Antihypertensive effects of Undaria pinnatifida (wakame) peptide on blood pressure in spontaneously hypertensive rats

We examined the angiotensin I-converting enzyme (ACE) inhibitory activity and antihypertensive effect of the hot water extract of wakame, Undaria pinnatifida. Ten dipeptides were isolated from the extract by several steps of chromatography, and their amino acid sequences were Tyr-His, Lys-Trp, Lys-Tyr, Lys-Phe, Phe-Tyr, Val-Trp, Val-Phe, Ile-Tyr, Ile-Trp, and Val-Tyr. Both single administration and repeated oral administration of synthetic Tyr-His, Lys-Tyr, Phe-Tyr, and Ile-Tyr significantly decreased blood pressure in spontaneously hypertensive rats.     

Antihypertensive effect of tryptic hydrolysate of milk casein in spontaneously hypertensive rats

1. Repeated oral administrations of tryptic hydrolysate of bovine milk casein (CEI) showed antihypertensive effect in spontaneously hypertensive rats. 2. Single oral administration of CEI antagonized the pressor response to angiotensin I. 3. Bovine milk casein hydrolysate inhibited the angiotensin I-converting enzyme (ACE) activity. Three peptides with ACE-inhibiting activity were isolated from CEI. 4. It is suggested that ACE-inhibiting peptides in the tryptic hydrolysate milk casein are absorbed from the intestinal tract and produce an antihypertensive effect.     

Gastrointestinal enzyme production of bioactive peptides from royal jelly protein and their antihypertensive ability in SHR

In order to clarify the potential physiological function of royal jelly (RJ), we report here the gastrointestinal enzyme production of antihypertensive peptides from RJ. Intact RJ and its protein fraction did not retard the action of angiotensin I-converting enzyme (ACE) activity at all. However, development of ACE inhibition power of RJ was newly observed by pepsin hydrolysis (IC(50)=0.358 mg protein/mL), and the subsequent trypsin and chymotrypsin hydrolyses (IC(50)=0.099 mg protein/mL). Single oral administration of this gastrointestinal RJ hydrolysate (1 g/kg dose) in 10-week spontaneously hypertensive rat resulted in a significant reduction of systolic blood pressure of 22.7 plus minus 3.6 mmHg at 2 hr (P<0.05 vs. 0 hr by one-way ANOVA, n=7). Then, the RJ hydrolysate was fractionated with gel permeation chromatography to obtain the di- and tri-peptides (DTP) fraction. As a result of isolation from the DTP fraction by reversed phase-high performance liquid chromatography, eleven ACE inhibitory peptides were isolated from the DTP-RJ hydrolysate. Some of the ACE inhibitors were derived from the RJ-glycoprotein; eight peptides with the IC(50) value of <10 &mgr;M were identified from natural resources for the first time. Consequently, RJ protein was thought to be a good resource of ACE inhibitory peptides produced by the gastrointestinal enzyme hydrolyses.     

Novel Angiotensin I-converting enzyme inhibitory peptides found in a thermolysin-treated elastin with antihypertensive activity

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC50 value of 531.6 μg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 μg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC50 value of 244 μM against ACE and may have potential use as a functional food.     

Novel Angiotensin I-Converting Enzyme Inhibitory Peptides Found in a Thermolysin-Treated Elastin with Antihypertensive Activity

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC₅₀ value of 531.6 µg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 µg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC₅₀ value of 244 µM against ACE and may have potential use as a functional food.     

