Angiotensin I converting enzyme (ACE) inhibitory activity of hetero-chitooligosaccharides prepared from partially different deacetylated chitosans

Angiotensin I converting enzyme (ACE) inhibitory activity of hetero-chitooligosaccharides (hetero-COSs) prepared from partially different deacetylated chitosans was investigated. Partially deacetylated chitosans, 90, 75, and 50% deacetylated chitosan, were prepared from crab chitin by N-deacetylation with 40% sodium hydroxide solution for durations. In addition, nine kinds of hetero-COSs with relatively high molecular masses (5000-10 000 Da; 90-HMWCOSs, 75-HMWCOSs, and 50-HMWCOSs), medium molecular masses (1000-5000 Da; 90-MMWCOSs, 75-MMWCOSs, and 50-MMWCOSs), and low molecular masses (below 1000 Da; 90-LMWCOSs, 75-LMWCOSs, and 50-LMWCOSs) were prepared using an ultrafiltration membrane bioreactor system. ACE inhibitory activity of hetero-COSs was dependent on the degree of deacetylation of chitosans. 50-MMWCOSs that are COSs hydrolyzed from 50% deacetylated chitosan, the relatively lowest degree of deacetylation, exhibited the highest ACE inhibitory activity, and the IC(50) value was 1.22 +/- 0.13 mg/mL. In addition, the ACE inhibition pattern of the 50-MMWCOSs was investigated by Lineweaver-Burk plots, and the inhibition pattern was found to be competitive.     

Angiotensin I converting enzyme inhibitory peptides from in vitro pepsin-pancreatin digestion of soy protein

Angiotensin I converting enzyme (ACE) inhibitory activity was determined in the soy protein isolate (SPI) digest produced by in vitro pepsin-pancreatin sequential digestion. The inhibitory activity was highest within the first 20 min of pepsin digestion and decreased upon subsequent digestion with pancreatin. An IC(50) value of 0.28 +/- 0.04 mg/mL was determined after 180 min of digestion, while no ACE inhibitory activity was measured for the undigested SPI at 0.73 mg/mL. Chromatographic fractionation of the SPI digest resulted in IC(50) values of active fractions ranging from 0.13 +/- 0.03 to 0.93 +/- 0.08 mg/mL. Although many of the fractions showed ACE inhibition, peptides with lower molecular masses and higher hydrophobicities were most active. The findings show that many different peptides with ACE inhibitory activities were produced after in vitro pepsin-pancreatin digestion of SPI and lead to the speculation that physiological gastrointestinal digestion could also yield ACE inhibitory peptides from SPI.     

Affinity purification of angiotensin converting enzyme inhibitory peptides using immobilized ACE

A lung extract rich in angiotensin converting enzyme (ACE) and pure ACE were immobilized by reaction with the activated support 4 BCL glyoxyl-agarose. These immobilized ACE derivatives were used for purification of ACE inhibitory peptides by affinity chromatography. The immobilized lung extract was used to purify inhibitory peptides from sunflower and rapeseed protein hydrolysates that had been obtained by treatment of protein isolates with alcalase. The ACE binding peptides that were retained by the derivatives were specifically released by treatment with the ACE inhibitor captopril and further purified by reverse-phase C18 HPLC chromatography. Inhibitory peptides with IC50 50 and 150 times lower than those of the original sunflower and rapeseed hydrolysates, respectively, were obtained. The derivative prepared using pure ACE was used for purification of ACE inhibitory peptides from the same type of sunflower protein hydrolysate. ACE binding peptides were released from the ACE-agarose derivatives by treatment with 1 M NaCl and had an IC50 a little higher than those obtained using immobilized extract and elution with captopril. Affinity chromatography facilitated the purification of ACE inhibitory peptides and potentially other bioactive peptides present in food proteins.     

