Enzyme Inhibitory Peptides Research Articles

Purification, Identification, and Inhibitory Mechanisms of a Novel ACE Inhibitory Peptide from Torreya grandis.

Torreya grandis meal has a high protein content and an appropriate amino acid ratio, making it an excellent protein source for producing ACE inhibitory peptides. To promote its application in food, medicine, and other fields, an alkaline protease hydrolysate of Torreya grandis was used in this study to isolate and identify a novel angiotensin-converting enzyme inhibitory peptide, VNDYLNW (VW-7), using ultrafiltration, gel chromatography purification, LC-MS/MS, and in silico prediction. The results show that the IC50 value of VW-7 was 205.98 µM. The Lineweaver-Burk plot showed that VW-7 had a mixed-type inhibitory effect on ACE. Meanwhile, according to the results of molecular docking, VW-7 demonstrated a strong affinity for ACE (binding energy -10 kcal/mol). VW-7 was bound to ACE through multiple binding sites. In addition, VW-7 could remain active during gastrointestinal digestion in vitro. Nitric oxide (NO) generation in human endothelial cells could rise after receiving a pretreatment with VW-7. These results indicated that Torreya grandis meal protein can be developed into products with antihypertensive function, and VW-7 has broad application prospects in the field of antihypertensive.

Anchovy’s protein as a potential precursor of Angiotensin-I Converting Enzyme (ACE) inhibitory peptide and Dipeptidyl Peptidase-IV (DPP-IV) inhibitory peptide by an in silico approach

Protein from fish is known as the precursor of biologically active peptides that exert various health benefits such as antihypertensive, antitumor, and immunomodulatory properties. Hence, this study aimed to perform an extraction of anchovy, LC-MS/MS analysis and in silico evaluation of the major proteins in anchovies as potential precursors of biologically active peptides in addition to determining whether such peptides can be released by selected proteolytic enzymes. Anchovy was subjected to protein extraction followed by total soluble protein concentration determination using Bradford assay. The sample was subjected to in-solution trypsin digestion, which was then analysed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). A bioinformatic approach by PEAKS Studio was used to identify the protein. A total of four anchovy’s proteins which are myosin light chain 1, V(D)J recombination-activating protein, 60 kDa chaperonin and heat shock protein 90AA1 were identified. Then, the identified proteins were subjected to in silico approach using the BIOPEP database. The biological potential of the theoretically released angiotensin-I converting enzyme (ACE) inhibitory peptides and dipeptidyl peptidase (DPP)-IV inhibitory peptides were predicted by determining the frequency of occurrence of fragments with a given activity. 60 kDa chaperonin and heat shock protein 90AA1 predicted the highest number of biological activities for ACE inhibitory peptides (284 and 264 fragments) and DPP-IV inhibitory peptides (395 and 409 fragments). The most promising enzyme for the generation of bioactive peptides from anchovy protein was anticipated to be pepsin (pH > 2), which theoretically released a high number of DPP-IV inhibitory peptides and ACE inhibitory peptides through the action of in silico proteolysis. Overall, this work highlighted that anchovy protein could be a promising precursor of bioactive peptides that have ACE and DPP-IV inhibitory activities for developing functional food or nutraceutical products.

Chitosan‐coated nanoliposomes for the enhanced stability of walnut angiotensin‐converting enzyme (ACE) inhibitory peptide