The antihypertensive activity of angiotensin-converting enzyme inhibitory peptide containing in bovine lactoferrin

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the bovine lactoferrin hydrolysate using peptic hydrolysis by 2-step of reverse-phase high-performance liquid chromatography. This peptide was identified as Leu-Arg-Pro-Val-Ala-Ala and it produced a concentration-dependent inhibition of ACE activity in vitro with an IC50 value of about 4.14 microM. Also, this inhibition was identified as noncompetitive from the Lineweaver-Burk plot. Moreover, the antihypertensive activity of Leu-Arg-Pro-Val-Ala-Ala was investigated by the intravenous injection into spontaneously hypertensive rats (SHRs). A dose-dependent reduction of systolic blood pressure by this peptide was observed at 60 min after injection and it maximally decreased the blood pressure at a rate of 1 nmol/ml/kg. The blood pressure lowering activity of this peptide was calculated as 210% of captopril (10 pmol/ml/kg) that was used as positive control. Otherwise, identification of this peptide in the blood of SHRs was carried out chromatographically. Reduction of blood pressure coincides with the peak peptide concentration in the serum. Thus, we conclude that this peptide inhibits ACE activity in vitro and lowers systolic blood pressure in spontaneously hypertensive rat.     

New antihypertensive peptides isolated from rapeseed

Four potent angiotensin converting enzyme (ACE) inhibitory peptides, IY, RIY, VW and VWIS, were isolated from subtilisin digest of rapeseed protein. Among them RIY and VWIS are new peptides with IC(50) 28 and 30 microM, respectively. All isolated peptides lowered blood pressure of spontaneously hypertensive rats (SHR) following oral administration. The maximum effect in the case of RIY was observed 4h after administration, while maximum effect of other peptides on blood pressure occurred 2h after administration. Furthermore, the antihypertensive effect of RIY was observed even in old rats, in which ACE inhibitors become less effective, suggesting that a different mechanism other than ACE inhibition is also involved in lowering blood pressure by this peptide. Subtilisin digest of rapeseed protein also significantly lowered blood pressure of SHR after oral administration of a single dosage 0.15 g/kg, exerting maximum antihypertensive effect 4h after administration. This digest appears promising as a functional food, which may be useful in the prevention and treatment of hypertension.     

Antihypertensive effect of angiotensin I-converting enzyme inhibitory peptides from a sesame protein hydrolysate in spontaneously hypertensive rats

Sesame peptide powder (SPP) exhibited angiotensin I-converting enzyme (ACE) inhibitory activity, and significantly and temporarily decreased the systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs) by a single administration (1 and 10 mg/kg). Six peptide ACE inhibitors were isolated and identified from SPP. The representative peptides, Leu-Val-Tyr, Leu-Gln-Pro and Leu-Lys-Tyr, could competitively inhibit ACE activity at respective Ki values of 0.92 microM, 0.50 microM, and 0.48 microM. A reconstituted sesame peptide mixture of Leu-Ser-Ala, Leu-Gln-Pro, Leu-Lys-Tyr, Ile-Val-Tyr, Val-Ile-Tyr, Leu-Val-Tyr, and Met-Leu-Pro-Ala-Tyr according to their content ratio in SPP showed a strong antihypertensive effect on SHR at doses of 3.63 and 36.3 microg/kg, which accounted for more than 70% of the corresponding dosage for the SPP-induced hypotensive effect. Repeated oral administration of SPP also lowered both SBP and the aortic ACE activity in SHR. These results demonstrate that SPP would be a beneficial ingredient for preventing and providing therapy against hypertension and its related diseases.     

Antihypertensive Effects of Peptides in Autolysate of Bonito Bowels on Spontaneously Hypertensive Rats

An autolysate of bonito bowels was treated with ultrafiltration, loose R0 concentration, ion-exchange chromatography, and reverse phase chromatography to increase its potency to inhibit angiotensin I-converting enzyme (ACE) activity by 16-fold. Oral administration of the partially purified autolysate decreased the systolic blood of spontaneously hypertensive rats (SHR) in a dose-dependent manner at the doses of 1g peptides/kg or higher. The relationship between the antihypertensive activity (in vivo) of the partially purified preparation and its ACE inhibitory activity (in vitro) in comparison with previously reported ACE inhibitory peptides is discussed.     

