACE) inhibitors is associated with a low rate of adverse side effects and is the preferred class of antihypertensive agents when treating patients with concurrent secondary diseases or cardiovascular diseases (Zaman et al. 2002). ACE (peptidyldipeptide hydrolase, EC 3.4.15.1) is a dipeptide-liberating Zn-containing exopeptidase, which removes a dipeptide from the C-terminus of angiotensin I to form angiotensin II, a very hypertensive compound. Several ACE inhibitory peptides were isolated from food proteins and exhibited generally to reduce blood pressures of SHRs (Martínez-Maqueda et al. 2012). Fujita et al. (2000) found that the octapeptides of FFGRCVSP (IC 50 = 0.4 μM) and ERKIKVYL (IC 50 = 1.2 μM) were potent ACE inhibitors, but none of them were effective in spontaneously hypertensive rats (SHRs) to reduce the blood pressure. These potential ACE inhibitory peptides were further hydrolyzed by the rat's gastrointestinal proteases and then lose their antihypertensive effects on SHR in vivo. We reported that yam dioscorin and its peptic hydrolysates exhibited ACE inhibitory activity (Hsu et al. 2002) and antihypertensive activity (Lin et al. 2006) using SHRs as models, however, the active peptides are not really isolated until now. In this study, using angiotensin converting enzyme inhibition as preliminary screenings, two out of twenty-three synthesized peptides from a computer-aided simulation of pepsin hydrolysis of yam dioscorin were selected for further ex vivo and in vivo experiments and captopril was used for comparisons. It is suggested that KTCGY and KRIHF show vasodilating effects and can reduce SHR's systolic blood pressure (SBP) which may contribute important roles in yam dioscorin for regulating blood pressure in vivo.

Materials
The 23

ACE inhibitory assay screenings
The ACE inhibitory activity was measured following the previous reports with some modifications (Hsu et al. 2002). Each synthesized peptide was dissolved in DMSO to 4 mM as stocks. The 1 ml, 0.5 mM FAPGG (dissolved in 50 mM Tris-HCl buffer, pH 7.5, containing 0.3 M NaCl) was mixed with 12.2 μl peptide solution, and then twenty μl (20 μU) of commercial ACE (stock solution, 1U/ ml) was added. The 0.1% DMSO solution was used instead of sample solution for blank experiments (ΔA blank /min). The decreased absorbance at 345 nm (ΔA sample /min) was recorded during 90 sec at room temperature. The ACE inhibition (%) was calculated as followed: [1 -(ΔA sample /min ÷ ΔA blank /min)] × 100.
Effects of KTCGY, KTCGYKTCGY, TCGYTCGY, and KRIHF on phenylephrine-induced tensions in rat thoracic aortic ring ex vivo Effects of four peptides and captopril on vascular tension were examined following the previous method (Wang et al. 2001). Male Sprague-Dawley rats were killed by decapitation, and sections of the thoracic aorta between the aortic arch and the diaphragm were excised carefully and placed in a Petri dish with oxygenated Krebs' buffer (120 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl 2 , 1 mM MgSO 4 , 27 mM NaHCO 3 , 1 mM KH 2 PO 4 , 10 mM glucose, pH 7.4, with 95% O 2 , 5% CO 2 ) and gently dissected free of fat and connective tissue. The endothelium was removed by gently rubbing the intimal layer of the vessel with a wire. The isolated aortic rings, 3 to 4 mm in length with or without the endothelium, were fixed isometrically in organ chambers under passive tension (1.8 g) for 60 min. Changes in vascular tension were recorded with a polygraph (Gould, model 2400, Valley View, OH, USA) via a force displacement transducer (Grass FT03, Quincy, MA, USA). After equilibration, the near maximal concentration was induced by PE (0.3 μM). When the rings achieved a stable tension, the acetylcholine (3 μM) was added to the bath to assess endothelial integrity. The relaxing effects of KTCGY, KTCGYKTCGY, TCGYTCGY, KRIHF, and captopril on PE (0.3 μM) pre-contracted endothelium-intact or endothelium-denuded aortic rings were examined. When PE treatments reached a steady plateau (considered as 100%), KTCGY, KTCGYKTCGY, TCGYTCGY, KRIHF, and captopril were then tested at cumulative concentrations (0.1 to 100 μM) for their ability to induce aortic ring relaxations.

Antihypertensive Effects of KTCGY, KRIHF, and captopril in SHRs
Experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Taipei Medical University (LAC-95-0076, LAC-97-0117, and LAC-100-0038). Male SHRs (18-28 weeks old; from National Laboratory Animal Center, Taipei, Taiwan) were housed individually in steel cages kept at 24°C under a 12-h light-dark cycle, with free access to water and a standard mouse/rat chow (Prolab® RMH2500, 5P14 Diet, PMI Nutrition International Brentwood, MO, USA). SHRs were randomly divided into 4 groups (N = 6 of each group), KTCGY or KRIHF at concentration of 10 mg/kg and 20 mg/kg was orally administered to the SHRs, and the SBP were measured after 0, 2, 4, 6, 8, and 24 h by using an indirect tail-cuff blood pressure meter (BP98-A, Softron, Tokyo, Japan). Distilled water (0.5 ml) was administered to the SHRs in the blank group. The captopril (10 mg/kg) was used as the positive control.