This study encapsulated walnut angiotensin-converting enzyme (ACE) inhibitory peptides within nanoliposomes and then modified them with chitosan. The resulting effect of the nanoliposome loading and chitosan coating on physicochemical characteristics, stability, bioactivity, chemical structure, and morphology of the encapsulated peptides was assessed. The resulting particle size and polymer dispersity index revealed that the chitosan-coated nanoliposomes loaded with walnut ACE inhibitory peptides (WAIP) (CL-P) exhibited higher physical stability compared with the nanoliposomes loaded with WAIP (L-P). The encapsulation efficiency (EE) of CL-P increased from 73.32% to 76.13% after chitosan modification, and the EE of L-P and CL-P could be maintained by storage at 4°C. In addition, the antioxidant activity and ACE inhibitory activity of the peptides were effectively protected by L-P and CL-P during storage. Fourier transform infrared spectroscopy showed that the nanoliposomes were bound in ionic form with both the peptides and chitosan. Transmission electron micrographs indicated the presence of vesicle-like carriers with a reservoir-type structure. This study highlights the potential of nanoliposomes and their modification with chitosan to increase the stability and bioactivity retention of ACE inhibitory peptides. PRACTICAL APPLICATION: Chitosan-coated nanoliposomes loaded with walnut ACE inhibitory peptides were prepared in this study. Chitosan coating increased nanoliposomes' encapsulation efficiency and provided higher physical stability. In addition, the bioactivity of the walnut ACE peptides was effectively protected during storage. This study was relevant for improving the storage and transportation used for nanoliposome systems applied in the food and health product industry.

Isolation and identification of angiotensin-converting enzyme inhibitory peptides from Tartary buckwheat albumin.

Tartary buckwheat protein peptides have been shown to be able to inhibit angiotensin-converting enzyme (ACE), but the exact protein type has been less studied for ACE activity inhibition, and only a few types of ACE inhibitory peptides have been reported. In this study, we purified and identified ACE inhibitory peptides from albumin hydrolysate (AH). Albumin, globulin, prolamin and glutelin were extracted from Tartary buckwheat, and their ACE active peptides were obtained by a pepsin-trypsin sequential hydrolysis process. All four hydrolysates exhibited ACE inhibitory activity, and AH displayed the strongest ACE inhibition activity and the highest peptide yield (82.28%). At 0.2 mg mL-1 , the inhibition rate of AH was 79.89%, followed by globulin hydrolysate at 71.84%, while prolamin hydrolysate and glutelin hydrolysate showed lower inhibition rates. The peptides with the highest inhibition rate were then isolated from AH using gel filtration chromatography and reversed-phase high-performance liquid chromatography, and identified using nanoscale high-performance liquid chromatography-tandem mass spectrometry. After isolation and purification, 42 ACE inhibitory peptides were identified in the fraction with the highest inhibition rate, 14 of which were completely novel discoveries in this study. These 14 peptides showed potent ACE inhibitory effects through computer analysis. Tartary buckwheat albumin can be used as a good source of ACE inhibitory peptides and can be further developed and utilized as edible supplements or drugs. © 2023 Society of Chemical Industry.

Revealing the Sequence Characteristics and Molecular Mechanisms of ACE Inhibitory Peptides by Comprehensive Characterization of 160,000 Tetrapeptides.

Chronic diseases, such as hypertension, cause great harm to human health. Conventional drugs have promising therapeutic effects, but also cause significant side effects. Food-sourced angiotensin-converting enzyme (ACE) inhibitory peptides are an excellent therapeutic alternative to pharmaceuticals, as they have fewer side effects. However, there is no systematic and effective screening method for ACE inhibitory peptides, and the lack of understanding of the sequence characteristics and molecular mechanism of these inhibitory peptides poses a major obstacle to the development of ACE inhibitory peptides. Through systematically calculating the binding effects of 160,000 tetrapeptides with ACE by molecular docking, we found that peptides with Tyr, Phe, His, Arg, and especially Trp were the characteristic amino acids of ACE inhibitory peptides. The tetrapeptides of WWNW, WRQF, WFRV, YYWK, WWDW, and WWTY rank in the top 10 peptides exhibiting significantly high ACE inhibiting behaviors, with IC50 values between 19.98 ± 8.19 μM and 36.76 ± 1.32 μM. Salt bridges, π-π stacking, π-cations, and hydrogen bonds contributed to the high binding characteristics of the inhibitors and ACE. Introducing eight Trp into rabbit skeletal muscle protein (no Trp in wide sequence) endowed the protein with a more than 90% ACE inhibition rate, further suggesting that meat with a high content of Trp could have potential utility in hypertension regulation. This study provides a clear direction for the development and screening of ACE inhibitory peptides.