Antihypertensive effect of ACE inhibitory oligopeptides from chicken egg yolks

Oligopeptides of 1 KDa or less were obtained by hydrolysis of chicken egg yolks with a crude enzyme, and by dialysis with a semipermeable membrane filter. Since the extracted peptides had an inhibitory action on the activity of angiotensin I-converting enzyme (ACE) in vitro, they were orally administered at 20, 100 and 500 mg/kg body weight to spontaneously hypertensive rats (SHR) for 12 weeks to analyze the physiological role on cardiovascular functions. The administered oligopeptides suppressed the development of hypertension at all dosages. After 12 weeks at 500 mg/kg body weight, the values for systolic, mean, and diastolic blood pressure were approximately 10% less in SHRs administered than controls. Furthermore, serum ACE activity of the peptide-administered groups was significantly lower than that of the control group in a dose-related manner. Our results imply that oligopeptides extracted from hen's egg yolks could potentially suppress the development of hypertension in SHR, and this effect might be induced by the inhibition of ACE activity.     

Effect of an excess intake of casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro in subjects with normal blood pressure, high-normal blood pressure, or mild hypertension

A double-blind, placebo-controlled, randomized clinical trial was conducted to evaluate the effects of ingesting an excess of tablets containing casein hydrolysate, incorporating angiotensin I-converting enzyme (ACE) inhibitory peptides such as Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), in subjects with blood pressure ranging from normal to mild hypertension. A total of 48 subjects were given either 5 times more than the effective amount of casein hydrolysate or a placebo in tablet form for 4 weeks. In the active group, systolic blood pressure (SBP) decreased significantly as compared with the placebo group. In stratified analysis, however, this antihypertensive effect was not found in normotensive subjects. In addition, neither an acute or nor an excessive reduction in blood pressure nor clinically important adverse events were observed in this study. These findings suggest that intake of a 5-fold excess of tablets containing casein hydrolysate can lead to a mild improvement in hypertension without side effects.     

Effect of an Excess Intake of Casein Hydrolysate Containing Val-Pro-Pro and Ile-Pro-Pro in Subjects with Normal Blood Pressure,High-Normal Blood Pressure,or Mild Hypertension

A double-blind, placebo-controlled, randomized clinical trial was conducted to evaluate the effects of ingesting an excess of tablets containing casein hydrolysate, incorporating angiotensin I-converting enzyme (ACE) inhibitory peptides such as Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), in subjects with blood pressure ranging from normal to mild hypertension. A total of 48 subjects were given either 5 times more than the effective amount of casein hydrolysate or a placebo in tablet form for 4 weeks. In the active group, systolic blood pressure (SBP) decreased significantly as compared with the placebo group. In stratified analysis, however, this antihypertensive effect was not found in normotensive subjects. In addition, neither an acute or nor an excessive reduction in blood pressure nor clinically important adverse events were observed in this study. These findings suggest that intake of a 5-fold excess of tablets containing casein hydrolysate can lead to a mild improvement in hypertension without side effects.     

Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatifida)

A peptide fraction having activity against angiotensin I-converting enzyme (ACE) was separated from the peptic digest of protein prepared from wakame (Undaria pinnatifida) by ion-exchange chromatographies and gel-filtration. Fractions with high ACE inhibitory activity were combined and further chromatographed on a reverse-phase column to yield four tetrapeptides with ACE inhibitory properties. These tetrapeptides were identified by sequence analysis and fast atom bombardment mass spectrometry as Ala-Ile-Tyr-Lys (IC(50): 213 microM), Tyr-Lys-Tyr-Tyr (64.2 microM), Lys-Phe-Tyr-Gly (90.5 microM), and Tyr-Asn-Lys-Leu (21 microM). Each tetrapeptide was synthesized and its antihypertensive activity was determined after oral administration in spontaneously hypertensive rats. The blood pressure significantly decreased after tetrapeptide ingestion. The present study demonstrated that dietary wakame may have beneficial effects on hypertension.     

