Kinetics of acetylcholinesterase inhibition by hemp seed protein-derived peptides.

The aim of this work was to enhance the acetylcholinesterase (AChE)-inhibitory activity of a pepsin-produced hemp seed protein hydrolysates (HPH) through reverse-phase HPLC separation followed by identification of peptide sequences present in the most active fraction. The HPH was separated into eight fractions (F1-F8) with F7 exhibiting significantly (p < 0.05) the strongest (97.5%) in vitro inhibition of electric eel AChE (eeAChE) activity in comparison to 53.8% for HPH. The HPH consisted mostly of low molecular weight peptides of < 11 amino acid residues and most contained at least one hydrophobic amino acid. Kinetics of enzyme inhibition revealed a mixed-type inhibition of eeAChE activity by HPH whereas F7 acted through an uncompetitive mode; in contrast inhibition of human AChE by HPH and F7 was uncompetitive. The stronger inhibitory potency of the F7 peptides fraction against both enzymes was confirmed through reduced maximal velocity, catalytic efficiency, and inhibition constant values when compared to the HPH. PRACTICAL APPLICATIONS: The use of natural products for the prevention or treatment of human diseases continues to be an area of intense research activities. Food protein-derived peptides obtained through enzymatic hydrolysis of hemp seed proteins were shown in vitro to be strong inhibitors of activities of both the eel and human forms of acetylcholinesterase (AChE). AChE is an important therapeutic target because excessive activity of this enzyme is a causative factor of neurodegenerative diseases such as dementia and Alzheimer's. This work showed that peptides in the most active fraction are small in sizes, which may favor their absorption into blood circulation and possible permeation of the blood-brain barrier. Therefore, the hemp seed peptides are potential agents that can be used to formulate functional foods and nutraceuticals against neurodegenerative diseases.

Effects of thermal processing on the nutritional and functional properties of defatted conophor nut (Tetracarpidium conophorum) flour and protein isolates.

Conophor nut (Tetracarpidium conophorum) was processed using different heat treatments to explore its full potential as food ingredients. The raw, boiled, and toasted nuts were defatted and the proteins isolated by alkaline solubilization and isoelectric precipitation. The variously processed nuts were analyzed for the proximate and amino acid compositions, and functional properties. The protein contents of the isolate ranges between 86.86 g/100g and 87.74 g/100 g, about 1.5-fold higher than those of the defatted flour samples. The essential amino acids of the isolates ranged between 40.57%-41.55%. Glutamic acid, aspartic acid, and arginine were the most predominant amino acids, while methionine and lysine were the first and second limiting amino acids, respectively. The protein efficiency ratio, biological values as well as the functional properties of the proteins were improved with processing. These properties may enhance the potential use of conophor nut protein isolates as high-quality protein ingredient in food systems.

Inhibitory properties of bambara groundnut protein hydrolysate and peptide fractions against angiotensin-converting enzymes, renin and free radicals.

BACKGROUND: An increased rate of high blood pressure has led to critical human hypertensive conditions in most nations. In the present study, bambara protein hydrolysates (BPHs) obtained using three different proteases (alcalase, trypsin and pepsin) and their peptide fractions (molecular weight: 10, 5, 3 and 1 kDa) were investigated for antihypertensive and antioxidant activities. RESULTS: Alcalase hydrolysate contained the highest amount of low molecular weight (LMW) peptides compared to pepsin and trypsin hydrolysates. LMW peptides fractions (<1 kDa) exhibited the highest inhibitory activity against angiotensin-converting enzyme (ACE) for all the enzymes hydrolysates. For renin inhibition, alcalase hydrolysate showed the highest inhibition at 59% compared to other hydrolysates and their corresponding membrane fractions. The antioxidant power of bambara protein hydrolysates and peptide fractions was evaluated through the inhibition of linoleic acid peroxidation and ABTS scavenging activity. Among the hydrolysates, alcalase exhibited the highest inhibition of linoleic acid oxidation. Furthermore, all BPHs were able to scavenge ABTS•+ to a three-fold greater extent compared to the isolate. CONCLUSION: BPH and LMW peptide fractions could potentially serve as useful ingredients in the formulation of functional foods and nutraceuticals against high blood pressure and oxidative stress. © 2016 Society of Chemical Industry.

Antihypertensive Properties of a Pea Protein Hydrolysate during Short- and Long-Term Oral Administration to Spontaneously Hypertensive Rats.