Purification and molecular docking of angiotensin converting enzyme-inhibitory peptides derived from corn gluten meal hydrolysate and from in silico gastrointestinal digestion

Corn gluten meal (CGM) was hydrolyzed by Alcalase with the degree of hydrolysis (DH) and protein recovery of 28.7 % and 67.7 %, respectively. Ultrafiltration revealed that the 3–10 kDa fraction (IC50 =15.98 µg/mL) involved potent ACE inhibitory peptides. The peptides in the fraction were further separated by size exclusion chromatography and reverse-phase chromatography. The novel peptide DLLGCS was identified to be the most active ACE inhibitor (IC50 =39.0 μM). The in silico digested peptide CS was approximately 4.5 times higher than its parent counterpart (IC50 =8.5 µM). Peptides CS and DLLGCS showed an uncompetitive inhibitor characteristic with Ki values of 12.52 and 11.40 µM, respectively. Based on molecular docking, both peptides interacted with the nonactive site of ACE, primarily via hydrogen bonds at Arg522, Asp121, Glu123 and Lys118 residues for DLLCGS and Glu162, Ser147 and Glu349 residues for CS. Therefore, peptides originated from CGM hydrolysates could exhibit a higher degree of inhibition against ACE enzyme upon gastrointestinal digestion, which is likely to be a potential source of bioactive peptides with ACE inhibitory activity.

In-situ and real-time monitoring of two-stage enzymatic preparation of ACE inhibitory peptides from Cordyceps militaris medium residues by ultrasonic-assisted pretreatment

A protocol for the preparation of angiotensin-Ⅰ-converting enzyme (ACE) inhibitory peptides from Cordyceps militaris medium residues (CMMR) was established by two-stage enzymatic hydrolysis (amylase and protease). In a combination (mono-, dual-, and tri-frequency) of five different frequencies (20, 28, 35, 40 and 50 kHz), ultrasound-assisted pretreatment increased ACE inhibition rate in hydrolysate by 63.30 % under the mode of 20/28 kHz. Afterwards, near-infrared (NIR) spectrometer combined with spectral preprocessing methods and multivariate analysis like partial least square (PLS), synergy interval-PLS (Si-PLS), random frog-PLS (RF-PLS) and competitive adaptive reweighted sampling (CARS-PLS) was used to monitor the ACE inhibitory activity. The performance of models was evaluated by the correlation coefficient (Rp) and root mean square error (RMSEP). CARS-PLS models achieved optimal results for both amylase and protease hydrolysis with Rp = 0.9693, RMSEP = 0.01 for the former and Rp = 0.9454, RMSEP = 0.03 for the latter. NIR spectrometer combined with CARS-PLS models may be employed for in-situ and real-time monitoring of the preparation of ACE inhibitory peptides under ultrasonic-assisted pretreatment.

Preparation and activity evaluation of angiotensin-I converting enzyme inhibitory peptides from protein hydrolysate of mulberry leaf.