Blood pressure-depressing activity of a peptide derived from silkworm fibroin in spontaneously hypertensive rats

Peptides showing inhibitory activity against the angiotensin I-converting enzyme (ACE) were investigated from the fibroin fraction of discarded silk fabric. Fibroin, which was hydrolyzed with alcalase after partial hydrolysis with hot aqueous 40% CaCl(2), released two major active peptides showing ACE-inhibitory activity. The two peptides were identified as glycyl-valyl-glycyl-tyrosine (GVGY) and glycyl-valyl-glycyl-alanyl-glycyl-tyrosine (GVGAGY) by analyses with a protein sequencer and LC/MS/MS. GVGY, whose ACE-inhibitory activity has not previously been reported, showed a blood pressure-depressing effect on spontaneously hypertensive rat (SHR).     

Analysis of novel angiotensin-I-converting enzyme inhibitory peptides from protease-hydrolyzed marine shrimp Acetes chinensis.

Acetes chinensis is an underutilized shrimp species thriving in the Bo Hai Gulf of China. In a previous study, we had used the protease from Bacillus sp. SM98011 to digest this kind of shrimp and found that the oligopeptide-enriched hydrolysate possessed antioxidant activity and high angiotensin I-converting enzyme (ACE) inhibitory activity with an IC50 value of 0.97 mg/ml. In this paper, by ultrafiltration, gel permeation chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC), five peptides with high ACE inhibitory activity were purified from the shrimp hydrolysates and their sequences were identified by amino acid composition analysis and molecular weight (MW) analysis. Three of them, FCVLRP (a), IFVPAF (f) and KPPETV (j), were novel ACE inhibitory peptides. Their IC50 values were 12.3 microM, 3.4 microM and 24.1 microM, respectively, and their recoveries were 30 mg/100 g (solid basis of shrimp), 19 mg/100 g and 33 mg/100 g, respectively. Lineweaver-Burk plots for the three novel peptides showed that they are all competitive inhibitors. To test the ACE inhibitory activity of peptide a, f, j after they were digested by digestive enzymes in vivo, 12 derived peptides from FCVLRP and IFVPAF were synthesized based on their amino acid sequences and the cleavage sites of digestive enzymes. No digestive enzyme cleavage site was found in KPPETV. The IC50 values of the derived peptides were determined and the result showed that except for VPAF, FC and FCVL, the ACE inhibitory activity of the other nine derived peptides did not significantly change when compared with their original peptides. Surprisingly, five peptides had lower IC50 values than their original peptides, particularly for RP (IC50 value = 0.39 microM), which is about 30 times lower than its original peptide and almost the lowest IC50 value for ACE inhibitory peptides reported. Therefore, the novel peptides identified from A. chinensis hydrolysates probably still maintain a high ACE inhibitory activity even if they are digested in vivo. This is the first report about novel ACE inhibitory peptides from hydrolysates of marine shrimp A. chinensis. The novel peptides from hydrolysate of A. chinensis and some of their derived peptides with high ACE inhibitory activity probably have potential in the treatment of hypertension or in clinical nutrition.     

Sugar Nucleotides from the Fungus Aureobasidium

Peptides showing inhibitory activity against the angiotensin I-converting enzyme (ACE) were investigated from the fibroin fraction of discarded silk fabric. Fibroin, which was hydrolyzed with alcalase after partial hydrolysis with hot aqueous 40% CaCl₂, released two major active peptides showing ACE-inhibitory activity. The two peptides were identified as glycyl-valyl-glycyl-tyrosine (GVGY) and glycyl-valyl-glycyl-alanyl-glycyl-tyrosine (GVGAGY) by analyses with a protein sequencer and LC/MS/MS. GVGY, whose ACE-inhibitory activity has not previously been reported, showed a blood pressure-depressing effect on spontaneously hypertensive rat (SHR).     