This study investigated short-term (24 h) and long-term (5 wk) systolic blood pressure (SBP)-lowering effects in spontaneously hypertensive rats (SHR) of a 5 kDa membrane pea protein hydrolysate permeate (PPH-5) produced through thermoase hydrolysis of pea protein isolate (PPI). Amino acid analysis showed that the PPH-5 had lower contents of sulfur-containing amino acids than the PPI. Size-exclusion chromatography indicated mainly low molecular weight (<10 kDa) peptides in PPH-5 but not in the PPI. The PPH-5 had renin and angiotensin converting enzyme inhibition IC50 values of 0.57 and 0.10 mg/mL (P < 0.05), respectively, and consisted mainly of peptides with 2 to 6 amino acids. Mass spectrometry analysis revealed mainly hydrophilic tetrapeptide sequences. After a single oral administration (100 mg/kg body weight) to SHR, the unheated PPI showed weakest (P < 0.05) SBP-lowering effect with a -4 mm Hg maximum when compared to -25 mm Hg for heat-treated PPI and -36 mm Hg for PPH-5. Incorporation of the PPH-5 as 0.5% or 1% (w/w) casein substitute in the SHR diet produced maximum SBP reductions of -22 or -26 mm Hg (P < 0.05), respectively after 3 wk. In comparison, the unhydrolyzed PPI produced a maximum SBP reduction of -17 mm Hg also after 3 wk. Potency of the pea products decreased in the 4th and 5th wk, though SBP values of the treated rats were still lower than the untreated control. We conclude that the antihypertensive potency of PPH-5 may have been due to the presence of easily absorbed hydrophilic peptides.

Structural and Antihypertensive Properties of Enzymatic Hemp Seed Protein Hydrolysates.

The aim of this work was to produce antihypertensive protein hydrolysates through different forms of enzymatic hydrolysis (2% pepsin, 4% pepsin, 1% alcalase, 2% alcalase, 2% papain, and 2% pepsin + pancreatin) of hemp seed proteins (HSP). The hemp seed protein hydrolysates (HPHs) were tested for in vitro inhibitions of renin and angiotensin-converting enzyme (ACE), two of the enzymes that regulate human blood pressure. The HPHs were then administered orally (200 mg/kg body weight) to spontaneously hypertensive rats and systolic blood pressure (SBP)-lowering effects measured over a 24 h period. Size exclusion chromatography mainly showed a 300-9560 Da peptide size range for the HPHs, while amino acid composition data had the 2% pepsin HPH with the highest cysteine content. Fluorescence spectroscopy revealed higher fluorescence intensities for the peptides when compared to the unhydrolyzed hemp seed protein. Overall, the 1% alcalase HPH was the most effective (p < 0.05) SBP-reducing agent (-32.5 ± 0.7 mmHg after 4 h), while the pepsin HPHs produced longer-lasting effects (-23.0 ± 1.4 mmHg after 24 h). We conclude that an optimized combination of the fast-acting HPH (1% alcalase) with the longer-lasting HPHs (2% and 4% pepsin) could provide daily effective SBP reductions.

A novel hemp seed meal protein hydrolysate reduces oxidative stress factors in spontaneously hypertensive rats.

This report shows the antioxidant effects of a hemp seed meal protein hydrolysate (HMH) in spontaneously hypertensive rats (SHR). Defatted hemp seed meal was hydrolyzed consecutively with pepsin and pancreatin to yield HMH, which was incorporated into rat feed as a source of antioxidant peptides. Young (8-week old) SHRs were divided into three groups (8 rats/group) and fed diets that contained 0.0%, 0.5% or 1.0% (w/w) HMH for eight weeks; half of the rats were sacrificed for blood collection. After a 4-week washout period, the remaining 20-week old SHRs were fed for an additional four weeks and sacrificed for blood collection. Plasma total antioxidant capacity (TAC) and superoxide dismutase (SOD), catalase (CAT) and total peroxides (TPx) levels were determined. Results showed that plasma TAC, CAT and SOD levels decreased in the older 20-week old SHRs when compared to the young SHRs. The presence of HMH in the diets led to significant (p < 0.05) increases in plasma SOD and CAT levels in both young and adult SHR groups; these increases were accompanied by decreases in TPx levels. The results suggest that HMH contained antioxidant peptides that reduced the rate of lipid peroxidation in SHRs with enhanced antioxidant enzyme levels and total antioxidant capacity.

Thermoase-derived flaxseed protein hydrolysates and membrane ultrafiltration peptide fractions have systolic blood pressure-lowering effects in spontaneously hypertensive rats.

Thermoase-digested flaxseed protein hydrolysate (FPH) samples and ultrafiltration membrane-separated peptide fractions were initially evaluated for in vitro inhibition of angiotensin I-converting enzyme (ACE) and renin activities. The two most active FPH samples and their corresponding peptide fractions were subsequently tested for in vivo antihypertensive activity in spontaneously hypertensive rats (SHR). The FPH produced with 3% thermoase digestion showed the highest ACE- and renin-inhibitory activities. Whereas membrane ultrafiltration resulted in significant (p < 0.05) increases in ACE inhibition by the <1 and 1-3 kDa peptides, only a marginal improvement in renin-inhibitory activity was observed for virtually all the samples after membrane ultrafiltration. The FPH samples and membrane fractions were also effective in lowering systolic blood pressure (SBP) in SHR with the largest effect occurring after oral administration (200 mg/kg body weight) of the 1-3 kDa peptide fraction of the 2.5% FPH and the 3-5 kDa fraction of the 3% FPH. Such potent SBP-lowering capacity indicates the potential of flaxseed protein-derived bioactive peptides as ingredients for the formulation of antihypertensive functional foods and nutraceuticals.