Angiotensin-I converting enzyme (ACE) inhibitory peptides drew wide attention in the food industry because of their natural reliability, non-toxicity, and safety. However, the characteristics of ACE inhibitory peptides obtained from protein hydrolysate of mulberry leaf prepared by Flavourzyme were still unclear. Based on the single-factor test, the Plackett-Burman test and response surface test were used to determine the key factors affecting the ACE inhibition rate in mulberry leaf protein hydrolysate and the optimum conditions of enzymatic hydrolysis. The results showed that the optimum technical parameters were as follows: the ratio of material to liquid is 1: 25 (w / v, g/mL), the Flavourzyme to substrate ratio was 3,000 U/g, the temperature of enzymatic hydrolysis was 50°C, pH was 6.3, and the time of enzymatic hydrolysis was 2.9 h. The ACE inhibitory peptides in the mulberry leaf protein hydrolysates were purified by ultrafiltration and gel filtration, aiming to obtain the highest active component. The 12 peptide sequences were identified by reverse liquid chromatography-mass spectrometry, and then, they were docked to the crystal structure of human angiotensin-I converting enzyme (1O8A), and the interaction mechanisms of 12 peptide sequences and 1O8A were analyzed. The docking results showed that among the 12 peptide sequences, ERFNVE (792.37 Da), TELVLK (351.72 Da), MELVLK (366.72 Da), and FDDKLD (376.67 Da), all had the lowest docking energy, and inhibition constant. The chemosynthetic ERFNVE (IC50: 2.65 mg/mL), TELVLK (IC50: 0.98 mg/mL), MELVLK (IC50:1.90 mg/mL) and FDDKLD (IC50:0.70 mg/mL) demonstrated high ACE-inhibitory activity with competitive inhibition mode. These results indicated that the ACE-inhibiting peptides from mulberry leaf protein hydrolyzed (FHMP) had the potential activities to inhibit ACE and could be used as functional food or drugs to inhibit ACE. This work provides positive support for mining the biological activity of mulberry leaves in the treatment of hypertension.

In silico identification of novel ACE and DPP-IV inhibitory peptides derived from buffalo milk proteins and evaluation of their inhibitory mechanisms.

The capacity of buffalo milk proteins to release bioactive peptides was evaluated and novel bioactive peptides were identified. The sequential similarity between buffalo milk proteins and their cow counterparts was analysed. Buffalo milk proteins were simulated to yield theoretical peptides via in silico proteolysis. The potential of selected proteins to release specific bioactive peptides was evaluated by the A value obtained from the BIOPEP-UWM database (Minkiewicz et al. in Int J Mol Sci 20(23):5978, 2019). Buffalo milk protein is a suitable precursor to produce bioactive peptides, particularly dipeptidyl peptidase IV (DPP-IV) and angiotensin I-converting enzyme (ACE) inhibitory peptides. Two novel ACE inhibitory peptides (KPW and RGP) and four potential DPP-IV inhibitory peptides (RGP, KPW, FPK and KFTW) derived from in silico proteolysis of buffalo milk proteins were screened using different integrated bioinformatic approaches (PeptideRanker, Innovagen, peptide-cutter and molecular docking). The Lineweaver-Burk plots showed that KPW (IC50 = 136.28 ± 10.77μM) and RGP (104.72 ± 8.37μM) acted as a competitive inhibitor against ACE. Similarly, KFTW (IC50 = 873.92 ± 32.89μM) was also a competitive inhibitor of DPP-IV, while KPW and FPK (82.52 ± 10.37 and 126.57 ± 8.45μM, respectively) were mixed-type inhibitors. It should be emphasized that this study does not involve any clinical trial.

Purification, molecular docking and in vivo analyses of novel angiotensin-converting enzyme inhibitory peptides from protein hydrolysate of moth bean (Vigna aconitifolia (Jacq.) Màrechal) seeds

The moth bean is a high-protein food legume. Enzymatic hydrolysates of food proteins demostrate health benefits. Search for diet related food protein hydrolysates is therefore within the scope of functional foods. Present study asertains to produce, screen and identify natural ACE-I inhibitory peptides derived from moth bean seed protein hydrolysates. The extracted protein was hydrolysed using alcalase, chymotrypsin, flavourzyme, papain, pepsin and trypsin respectively. Alcalase achieved the greatest degree of hydrolysis and ACE inhibition. The highest ACE-I inhibitory activity was exhibited by the peptide with the lowest molecular weight i.e. <3 kDa (IC50 11.19 ± 0.15 μg/mL). This was further separated by FPLC, followed by mass spectrometry. Molecular docking analysis showed the peptides IAWDFR and ADLPGLK bind to active sites whereas DKPWWPK and AVIPNAPNLR to non-active sites of the ACE molecule. In vivo administration of MBP hydrolysate to dexamethasone-induced hypertensive rats reduced their systolic blood pressure (125 ± 0.76 mmHg) compared with positive control (155 ± 3.13 mmHg). Moth bean protein peptides exhibit functional nutraceutical properties with adequate antihypertensive activity.