Antihypertensive Effect of Angiotensin I-Converting Enzyme Inhibitory Peptides from a Sesame Protein Hydrolysate in Spontaneously Hypertensive Rats

Sesame peptide powder (SPP) exhibited angiotensin I-converting enzyme (ACE) inhibitory activity, and significantly and temporarily decreased the systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs) by a single administration (1 and 10 mg/kg). Six peptide ACE inhibitors were isolated and identified from SPP. The representative peptides, Leu-Val-Tyr, Leu-Gln-Pro and Leu-Lys-Tyr, could competitively inhibit ACE activity at respective Ki values of 0.92 μM, 0.50 μM, and 0.48 μM. A reconstituted sesame peptide mixture of Leu-Ser-Ala, Leu-Gln-Pro, Leu-Lys-Tyr, Ile-Val-Tyr, Val-Ile-Tyr, Leu-Val-Tyr, and Met-Leu-Pro-Ala-Tyr according to their content ratio in SPP showed a strong antihypertensive effect on SHR at doses of 3.63 and 36.3 μg/kg, which accounted for more than 70% of the corresponding dosage for the SPP-induced hypotensive effect. Repeated oral administration of SPP also lowered both SBP and the aortic ACE activity in SHR. These results demonstrate that SPP would be a beneficial ingredient for preventing and providing therapy against hypertension and its related diseases.     

Novel angiotensin I-converting enzyme inhibitory peptides isolated from Alcalase hydrolysate of mung bean protein.

Mung bean protein isolates were hydrolyzed for 2 h by Alcalase. The generated hydrolysate showed angiotensin I-converting enzyme (ACE) inhibitory activity with the IC(50) value of 0.64 mg protein/ml. Three kinds of novel ACE inhibitory peptides were isolated from the hydrolysate by Sephadex G-15 and reverse-phase high performance liquid chromatography (RP-HPLC). These peptides were identified by amino acid composition analysis and matrix assisted-laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), as Lys-Asp-Tyr-Arg-Leu, Val-Thr-Pro-Ala-Leu-Arg and Lys-Leu-Pro-Ala-Gly-Thr-Leu-Phe with the IC(50) values of 26.5 microM, 82.4 microM and 13.4 microM, respectively.     

Vasodilating effect of di-peptides in thoracic aortas from spontaneously hypertensive rats

In this study, we found that antihypertensive di-peptide Val-Tyr (VY) showed a vascular relaxation effect in KCl-induced contraction of thoracic aorta rings from 18-week-old spontaneously hypertensive rats among di-peptides of VY, Ile-Tyr, and Tyr-Val irrespective of their angiotensin I-converting enzyme inhibitory activity. The effect was endothelium-independent, and was closely associated with vascular responses in the vascular smooth muscle layer.     

Latent production of angiotensin I-converting enzyme inhibitors from buckwheat protein.

The latent production of angiotensin I-converting enzyme (ACE) Inhibitors from tartary buckwheat (BW) was investigated, and the peptides responsible for ACE inhibition characterized. Intact buckwheat was found to exhibit ACE inhibitory activity having an IC50 value of 3.0 mg/ml. The activity of the protein fraction (IC50: 0.36 mg protein/ml) was not enhanced by pepsin treatment. Pepsin, followed by chymotrypsin and trypsin hydrolysis, resulted in a significant increase in the ACE inhibitory activity (IC50: 0.14 mg protein/ml). The rutin contained in the buckwheat did not exhibit any ACE inhibition. A single oral administration of BW digest lowered the systolic blood pressure of a spontaneously hypertensive rat. Thus, BW proteins offer a potential resource for producing ACE inhibitory peptides during the digestion process. From the di-/tri-peptide fraction (DTPF) of the BW digest, inhibitory peptides were identified. The magnitude (%) of the total ACE inhibitory contribution of each identified peptide, relative to the overall inhibition of the DTPF, was about 41%.