Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from the marine rotifer, Brachionus rotundiformis. ACE inhibitory peptides were separated from rotifer hydrolysate prepared by Alcalase, α-chymotrypsin, Neutrase, papain, and trypsin. The Alcalase hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The IC₅₀ value of Alcalase hydrolysate for ACE inhibitory activity was 0.63 mg/ml. We attempted to isolate ACE inhibitory peptides from Alcalase prepared rotifer hydrolysate using gel filtration on a Sephadex G-25 column and high performance liquid chromatography on an ODS column. The IC₅₀ value of purified ACE inhibitory peptide was 9.64 μM, and Lineweaver–Burk plots suggest that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. Amino acid sequence of the peptide was identified as Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, with a molecular weight 1538 Da. The results of this study suggest that peptides derived from rotifers may be beneficial as anti-hypertension compounds in functional foods resource.     

Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate

Isolation of bioactive compounds and commercialization of marine microalgae sources are interesting targets in future marine biotechnology. Cultured biomass of the marine microalga, Nannochloropsis oculata, was used to purify angiotensin-I converting enzyme (ACE) inhibitory peptides using proteases including pepsin, trypsin, α-chymotrypsin, papain, alcalase, and neutrase. The pepsin hydrolysate exhibited the highest ACE inhibitory activity, compared to the other hydrolysates and then was separated into three fractions (F1, F2, and F3) using Sephadex G-25 gel filtration column chromatography. First fraction (F1) showed the highest ACE inhibitory activity and it was further purified into two fractions (F1-1 and F1-2) using reverse-phase high-performance liquid chromatography. The IC₅₀ value of purified ACE inhibitory peptides were 123 and 173 μM and identified as novel peptides, Gly-Met-Asn-Asn-Leu-Thr-Pro (GMNNLTP; MW, 728 Da) and Leu-Glu-Gln (LEQ; MW, 369 Da), respectively. In addition, nitric oxide production level (%) was significantly increased by the purified peptide (Gly-Met-Asn-Asn-Leu-Thr-Pro) compared to the purified peptide (Leu-Glu-Gln) and other treated pepsin hydrolysate fractions on human umbilical vein endothelial cells (HUVECs). Cell viability assay showed no cytotoxicity on HUVECs with the treated purified peptides and fractions. These results suggest that the isolated peptides from cultured marine microalga, N. oculata protein sources may have potentiality to use commercially as ACE inhibitory agents in functional food industry.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the Styela clava flesh tissue. Nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, α-chymotrypsin and papain) were used, and their respective enzymatic hydrolysates and an aqueous extract were screened to evaluate their potential ACE inhibitory activity. Among all of the test samples, Protamex hydrolysate possessed the highest ACE inhibitory activity, and the Protamex hydrolysate of flesh tissue showed relatively higher ACE inhibitory activity compared with the Protamex hydrolysate of tunic tissue. We attempted to isolate ACE inhibitory peptide from the Protamex hydrolysate of S. clava flesh tissue using ultrafiltration, gel filtration on a Sephadex G-25 column and high performance liquid chromatography (HPLC) on an ODS column. The purified ACE inhibitory peptide exhibited an IC₅₀ value of 37.1 μM and was identified as non-competitive inhibitor of ACE. Amino acid sequence of the peptide was identified as Ala-His-Ile-Ile-Ile, with a molecular weight 565.3 Da. The results of this study suggested that the peptides derived from enzymes-assisted extracts of S. clava would be useful new antihypertension compounds in functional food resource.     

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.     