Release of angiotensin I-converting enzyme inhibitory peptides from flaxseed (Linum usitatissimum L.) protein under simulated gastrointestinal digestion

The scope of this study was to determine the ability of flaxseed (Linum usitatissimum L.) proteins to release angiotensin I-converting enzyme inhibitory (ACEI) peptides during simulated gastrointestinal (GI) digestion using a static (SM; no absorption in the intestinal phase) and a dynamic model (DM; simultaneous absorption of digested products in the intestinal phase via passive diffusion). Gastric and gastric + small intestinal digests of flaxseed proteins of both models possessed ACEI activity. The ACEI activity of the gastric + small intestinal digest in the DM (IC(50) unabsorbed, 0.05 mg N/mL; IC(50) absorbed, 0.04 mg N/mL) was significantly higher (p < 0.05) than that of the SM (IC(50), 0.39 mg N/mL). Two peptides, a pentapeptide (Trp-Asn-Ile/Leu-Asn-Ala) and a hexapeptide (Asn-Ile/Leu-Asp-Thr-Asp-Ile/Leu), were identified in the most active ACEI fraction (0.5-1 kDa) of the absorbable flaxseed protein digest by de novo sequencing.     

Peptides with angiotensin I-converting enzyme (ACE) inhibitory activity from defibrinated,hydrolyzed bovine plasma

Defibrinated bovine plasma (DBP) was treated with the microbial protease Flavourzyme to obtain protein hydrolysates with various degrees of hydrolysis (DH). The angiotensin I-converting enzyme (ACE) inhibiting activity of the hydrolyzed protein was assessed with hippuryl-His-Leu as the substrate. The amount of hippuric acid released, due to uninhibited ACE activity, was determined by high-performance liquid chromatography. ACE inhibiting (ACEI) activity was found to increase with increasing DH; the 43% DH hydrolysate exhibited the highest activity and had an IC(50) of 1.08 mg/mL. Peptide fractions with high ACEI activity were isolated using size exclusion chromatography. The fraction that possessed the highest ACEI activity contained peptides with GYP, HL(I), HPY, HPGH, L(I)F, SPY, and YPH sequence motifs, as determined by reversed-phase liquid chromatography-tandem mass spectrometry using a novel immonium precursor-ion scanning technique. Some of these motifs correspond to sequences found in bovine serum albumin, a potential source of ACEI peptides in bovine plasma.     

Angiotensin converting enzyme inhibitory activity in commercial fermented products. Formation of peptides under simulated gastrointestinal digestion

The angiotensin converting enzyme (ACE)-inhibitory activity of several commercial fermented milks was evaluated. Most of these products showed moderate inhibitory activity, but a few exceptions were detected. The high ACE-inhibitory activity found in some cases could be related to the origin of the milk. Two of these products were subjected to an enzymatic hydrolysis process, which simulates physiological digestion, to study the influence of digestion on ACE-inhibitory activity. The activity did not significantly change or increase during simulated gastrointestinal digestion. The peptides generated from one selected product during simulated digestion were sequenced by tandem spectrometry. Most peptides found at the end of the simulated digestion were released after 30 min of incubation with the pancreatic extract. This suggests that physiological digestion promotes the formation of active peptides from the proteins present in these fermented products. The potential ACE-inhibitory activity of the identified peptides is discussed with regard to their amino acid sequences.     

A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate

Alaska pollack frame protein, which is normally discarded as an industrial byproduct in the processing of fish in plants, was hydrolyzed with pepsin. This was fractionated into five major types of Alaska pollack frame protein hydrolysates (APH-I, 10-30 kDa; APH-II, 5-10 kDa; APH-III, 3-5 kDa; APH-IV, 1-3 kDa; and APH-V, below 1 kDa) using an ultrafiltration membrane bioreactor system. Angiotensin I converting enzyme (ACE) inhibitory activities of the fractionated hydrolysates were investigated, and the fraction that exhibited the highest ACE inhibitory activity was further purified using consecutive chromatographic methods on SP-Sephadex C-25 column, Sephadex G-25 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column. Finally, we purified a novel ACE inhibitory peptide with an IC50 value of 14.7 microM, and the sequence of the peptide was Phe-Gly-Ala-Ser-Thr-Arg-Gly-Ala. In addition, the ACE inhibition pattern of the peptide was found to be noncompetitive.     