Structural and functional properties of hemp seed protein products.

The effects of pH and protein concentration on some structural and functional properties of hemp seed protein isolate (HPI, 84.15% protein content) and defatted hemp seed protein meal (HPM, 44.32% protein content) were determined. The HPI had minimum protein solubility (PS) at pH 4.0, which increased as pH was decreased or increased. In contrast, the HPM had minimum PS at pH 3.0, which increased at higher pH values. Gel electrophoresis showed that some of the high molecular weight proteins (>45 kDa) present in HPM were not well extracted by the alkali and were absent or present in low ratio in the HPI polypeptide profile. The amino acid composition showed that the isolation process increased the Arg/Lys ratio of HPI (5.52%) when compared to HPM (3.35%). Intrinsic fluorescence and circular dichroism data indicate that the HPI proteins had a well-defined structure at pH 3.0, which was lost as pH value increased. The differences in structural conformation of HPI at different pH values were reflected as better foaming capacity at pH 3.0 when compared to pH 5.0, 7.0, and 9.0. At 10 and 25 mg/mL protein concentrations, emulsions formed by the HPM had smaller oil droplet sizes (higher quality), when compared to the HPI-formed emulsions. In contrast at 50 mg/mL protein concentration, the HPI-formed emulsions had smaller oil droplet sizes (except at pH 3.0). We conclude that the functional properties of hemp seed protein products are dependent on structural conformations as well as protein concentration and pH.

Glycinyl-histidinyl-serine (GHS), a novel rapeseed protein-derived peptide has blood pressure-lowering effect in spontaneously hypertensive rats.

A novel antihypertensive peptide (Gly-His-Ser or GHS) with dual inhibition of angiotensin I-converting enzyme (ACE) and renin activities was isolated from the 3 kDa membrane ultrafiltration permeate of a pepsin+pancreatin rapeseed protein digest. The IC50 values of GHS were 0.52 ± 0.01 mg/mL and 0.32 ± 0.01 mg/mL for ACE and renin inhibitions, respectively, which are 1.5 times the ACE inhibition and 3.5 times the renin inhibition of the 3 kDa permeate. Oral administration (30 mg/kg body weight) to spontaneously hypertensive rats showed GHS to be an effective hypotensive agent with maximum blood pressure reduction of -17.29 ± 2.47 mmHg after 6 h. In contrast, the 3 kDa permeate exhibited a maximum of -21.29 ± 9.29 mmHg after 4 h, although at a relatively higher dose of 100 mg/kg body weight). GHS inhibited ACE and renin activities noncompetitively, but the renin inhibition became uncompetitive at a higher peptide concentration.

Antihypertensive and free radical scavenging properties of enzymatic rapeseed protein hydrolysates.

In this study, rapeseed protein isolate (RPI) was digested with various proteases to produce rapeseed protein hydrolysates (RPHs), which were then separated into different peptide fractions (<1, 1-3, 3-5, and 5-10kDa) by membrane ultrafiltration. Membrane fractionation showed that peptides with sizes <3 kDa had significantly (p<0.05) reduced surface hydrophobicity when compared to the RPHs and peptide fractions with sizes >3 kDa. In contrast, the <3 kDa peptides showed significantly (p<0.05) higher oxygen radical scavenging ability when compared to the >3 kDa peptides and RPHs. In vitro inhibition of angiotensin I-converting enzyme (ACE) was significantly (p<0.05) higher for the Thermolysin, Proteinase K and Alcalase RPHs when compared to the pepsin+pancreatin (PP) and Flavourzyme RPHs. The Alcalase RPH had significantly (p<0.05) higher renin inhibition among the RPHs, while with the exception of Thermolysin, the 5-10 kDa peptide fraction had the least renin-inhibitory ability when compared to the <5 kDa peptide fractions. Oral administration (100mg/kg body weight) of the RPHs and RPI to spontaneously hypertensive rats (SHR) showed the Alcalase RPH to be the most effective in blood pressure (BP) reduction (∼24 mm Hg) while Proteinase K RPH was the least effective (∼5 mm Hg) after 8h. However, the PP RPH had the most prolonged effect with BP reduction of ∼20 mm Hg after 24h of oral administration. We conclude that the strong BP-lowering ability of Alcalase and PP RPHs could be due to high resistance of the peptides to structural degradation coupled with high absorption rate within the gastrointestinal tract.