Biological activity from the gelatin hydrolysates of duck skin by-products

In this study, free radical scavengers and angiotensin I converting enzyme (ACE) inhibitors from the gelatin hyrdolysates of duck skin by-products were examined. Gelatin was obtained by pretreating duck skin by-products with acid and alkaline and hydrolysis using nine proteases (Alcalase, Collaganase, Flavourzyme, Neutrase, Protamex, papain, pepsin, trypsin and α-chymotrypsin). Of the various hydrolysates produced, the pepsin hydrolysate exhibited the highest free radical scavenging activity. The DPPH, hydroxyl and alkyl radical scavenging activity of pepsin was the most prominent with IC₅₀ values of 1.230, 0.554 and 1.193 mg/ml respectively, which were measured using an electron spin resonance (ESR) spectrometer. However, when the gelatin was hydrolyzed as a combination of two enzymes, Collaganase and pepsin, the DPPH, hydroxyl and alkyl radical scavenging activity increased as the IC₅₀ decreased to 0.632, 0.222 and 0.708 mg/ml, respectively. In addition, the ability of pepsin hydrolysates from the gelatin of duck skin by-products to inhibit oxidative damage to DNA was assessed in vitro by measuring the conversion of supercoiled pBR322 plasmid DNA to the open circular form. The enzymatic hydrolysates from the gelatin of duck skin by-products significantly protected hydroxyl radical-induced DNA damage in a dose-dependent manner, while also inhibiting the ACE activity of the α-chymotrypsin hydrolysates.These results indicate that enzymatic hydrolysates from the gelatin of duck skin by-products may be a beneficial ingredient in functional foods and/or pharmaceuticals.     

Novel whey-derived peptides with inhibitory effect against angiotensin-converting enzyme: in vitro effect and stability to gastrointestinal enzymes

Whey protein concentrate (WPC) was subjected to enzymatic hydrolysis by proteases from the flowers of Cynara cardunculus, and the resulting angiotensin-converting enzyme (ACE)-inhibitory effect was monitored. The whole WPC hydrolysate exhibited an IC₅₀ value of 52.9 ± 2.9 μg/mL, whereas the associated peptide fraction with molecular weight below 3 kDa scored 23.6 ± 1.1 μg/mL. The latter fraction was submitted to RP-HPLC, and 6 fractions were resolved that exhibited ACE-inhibitory effects. Among the various peptides found, a total of 14 were identified via sequencing with an ion-trap mass spectrometer. Eleven of these peptides were synthesized de novo - to validate their ACE-inhibitory effect, and also to ascertain their stability when exposed to simulated gastrointestinal digestion. Among them, three novel, highly potent peptides were found, corresponding to α-lactalbumin f(16-26) - with the sequence KGYGGVSLPEW, α-lactalbumin f(97-104) with DKVGINYW, and β-lactoglobulin f(33-42) with DAQSAPLRVY; their IC₅₀ values were as low as 0.80 ± 0.1, 25.2 ± 1.0 and 13.0 ± 1.0 μg/mL, respectively. None of them remained stable in the presence of gastrointestinal enzymes: they were partially, or even totally hydrolyzed to smaller peptides - yet the observed ACE-inhibitory effects were not severely affected for two of those peptides.     

Angiotensin I-converting enzyme inhibitory oligo-tyrosine peptides synthesized by α-chymotrypsin

Oligo-tyrosine peptides with degrees of polymerization ranging from 2 to 5 could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media, although the peptide yield was low due to a preferential hydrolysis of the substrate. It was also confirmed that α-chymotrypsin efficiently converted tyrosine tetramer to the dimer which was resistant to the digestion. Both Tyr-Tyr and Tyr-Tyr-Tyr showed high inhibitory activity for angiotensin I-converting enzyme from rabbit lung, and their IC₅₀ values were 34 μM and 51 μM, respectively. These two peptides exhibited a mix of competitive and noncompetitive inhibitions. Tyr-Tyr-Tyr was first recognized as an ACE inhibitor, suggesting that α-chymotrypsin could be applied to synthesis of novel potential materials for antihypertensive medicines.     

A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats

Marine Chlorella ellipsoidea protein was hydrolyzed using Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, α-chymotrypsin, pepsin and papain. Alcalase-proteolytic hydrolysate exhibited the highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight (below 5 kDa, 5–10 kDa and above 10 kDa). The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from marine C. ellipsoidea, which was composed of 4 amino acids, Val–Glu–Gly–Tyr (MW: 467.2 Da, IC₅₀ value: 128.4 μM), was isolated. Lineweaver–Burk plots suggest that the peptide purified acts as a competitive inhibitor against ACE and stable against gastrointestinal enzymes of pepsin, trypsin and α-chymotrypsin. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHRs) also revealed that oral administration of purified peptide can decrease systolic blood pressure significantly. The results suggest that marine C. ellipsoidea would be an attractive raw material for the manufacture of antihypertensive nutraceutical ingredients.     