Camel milk as an integrated food and its physical and chemical properties with therapeutic characteristics

A staple food in many regions of the world, especially in the arid and semi-arid regions, is camel's milk. Health-promoting ingredients found in camel's milk include lactoferrin, zinc, lactoactive peptides, and mono- and polyunsaturated fatty acids. Some significant human ailments, such as asthma, gastrointestinal issues, jaundice, tuberculosis, and asthma, may be treated with the use of these drugs. Compared to cow's milk, camel's milk has a more varied composition. In camels, nutrition, breed, age, and lactation stage have a greater impact on milk composition. The ratio of components in camel's milk varies greatly depending on the region and season. Along with having a large number of soluble proteins, camel's whey protein contains native proteases such chymotrypsin A and cathepsin D. These whey proteins have distinctive qualities, such as the technical, physiological, chemical, physical, and biological traits that are advantageous in the food application, in addition to their high nutritional value. Camel's milk proteins are hydrolyzed to create bioactive peptides, which have an impact on the body's primary organ systems and give them physiological activities. The angiotensin-I-converting enzyme (ACE) inhibitory peptides, antidiabetic, antibacterial, antioxidant, and anticholesterol properties of camel milk.

Characterization and biological function analysis of endogenous peptides derived from donkey colostrum proteins.

Donkey colostrum, due to its abundance of active ingredients, including lysozyme, proteins, and peptides, is essential for the growth and immune defence of newborns. However, research on endogenous peptides in donkey colostrum is inadequate. This study analysed the profiles of endogenous peptides, their potential bioactivity, and the enzymes that generated these peptides using two different strategies. A total of 6202 endogenous peptides were characterised through a database search, while an additional 2997 peptides were identified de novo. Among the 1142 proteins identified, trypsin and plasmin demonstrated the highest bioactivities. Furthermore, a bioinformatics-based screening identified antioxidant peptides, angiotensin I-converting enzyme inhibitory peptides, and dipeptidyl peptidase IV inhibitory peptides as the three most active peptides. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted. These findings enhance our knowledge of endogenous peptides in donkey colostrum and provide crucial information regarding these peptides as nutritional factors for the future development of functional foods derived from donkey sources.

Unlocking the bioactivity of meat proteins: Comparison of meat and meat hydrolysate via simulated gastrointestinal digestion

Understanding the functional attributes of meat proteins is crucial for determining their nutritional benefits. Depending on the form in which meat proteins are available, the digestive process can release peptides which are valuable for nutrition and may also possess bioactive properties, affecting physiology. Liquid chromatography – mass spectrometry (LC-MS) was used to quantitatively compare the molecular peptide features (representing non-redundant peptides), during the different stages of a simulated gastrointestinal digestion process of a minimally processed powdered meat and its enzymatically produced hydrolysate. Results from a principal component analysis (PCA) indicated that the hydrolysate did not undergo extensive additional digestion whereas the powdered meat was digested both at the gastric and in the intestinal phases. Bioactive peptide sequence prediction identified the meat hydrolysate but not the meat powder as the only source of exact and partial bioactive matches in the angiotensin-I converting enzyme and dipeptidyl peptidase IV inhibition categories. Also, a higher source of cryptides (encrypted bioactive peptides), indicated that meat hydrolysates are potentially a better substrate for the release of these enzyme inhibitory peptides. These observations thus suggest that pre-digestion of a complex food matrix such as meat, may enhance its bioavailability following oral consumption early in the digestion process. SignificanceThis work highlights enzymatic hydrolysis of meat proteins prior to ingestion allows for potentially higher bioavailability of bioactive peptides that inhibit angiotensin-I converting enzyme and dipeptidyl peptidase IV, thus possibly aiding high blood pressure and type 2 diabetes management.