Statistical analysis
Data were expressed as mean ± S.D. For animal experiments, the differences between the blank and the experimental group at the same time was analyzed using Student's t-test, and the P-value of less than 0.05 (*), 0.01 (**), and 0.001 (***) were recognized as different significantly. The statistical analysis was performed using the SigmaPlot software 10.0.

ACE inhibitory assay screenings
The deduced sequences of dioscorin A (UniProtKB/ TrEMBL:Q9M519) were selected for computer-aided simulation of pepsin hydrolysis (pH > 2). There were sixty-four peptide fragments and seven amino acid residues (A, L, Q, E, F, Y, and W) were released from dioscorin A in a computer-aided simulation hydrolysis (Additional file 1: Figure S1). The deduced sequences of dioscorin B (UniProtKB/TrEMBL:Q9M501) were selected for simulation of pepsin hydrolysis (pH > 2). There were sixty-five peptide fragments and eight amino acid residues (A, I, L, Q, E, F, Y, and W) were released from dioscorin B in a computer-aided simulation hydrolysis (Additional file 1: Figure S2). Following the general rules which the peptide contained aromatic or branched-chain aliphatic amino acids closed to the COOH-terminus for ACE inhibitions (Cheung et al. 1980), the twenty-three peptides were selected for syntheses in order to test ACE inhibitory activity. In assay system, the substrate FAPGG was hydrolyzed by ACE to generate FAP and dipeptide GG released, and the absorbance at 345 nm was decreased. From the results of Figure 1, under the same concentration of 40 μM for each peptide, the KTCGY (No.4) and KRIHF (No. 7) showed the first two potent ACE inhibitory peptides among 23 synthesized peptides. The KTCGY and KRIHF with higher than 50% ACE inhibitory activity were selected for further ex vivo and in vivo antihypertensive experiments. Table 1 showed the information of KTCGY and KRIHF in yam dioscorin.