Purification and molecular docking study of angiotensin I-converting enzyme (ACE) inhibitory peptides from hydrolysates of marine sponge Stylotella aurantium

Angioteinsin I-converting enzyme (ACE) inhibitory peptide was isolated from marine sponge (Stylotella aurantium) hydrolysate prepared by various hydrolysis enzymes. The peptic hydrolysate exhibited highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight. The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. The amino acid sequences of the purified peptides were identified to be Tyr-Arg (337.2 Da), and Ile-Arg (287.2 Da). The purified peptides from marine sponge had an IC₅₀ value of 237.2 μM and 306.4 μM, respectively. The molecular docking study revealed that ACE inhibitory activity of the purified peptides was mainly attributed to the hydrogen bond interactions and Pi interaction between the dipeptides and ACE. The results suggest that marine sponge, S. aurantium would be an attractive raw material for the manufacture of anti-hypertensive nutraceutical ingredients.     

Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks (<Emphasis Type="Italic">Lassi</Emphasis>) Fermented by <Emphasis Type="Italic">Lactobacillus acidophillus</Emphasis> with Consideration of Incubation Period and Simmering Treatment

The lassi, fermented milks product containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus acidophilus NCDC-15 and the incubation period and simmering effect was also optimized for production of ACE-inhibitory peptides. The time–temperature combination for the heat treatment was optimized using RSM. The biological activity was measured in the supernatant of the fermented milk after centrifugation. The lowest IC₅₀ values for the inhibition of angiotensin-converting enzyme (ACE) was found 28.9 ± 0.95 μg protein/ml in the supernatant of milk fermented by L. acidophilus and heated at 78 °C for 10 h. The fractions which showed the highest ACE-inhibitory indexes were further purified by different techniques including solid phase extraction, RP-HPLC and FPLC and the related peptides were identified by LC–MS/MS using the Ultimate 3000 nano HPLC system (Dionex) coupled to a 4000 Q TRAP electro-spray ionization mass spectrometry. The high ACE-inhibitory activity containing fractions of the milk fermented by L. acidophilus contained the sequences of b-casein (b-CN) fragment. The fraction-III showed minimum IC₅₀ value i.e. 14.57 ± 0.72 μg/ml compared with fraction-I and fraction-II. Among these peptides 14 peptides have been identified from the fraction-I of the lassi prepared from L. acidophilus i.e. β-CN f47–56, β-CN f47–57, β-CN f199–209, β-CN f176–182, β-CN f176–183, β-CN f176–184, β-CN f1–7, β-CN f57–68, β-CN f166–175, β-CN f195–206, β-CN f195–207, β-CN f195–209, β-CN f94–106 and β-CN f169–176 showed partially or completely homology to that the milk protein bioactive peptides having ACE inhibitory. The two peptides KVLPVPQK (β-CN f169–176) and YQEPVLGPVRGPFPIIV (β-CN f193–209) have the same sequence as ACE inhibitory peptides (Maeno et al. in J Dairy Sci 79(8):1316–1321, 1996; Yamamoto et al. in J Dairy Sci 77:917–922, 1994b).     

ACE inhibitory,hypotensive and antioxidant peptide fractions from Mucuna pruriens proteins

Hydrolysates and peptide fractions obtained from Mucuna pruriens protein concentrate were studied for their angiotensin converting enzyme (ACE) inhibitory, hypotensive and antioxidant activities. The hydrolysate obtained by pepsin–pancreatin (HPP) was the most active with an ACE IC₅₀ value of 19.5 μg/mL, a Trolox equivalent antioxidant capacity (TEAC) value of 102.8 mM/mg and a ferric reducing power (FRP) IC₅₀ of 67.2 μg/mL. At a dose of 5 mg/kg HPP decrease systolic (32.2%) and diastolic (37%) blood pressure in rats more pronounced than Captopril. The peptide fraction <1 kDa from HPP was the most active with an ACE inhibitory of 10.2 μg/mL (IC₅₀), a TEAC value of 709.8 mM/mg and a FRP IC₅₀ of 54.9 μg/mL. These results indicate that the hydrolysates and peptide fractions of M. pruriens would be used as nutraceuticals ingredients for preventing and providing therapy against hypertension and diseases related to oxidative damage.     