ACE Inhibitory Peptides Derived from Muscovy Duck (Cairina moschata) Plasma.

In this study, angiotensin-converting enzyme inhibitory peptides (ACE-IPs) derived from Muscovy duck (Cairina moschata) plasma hydrolysate (MDPH) were investigated. According to the general research protocol for bioactive peptides, the crude ACE-IPs of Muscovy duck plasma were separated and purified by ultrafiltration, gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). Then the components with the highest ACE inhibition potential were selected for identification. Finally, the inhibition mechanism was explored by molecular docking and in silico simulated digestion. A total of 121 peptides was detected, and five were screened for synthesis verification and molecular docking. The peptide VALSSLRP revealed high ACE inhibitory activity (91.67 ± 0.73%) because this peptide bound tightly to the S1' pocket and formed 3 hydrogen bonds. Meaningfully, this work provides some new information about the generation of ACE-IPs derived from duck blood plasma.

The Molecular Docking and Inhibition Kinetics of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Soft-Shelled Turtle Yolk

The soft-shelled turtle yolk (SSTY) protein hydrolysate contains a potential source of bioactive peptides. Our previous study found that five SSTY peptides (WLQL, LPSW, LPLF, VPGLAL and LVGLPL) showed moderate to high dipeptidyl peptidase IV (DPP-IV) inhibitory activities. This study further investigated their angiotensin-I-converting enzyme (ACE) inhibitory activity. Consequently, WLQL was identified as the most potent ACE inhibitory peptide with a remarkably low IC50 value (16.87 ± 0.54 µM). The Lineweaver–Burk plot analysis was performed for the characterization of the peptide’s inhibition mode and the inhibition kinetics was rationalized using the molecular docking simulation. The result revealed that WLQL would dock into the S1 pockets of ACE, while LPSW interacted with ACE’s secondary binding site. Further evaluation of the peptides’ stability against ACE involved a pre-incubation experiment. After 3 h of pre-incubation with ACE, the four peptides were hydrolyzed into smaller fragments with varying degrees, suggesting that they are substrate-type inhibitors. In contrast, LVGLPL can tolerate hydrolysis by ACE and act as a true inhibitor.

Angiotensin-converting enzyme inhibitory peptides and isoflavonoids from soybean [Glycine max (L.) Merr.

Angiotensin-converting enzyme I (ACE I) is a zinc-containing metallopeptidase involved in the renin-angiotensin system (RAAS) that helps in the regulation of hypertension and maintains fluid balance otherwise, which results in cardiovascular diseases (CVDs). One of the leading reasons of global deaths is due to CVDs. RAAS also plays a central role in maintaining homeostasis of the CV system. The commercial drugs available to treat CVDs possess several fatal side effects. Hence, phytochemicals like peptides having plant-based origin should be explored and utilized as alternative therapies. Soybean is an important leguminous crop that simultaneously possesses medicinal properties. Soybean extracts are used in many drug formulations for treating diabetes and other disorders and ailments. Soy proteins and its edible products such as tofu have shown potential inhibitory activity against ACE. Thus, this review briefly describes various soy proteins and products that can be used to inhibit ACE thereby providing new scope for the identification of potential candidates that can help in the design of safer and natural treatments for CVDs.

Impact of Gastrointestinal Digestion Simulation on the Formation of Angiotensin-I-Converting Enzyme Inhibitory (ACE-I) Peptides from Germinated Lamtoro Gung Flour.