Isolation and antihypertensive effect of angiotensin I-converting enzyme (ACE) inhibitory peptides from spinach Rubisco

Four new inhibitory peptides for angiotensin I-converting enzyme (ACE), that is, MRWRD, MRW, LRIPVA, and IAYKPAG, were isolated from the pepsin-pancreatin digest of spinach Rubisco with the use of HPLC. IC(50) values of individual peptides were 2.1, 0.6, 0.38, and 4.2 microM, respectively. MRW and MRWRD had an antihypertensive effect after oral administration to spontaneously hypertensive rats. Maximal reduction occurred 2 h after oral administration of MRW, whereas MRWRD showed maximal decrease 4 h after oral administration at doses of 20 and 30 mg/kg, respectively. IAYKPAG also exerted antihypertensive activity after oral administration at the dose of 100 mg/kg, giving a maximum decrease 4 h after oral administration. IAYKP, IAY, and KP, the fragment peptides of IAYKPAG, also exerted antihypertensive activity. LRIPVA [corrected] did not show any antihypertensive effect at a dose of 100 mg/kg despite its potent ACE-inhibitory activity.     

His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo.

It has been reported that soybean peptide fractions isolated from Korean fermented soybean paste exert angiotensin I converting enzyme (ACE) inhibitory activity in vitro. In this study, further purification and identification of the most active fraction inhibiting ACE activity were performed, and its antihypertensive activity in vivo was confirmed. Subsequently, a novel ACE inhibitory peptide was isolated by preparative HPLC. The amino acid sequence of the isolated peptide was identified as His-His-Leu (HHL) by Edman degradation. The IC(50) value of the HHL for ACE activity was 2.2 microg/mL in vitro. Moreover, the synthetic tripeptide HHL (spHHL) resulted in a significant decrease of ACE activity in the aorta and led to lowered systolic blood pressure (SBP) in spontaneously hypertensive (SH) rats compared to control. Triple injections of spHHL, 5 mg/kg of body weight/injection resulted in a significant decrease of SBP by 61 mmHg (p < 0.01) after the third injection. These results demonstrated that the ACE inhibitory peptide HHL derived from Korean fermented soybean paste exerted antihypertensive activity in vivo.     

Purification of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide with an antihypertensive effect from loach (Misgurnus anguillicaudatus)

To isolate and characterize novel angiotensin I-converting enzyme (ACE) inhibitory peptide from loach (Misgurnus anguillicaudatus), six proteases, pepsin, α-chymotrypsin, bromelain, papain, alcalase, and Neutrase, were used to hydrolyze loach protein. The hydrolysate (LPH) generated by bromelain [ratio of enzyme to substrate, 3:1000 (w/w)] was found to have the highest ACE inhibitory activity (IC(50), 613.2 ± 8.3 μg/mL). Therefore, it was treated by ultrafiltration to afford fraction of LPH-IV (MW < 2.5 kDa) with an IC(50) of 231.2 ± 3.8 μg/mL, having higher activity than the other fractions. Then, LPH-IV was isolated and purified by consecutive purification steps of gel filtration chromatography and reverse-phase high-performance liquid chromatography to afford a purified peptide with an IC(50) of 18.2 ± 0.9 μg/mL, an increase of 33.7-fold in ACE inhibitory activity as compared with that of LPH. The purified peptide was identified as Ala-His-Leu-Leu (452 Da) by Q-TOF mass spectrometry and amino acid analyzer. An antihypertensive effect in spontaneously hypertensive rats revealed that oral administration of LPH-IV could decrease systolic blood pressure significantly.     