Discussion
The present ex vivo and in vivo data showed KTCGY and KRIHF with vasodilating effects against PE-induced endothelium-intact aortic tensions and lowering SHR's SBP which were derived from a computer-aided simulation of pepsin hydrolysis of the yam tuber dioscorin. The penta-peptide of KTCGY was positioned at amino acid residue 52 to residue 56 of dioscorin B protein (UniProtKB/TrEMBL:Q9M501, Fig. S2), and the pentapeptide of KRIHF was positioned at amino acid residue 118 to residue 122 of dioscorin A protein (UniProtKB/ TrEMBL:Q9M519, Fig. S1) and dioscorin B protein (UniProtKB/TrEMBL:Q9M501, Fig. S2). Conlan et al. (1995) first reported two classes of cDNA clones encoding major yam tuber storage proteins from Dioscorea cayenensis and named as dioscorin. We demonstrated that dioscorins from six cultivars of three Dioscorea species accounted over 90% of water-soluble proteins by immune stains (Hou et al. 2000). The processed yam tuber is a traditional Chinese medicine used as a juvenescent substance, with no apparent side effects after long-term uses; the tuber is involved in the top levels in a famous Chinese pharmacopoeia. It was recently reviewed that yam dioscorin and its peptic hydrolysates exhibited several biological activities in vitro and in vivo (Lu et al. 2012). It seems that yam or its major protein, dioscorin, may be potentials in developments of several aspects of functional food and needed further investigations.
From the present results of ACE inhibitory activity screenings (Figure 1 . These potent ACE inhibitory peptides were promised to the general rules which peptides contained aromatic or branched-chain aliphatic amino acids closed to the COOH-terminus for ACE inhibitions (Cheung et al. 1980). The dioscorin peptic hydrolysates were reported to exhibit ACE inhibitory activity (Hsu et al. 2002) and antihypertensive activity (Lin et al. 2006) using SHR as models. Therefore, the synthetic KTCGY and KRIHF were used for ex vivo and in vivo experiments. KTCGY and KRIHF showed endothelium-dependent aortic ring relaxations in PEinduced tensions (Figure 2A, D) which the vasodilating effects were similar to ACE inhibitor, captopril ( Figure 2E). The captopril is designed to inhibit ACE, one of important enzymes in renin-angiotensin system, to achieve antihypertension (Rubin et al. 1978). It was reported that captopril also showed endothelium-dependent rabbit aortic ring relaxations in PE-induced tensions, and the possible action mechanism might be from its superoxide radical scavenging activity of its structural sulfhydryl group (Mittra and Singh 1998). Hernández-Ledesma et al. (2005) reported that individual amino acids exhibited different oxygen radical absorbance capacity (ORAC) activities, such as Trp [W, 4.649 μmole trolox equivalents (TE)/ μmole], Tyr (Y, 1.574 μmole TE/μmole), Met (M, 1.126 μmole TE/μmole), Cys (0.1492 μmole TE/μmole), His (H, 0.073 μmole TE/μmole), and Phe (F, 0.0025 μmole TE/μmole), which a Trp-indole group, Tyr-phenolic group, His-imidazole group, and Cys-sulfhydryl group served as hydrogen donors to contribute ORAC activity. It was reported that the ovokinin (2-7) (a hexa-peptide, RADHPF) isolated from chymotryptic ovalbumin hydrolysates exhibited dose-dependently nitric oxide-stimulated vaso-relaxations in endothelium-intact PE-induced tensions of SHR's mesenteric artery, however, ovokinin (2-7) showed less effect on ACE inhibition (IC 50 higher than 1 mM) (Matoba et al. 1999). Therefore, we propose that vaso-relaxing peptides of KTCGY and KRIHF in Figure 3 Antihypertensive activity of (A) KTCGY and (B) KRIHF on systolic blood pressure of spontaneously hypertensive rats (SHRs). KTCGY or KRIHF at concentration of 10 mg/kg and 20 mg/kg was orally administered to the SHRs (6 rats/group), and the SBP were measured after 0, 2, 4, 6, 8, and 24 h by using an indirect tail-cuff blood pressure meter (BP98-A, Softron, Tokyo, Japan). Distilled water (0.5 ml) was administered to the SHRs in the blank group. The captopril (10 mg/kg) was used as the positive control. Data were expressed as mean ± S.D. For animal experiments, the differences between the blank and the experimental group at the same time was analyzed using Student's t-test, and the P-value of less than 0.05 (*), 0.01 (**), and 0.001 (***) were recognized as different significantly.
endothelium-intact aortic rings may involve radical scavenging and nitric oxide stimulating activities and need further investigations.
The KTCGYKTCGY and TCGYTCGY showed endothelium-intact and endothelium-denuded vaso-relaxing activities in PE-induced tensions which were different from the action mode of KTCGY of above-mentioned. The ovokinin (an octa-peptide, FRADHPFL) isolated from peptic hydrolysates of egg ovalbumin showed bradykinin B1 receptor antagonist and exhibited endothelium-independent canine mesenteric artery relaxations in prostaglandin F2α-induced tensions (Fujita et al. 1995). Recently, a series of experiments by Matsui team demonstrated that WH di-peptide showed endothelium-independent SD rat aortic ring relaxations in KCl-induced contractions by suppressing Ca 2+ influx (Tanaka et al. 2008), which the inhibition of extracellular calcium entry into vascular smooth muscle cells by blocking dihydropyridine-like L-type Ca 2+ channels (Wang et al. 2010), and the inhibition against voltage-dependent L-type Ca 2+ channels phosphorylation (Kobayashi et al. 2012). The HRW tri-peptide also showed endothelium-independent vaso-relaxing activities in PE-induced tensions by inhibiting extracellular Ca 2+ influx (Tanaka et al. 2009). Therefore, it is proposed that KTCGYKTCGY and TCGYTCGY with endotheliumindependent vaso-relaxing activities in PE-induced tensions might involve Ca 2+ influx and Ca 2+ channels and needed investigations further.
From the present results of in vivo SHR oral administration, both penta-peptides of KTCGY and KRIHF at doses of 10 and 20 mg/kg exhibited antihypertensive activities by lowering SBP, but not diastolic blood pressure (data not shown), among which KTCGY of 20 mg/kg exhibited the similar lowering SBP profile to captopril of 10 mg/kg after a single oral administration. However, the vasorelaxing peptide with antihypertensive activity is not necessary for potent ACE inhibition. Therefore, it might loss some antihypertensive peptides from ACE inhibitory screenings in the present study. The RF di-peptide (Kagebayashi et al. 2012) and IHRF tetra-peptide (Kontani et al. 2014) isolated from rice glutelin with lower ACE inhibition was reported to exhibit cholecystokinindependent vaso-relaxing and antihypertensive activities in SHR, which the RF di-peptide was the same as No. 17 synthesized peptide in Figure 1 of less ACE inhibitory activity in the present study from computer-aided simulation of pepsin hydrolysis of yam dioscorin A (residues of 134-135 and 158-159) and yam dioscorin B (residues of 158-159). However, these results provide evidences to support yam dioscorin after ingestion for blood pressure regulations.

Conclusions
The KTCGY and KRIHF show vasodilating effects and can reduce SHR's systolic blood pressure which may contribute important roles in yam dioscorin for regulating blood pressure in vivo and will be beneficial for antihypertension in functional food preparations.