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine α_S1-casein (α_S1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine α_S1-CN f1-6 and α_S2-CN f182-185 and f186-188; caprine β-CN f58-65 and α_S2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC₅₀) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC₅₀s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.     

ACE Inhibitory and Antioxidant Activities of Novel Peptides from <Emphasis Type="Italic">Scorpaena notata</Emphasis> By-product Protein Hydrolysate

This study describes the isolation of angiotensin I converting enzyme and antioxidative peptides from head protein hydrolysate of red scorpionfish (Scorpaena notata) prepared by treatment with a protease from the fungus Penicillium digitatum. After ultrafiltration, three peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high performance liquid chromatography (RP-HPLC) with a high purification yield of 2.22 mg of peptide/g of initial protein. Active peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS–MS), corresponding to the following sequences: Gln–Gln–Pro–His–Ser–Arg–Ser–Lys–Gly–Phe–Pro–Gly–Pro (1424.724 Da), Gly–Gln–Lys–Ser–Val–Pro–Glu–Val–Arg (1000.565 Da) and Val–Glu–Gly–Lys–Ser–Pro–Asn–Val (830.448 Da). Peptides D-I, E-I and F-I showed high angiotensin-I converting enzyme inhibitory activity with an IC₅₀ values of 0.98, 1.69 and 1.44 µM, respectively as well as a synergistic antioxidant activity between the different fractions. Thus, we have demonstrated that underutilized wastes can be valorized by production of peptides that can be used as potential therapeutic compounds active against oxidative stress and hypertension.     

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.     

Availability of tyrosine amide for α-chymotrypsin-catalyzed synthesis of oligo-tyrosine peptides

Oligo-tyrosine peptides such as Tyr-Tyr having angiotensin I-converting enzyme (ACE) inhibitory activity could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media. However, peptide yield in the reaction was below 10%. Since l-tyrosine amide showed highly nucleophilic activity for the deacylation of enzyme through which a new peptide bond was made, its application to the enzymatic peptide synthesis was evaluated in this study. Addition of tyrosine amide into the reaction produced Tyr-Tyr-NH₂, of which yield exceeded 130% on the basis of tyrosine ethyl ester. Although purified Tyr-Tyr-NH₂ did not inhibit ACE activity, α-chymotrypsin could act on the dipeptide amide and convert about 40% of it to Tyr-Tyr. The use of both ester and amide forms of tyrosine is expected to be a potent procedure for α-chymotrypsin-catalyzed synthesis of antihypertensive peptides.     

Antihypertensive Peptides Derived from Soy Protein by Fermentation

Soy protein is widely used as a nitrogen source in infant and adult formulations, both in an intact and hydrolyzed form. Here, the objective was to screen for maximum proteolytic activity in different strains of lactobacillus and use it for fermentation of soy protein to obtain Angiotensin converting-I-enzyme (ACE I) inhibitory peptides for its use as a nutraceutical. Based on the proteolytic activity, Lactobacillus casei spp. pseudoplantarum was selected. The two ACE inhibitory peptide fractions F2 and F3 were isolated having IC₅₀ values of 17 ± 0.63 and 30 ± 0.13 μg/ml respectively. The N-terminal sequence of peptide (F2) was determined to be Leu-Ile-Val-Thr-Gln (LIVTQ). The peptide analogues of LIVTQ were synthesized to study the effect of individual residues on ACE enzyme. LIVTQ and LIVT peptides show inhibition against ACE enzyme having an IC₅₀ value of 0.087 and 0.110 μM respectively. Our results depict that glutamine (Q) and threonine (T) residues have an important role in ACE inhibition.     

Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacific cod (Gadus macrocephalus) skin gelatin

Potent antioxidative peptides were purified from Pacific cod (Gadus macrocephalus) skin gelatin using alcalase, neutrase, papain, trypsin, pepsin, and α-chymotrypsin. Among them, the papain hydrolysate exhibited the highest antioxidant activity. Therefore, it was further purified and obtained two peptides with amino acid sequences of Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-Gly-Asn-Val (485.5 Da). The antioxidant activity of the purified peptides was performed by electron spin resonance technique. Moreover, their intracellular free radical scavenging activity using 2′,7′-dichlorofluorescin diacetate and the protective effect against oxidation-induced DNA damage were evaluated in mouse macrophages (RAW 264.7 cells). Furthermore, both peptides have shown potential angiotensin-I converting enzyme inhibitory effect. The present study demonstrated that the peptides derived from Pacific cod (G. macrocephalus) skin gelatin could be used in the food industry as functional ingredients with potent antioxidative and antihypertensive benefits.     

Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks Started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4

Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp. bulgaricus SS1 and L. lactis subsp. cremoris FT4. The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry. The most inhibitory fractions of the milk fermented by L. delbrueckii subsp. bulgaricus SS1 contained the sequences of β-casein (β-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82. Those from the milk fermented by L. lactis subsp. cremoris FT4 contained the sequences of β-CN f7-14, f47-52, and f169-175 and κ-CN f155-160 and f152-160. Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature. In particular, the β-CN f47-52 sequence had high homology with that of angiotensin-II. Some of these peptides were chemically synthesized. The 50% inhibitory concentrations (IC₅₀s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter). When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC₅₀s increased considerably. A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed. Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin.     

Antioxidant peptides isolated from the marine rotifer,Brachionus rotundiformis

Protein derived from the rotifer Brachionus rotundiformis was hydrolyzed using different proteases (Alcalase, α-chymotrypsin, Neutrase, papain, pepsin and trypsin) for production of antioxidant peptide. Antioxidant activities of hydrolysates were evaluated using DPPH radical scavenging activity. Peptic hydrolysate exhibited the highest antioxidative activity compared to other hydrolysates. To identify antioxidant peptides, peptic hydrolysate was purified using consecutive chromatographic methods, and antioxidant peptides were identified to be Leu-Leu-Gly-Pro-Gly-Leu-Thr-Asn-His-Ala (1076 Da), and Asp-Leu-Gly-Leu-Gly-Leu-Pro-Gly-Ala-His (1033 Da) by Q-TOF ESI mass spectroscopy. EC₅₀ values of purified peptides were 189.8 and 167.7 μM, respectively. Antioxidant activities of peptides purified from the rotifer protein hydrolysate were evaluated, with results showing that peptides significantly quenched free radicals.     

Inhibition of Angiotensin-Converting Enzyme Activity by Flavonoids: Structure-Activity Relationship Studies

Previous studies have demonstrated that certain flavonoids can have an inhibitory effect on angiotensin-converting enzyme (ACE) activity, which plays a key role in the regulation of arterial blood pressure. In the present study, 17 flavonoids belonging to five structural subtypes were evaluated in vitro for their ability to inhibit ACE in order to establish the structural basis of their bioactivity. The ACE inhibitory (ACEI) activity of these 17 flavonoids was determined by fluorimetric method at two concentrations (500 µM and 100 µM). Their inhibitory potencies ranged from 17 to 95% at 500 µM and from 0 to 57% at 100 µM. In both cases, the highest ACEI activity was obtained for luteolin. Following the determination of ACEI activity, the flavonoids with higher ACEI activity (i.e., ACEI >60% at 500 µM) were selected for further IC₅₀ determination. The IC₅₀ values for luteolin, quercetin, rutin, kaempferol, rhoifolin and apigenin K were 23, 43, 64, 178, 183 and 196 µM, respectively. Our results suggest that flavonoids are an excellent source of functional antihypertensive products. Furthermore, our structure-activity relationship studies show that the combination of sub-structures on the flavonoid skeleton that increase ACEI activity is made up of the following elements: (a) the catechol group in the B-ring, (b) the double bond between C2 and C3 at the C-ring, and (c) the cetone group in C4 at the C-ring. Protein-ligand docking studies are used to understand the molecular basis for these results.