Isoflavone content and its potential contribution to the antihypertensive activity in soybean Angiotensin I converting enzyme inhibitory peptides

A soybean angiotensin I converting enzyme (ACE) inhibitory peptide fraction was reported to have antihypertensive activity in a rat study. The purpose of the present study was to examine if the presence of isoflavones in the soybean ACE inhibitory peptide fraction may contribute to the blood-pressure-lowering property. The isoflavone concentration in soybean samples was analyzed on a C 18 reverse-phase column using a two-step gradient solvent system. Producing soybean hydrolysate led to a nearly 40% loss of isoflavones compared with the original soybean flour, but the isoflavone composition did not change and the dominant isoflavone chemicals remained as 6'-O-malonylgenistin and 6'-O-malonyldaidzin. Ion exchange chromatography affected significantly both the content and the composition of the isoflavones. The dominant isoflavones in the ion-exchanged fraction were aglycones and nonacylated isoflavones, accounting for 95.8% of the total amount of 987.7 microg/g. It was calculated that the isoflavone content in the soybean ACE inhibitory peptide fraction was 25 times less than the minimal effective isoflavone dose reported. In vitro study also showed that adding isoflavones into both soybean flour hydrolysate and soybean ACE inhibitory peptide samples to a concentration of as high as 31.5% (w/w) did not affect ACE inhibitory activity (IC 50 values). The findings along with previously published results indicated that the contribution of isoflavones in soybean ACE inhibitory peptide fraction to the blood-pressure-lowering property in a short-term feeding study might be negligible.     

Vascular effects, angiotensin I-converting enzyme (ACE)-inhibitory activity, and antihypertensive properties of peptides derived from egg white

In this study, we have identified novel antihypertensive peptides derived from egg-white proteins. The sequences YRGGLEPINF and ESIINF produced an acute blood-pressure-lowering effect in spontaneously hypertensive rats upon a single oral administration. Our results suggest that the antihypertensive action could be attributed to a vascular-relaxing mechanism that would occur in vivo independently of angiotensin I-converting enzyme (ACE) inhibition, because neither these peptides nor their main digestion fragments, except for the dipeptide YR, acted as ACE inhibitors in vitro. The vasodilator and antihypertensive activity of the sequences ESI and NF would explain the blood-pressure-lowering effect of ESIINF. With regard to YRGGLEPINF, in addition to NF, YR appeared as the main fragment responsible for its activity. The dipeptide YR, named kyotorphin and previously identified as an endogenous analgesic neuropeptide in the central nervous system, showed strong vasodilator and antihypertensive properties. The structure-activity features of the vasodilator peptides are discussed.     

Hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats

Angiotensin converting enzyme (ACE) inhibitory peptides prepared from soy protein by the action of alcalase enzyme was tested for its hypotensive effect on spontaneously hypertensive rats (SHR). Captopril, an ACE inhibitor used widely for hypertension treatment, was also applied in comparison. A significant (p < 0.05) decrease in systolic blood pressure of SHR was observed when soy ACE inhibitory peptides were orally administrated at three different dose levels (100, 500, and 1000 mg/kg of body weight/day), whereas little change occurred in the blood pressure of normotensive rats even at the highest dose. After a month-long feeding, blood pressure readings of SHR fell by approximately 38 mmHg from the original level at the lowest dose; a steadily and progressively hypotensive effect existed for these soy ACE inhibitory peptides administration groups. An obvious fluctuation was observed at the third week, although Captopril had a stronger hypotensive effect. The ACE activity of serum, aorta and lung, and lipid content of serum of SHR upon administration of soy ACE inhibitory peptides did not show a significant difference from that of the control group, whereas the serum ACE activity increased and the aorta ACE activity decreased significantly (p < 0.05) for the Captopril group. Serum Na(+) concentration decreased significantly in both the peptides-treated groups and the Captopril-treated group in comparison with the control group, whereas no lowering effect was observed for serum K(+) and serum Ca(2+) concentrations. These results suggested that the hypotensive effect of ACE inhibitory peptides derived from soy protein could be at least partly attributed to the action on salt/water balance.     

Release of flavonoids from lupin globulin proteins during digestion in a model system

Lupin seed globulin proteins form complexes with flavonoids, predominantly apigenin C-glycosides. Enzymes typical for the gastrointestinal tract were used to hydrolyze lupin seed globulins. Release of native flavonoids as a result of the proteolysis reaction was observed. Different analytical methods such as size exclusion chromatography, HPLC-MS, and fluorescence spectroscopy (steady-state fluorescence, fluorescence anisotropy, fluorescence lifetimes) were used for a detailed characterization of this phenomenon. Flavonoids liberated from lupin globulin proteins as a result of pancreatin-catalyzed digestion were bound by γ-conglutin resistant to this enzyme. Two possible mechanisms of this interaction may be suggested: hydrogen bonding between oligosaccharide chains of glycoproteins and the sugar moieties of the flavonoid glycosides or electrostatic attraction between positively charged γ-conglutin and flavonoids partially ionized at pH 7.5.     

Synthesis of angiotensin I-converting enzyme (ACE)-inhibitory peptides and gamma-aminobutyric acid (GABA) during sourdough fermentation by selected lactic acid bacteria

This article aimed at investigating the synthesis of angiotensin I-converting enzyme (ACE)-inhibitory peptides and gamma-aminobutyric acid (GABA) during sourdough fermentation of white wheat, wholemeal wheat, and rye flours. Sourdough lactic acid bacteria, selected previously for proteinase and peptidase activities toward wheat proteins or for the capacity of synthesizing GABA, were used. The highest ACE-inhibitory activity was found by fermenting flour under semiliquid conditions (dough yield 330) and, especially, by using wholemeal wheat flour. Fourteen peptides, not previously reported as ACE-inhibitory, were identified from the water/salt-soluble extract of wholemeal wheat sourdough (IC 50 0.19-0.54 mg/mL). The major part of the identified peptides contained the well-known antihypertensive epitope VAP. The synthesis of GABA increased when the dough yield was decreased to 160. The highest synthesis of GABA (258.71 mg/kg) was found in wholemeal wheat sourdough.     

Improved synthesis of lysine- and arginine-derived Amadori and Heyns products and in vitro measurement of their angiotensin I-converting enzyme inhibitory activity

The L-lysine- and L-arginine-derived Amadori and Heyns products consisting of N-(1-deoxy-d-fructos-1-yl)amino acid and N-(2-deoxy-d-glucos-2-yl)amino acid were prepared by reaction of d-fructose and d-glucose with l-lysine hydrochloride and l-arginine hydrochloride using commercial zinc powder as deprotonating reagent and also as catalyst precursor in a simple synthetic route in high yield. These compounds were screened for angiotensin I-converting enzyme (ACE) inhibitory activity using a high-throughput colorimetric assay (utilizing porcine kidney ACE). The IC(50) values fall in the range of 1030-1175 μM, with N(α)-(1-deoxy-d-fructos-1-yl)arginine showing the best IC(50) value (1030 ± 38 μM). This study demonstrates an improved synthetic method for simple Amadori and Heyns products and their moderate ACE inhibitor activity.     

Optimizing angiotensin I-converting enzyme inhibitory activity of Pacific hake (Merluccius productus) fillet hydrolysate using response surface methodology and ultrafiltration

The in vitro angiotensin I-converting enyzme (ACE) inhibitory activity of Pacific hake hydrolysates was investigated as a function of hydrolysis conditions, starting material variability, and ultrafiltration. Hake fillets were hydrolyzed using Protamex protease under various conditions of pH, hydrolysis time, and enzyme-to-substrate ratio (% E/S) according to a response surface methodology (RSM) central composite design. The hydrolysate produced at pH 6.5, 125 min, and 3.0% E/S had an IC 50 of 165 +/- 9 microg of total solids/mL. ACE-inhibitory activity was not significantly different (P < 0.05) for hydrolysates produced using higher time-enzyme combinations within the model or from fish of different catches. Ultrafiltration (10 kDa molecular mass cutoff) resulted in an IC50 value of 44 +/- 7 microg of peptides/mL, 2.5 times more potent than the commercial product PeptACE Peptides (IC50 = 114 +/- 8 microg of peptides/mL). These results suggest that hydrolysates prepared with minimal fractionation from Pacific hake, an undervalued fish, may be a commercially competitive source of ACE-inhibitory peptides.     

Bioaccessibility and antioxidant activity stability of phenolic compounds from extra-virgin olive oils during in vitro digestion

The impact of an in vitro procedure that mimics the physiochemical changes occurring in gastric and small intestinal digestion on the bioaccessibility and antioxidant activity of phenols from 10 extra-virgin olive oil samples was assessed. Extra-virgin olive oil phenols were totally extracted in the aqueous phase, which reproduces gastric fluids during the digestion procedure. A linear bioaccessibility model, based on tyrosol behavior in model oil samples, was used to estimate the bioaccessibility index (BI%) of extra-virgin olive oil phenols. The BI% varied amongst samples from a maximum of 90% to a minimum of 37%, thus indicating that only a fraction of phenols can be considered bioaccessible. The specific antioxidant activity of olive oil phenols proved to be negatively affected by the digestion procedure. By computing a principal component analysis, it was possible to show that differences in the potential bioactive effect of extra-virgin olive oil samples were related to different phenolic profiles.     

Retention of phenylalanine ammonia-lyase activity in wheat seedlings during storage and in vitro digestion

The retention of phenylalanine ammonia-lyase (PAL) activity in Red Spring wheat seedlings during storage and in vitro protein digestion was evaluated toward assessing the efficacy of plant PAL as a dietary supplement for patients suffering from the metabolic disease, phenylketonuria. Retention of PAL activity in freeze-dried wheat seedling tissues following three months of storage at -20 degrees C ranged from 62% in the leaf to 89% in root/residual seed tissues. After a 3-h two-stage ("gastric-intestinal") in vitro digestion, 36% and 42% recovery of PAL activity was associated with chopped fresh leaf and root/residual seed tissues respectively; however, no activity was recovered from freeze-dried tissues. High performance liquid chromatographic analysis of the residual phenylalanine (Phe) after in vitro digestion confirmed that the fresh tissues effected a significantly higher conversion of exogenous Phe than freeze-dried tissues. These results demonstrate that the plant cell walls provide protection of PAL during in vitro digestion. In cases where exogenous Phe (100 mg; 24 mM) was supplied to the tissues, the product of the reaction, trans-cinnamic acid, may have exerted a significant inhibitory effect on PAL activity.     

Evaluation of the stability and antioxidant activity of nanoencapsulated resveratrol during in vitro digestion

Resveratrol was encapsulated in oil-in-water food-grade nanoemulsions of subcellular size, produced by high-pressure homogenization. Physicochemical stability was evaluated under accelerated aging (high temperature and UV light exposure), as well as during simulated gastrointestinal digestion. Antioxidant activity was assessed at different stages of digestion by chemical assays and by an improved cellular assay, to measure exclusively the residual activity of resveratrol that penetrated inside Caco-2 cells. Results showed that the nanoemulsions based on soy lecithin/sugar esters and Tween 20/glycerol monooleate were the most physically and chemically stable, in terms of mean droplet size (always <180 nm) and resveratrol loading, during both accelerated aging and gastrointestinal digestion. These formulations also exhibited the highest chemical and cellular antioxidant activities, which was comparable to unencapsulated resveratrol dissolved in DMSO, suggesting that nanoencapsulated resveratrol, not being metabolized in the gastrointestinal tract, can be potentially absorbed through the intestinal wall in active form.