The germination of lamtoro gung has been shown to increase the angiotensin-I-converting enzyme inhibitory (ACE-I) activity in previous studies. The 48 h germinated flour had the highest ACE-I activity. Administration of the gastrointestinal digestion (GID) simulation with commercial enzymes was expected to increase the ACE-I activity. However, the GID simulation to increase ACE-I in the germinated lamtoro gung flour has not been found. Therefore, this study aimed to evaluate the GID simulation of ACE-I peptides in sprouted lamtoro gung flour. This study also identified and characterised the peptide with the ACE-I activity. The GID simulation was performed using commercial pepsin (pH 2) and pancreatin (pH 7.5). Both simulations occurred at 37 °C for 240 min. The degree of hydrolysis, peptide concentration, and ACE-I activity was analysed. Samples with the highest ACE-I activity were then fractionated and identified, to determine the peptide responsible for the ACE-I activity. The 180 min GID simulation in the test sample showed the highest ACE-I activity (89.70%). This result was supported by an increased degree of hydrolysis (DH) and peptide concentrations throughout the GID simulation. The <1 kDa peptide fraction had the highest inhibitory activity and had the most elevated peptide portion (54.69%). Peptide sequences containing crucial amino acids were found in the <1 kDa peptide fraction. PRPPKPP, PPPPPGARAP, and PFPPSNPPP had proline in the C and N terminal residues. The peptides obtained also had other biological activities, such as a DPP IV inhibitor, an alpha-glucosidase inhibitor, and antioxidative activity. Based on the toxicity prediction, those peptides are non-toxic and safe to consume.

Antidiabetic, angiotensin‐converting enzyme inhibitory and anti‐inflammatory activities of fermented camel milk and characterisation of novel bioactive peptides from lactic‐fermented camel milk with molecular interaction study

This study was to separate and purify antidiabetic and angiotensin‐converting enzyme (ACE) inhibitory peptides from Lactiplantibacillus plantarum KGL3A fermented camel milk. After 48 h of fermentation at 37°C, ɑ‐amylase inhibition, ɑ‐glucosidase inhibition, lipase inhibition and ACE inhibitory activities were 80.94%, 64.45%, 63.93%, and 77.53%, respectively in fermented camel milk. Optimisation of growth condition for the evaluation of maximum peptide production was evaluated by measuring proteolytic activity (O‐phthalaldehyde, OPA method) with different inoculation rates and incubation times and highest proteolytic activity (9.21 mg/mL) was observed after 48 h of fermentation at 2.5% rate of inoculation. The antidiabetic and ACE inhibitory activity of 3 kDa permeate fraction were higher as compared with other fractions. Purification of antidiabetic and ACE inhibitory peptides from fermented camel milks was performed through sodium dodecyl‐sulphate polyacrylamide gel electrophoresis, and 2‐dimensional polyacrylamide gel electrophoresis and maximum number of protein bands were present in between 25 and 10 kDa. The generated peptide sequences were matched with antihypertensive peptide database (AHTPDB) and BIOPEP databases for confirming the antidiabetic and ACE inhibitory activity. Peptides, that is. TDVMPQWW and MMSLVSLLLVGILFPTIQAK were having highest peptide ranker score among the all sequences. Furthermore, anti‐inflammatory activity (TNF‐α, IL‐6 and IL‐1β) of fermented camel milk was evaluated in macrophage cell line RAW 264.7 (Ralph and William's cell line). Furthermore, peptides were predicted to have improved binding affinity against Human Angiotensin converting enzyme (hACE) through molecular docking.

Soybean-Derived Tripeptide Leu-Ser-Trp (LSW) Protects Human Vascular Endothelial Cells from TNFα-Induced Oxidative Stress and Inflammation via Modulating TNFα Receptors and SIRT1.

Soybean is a rich source of high-quality proteins and an excellent food source of bioactive peptides. A tripeptide, Leu-Ser-Trp (LSW), was previously identified from soybean as an angiotensin-converting enzyme inhibitory peptide. In the present work, we further studied its antioxidant and anti-inflammatory activities in human vascular endothelial cells (EA.hy926) and elucidated the mechanisms underlying these biological activities. In tumor necrosis factor alpha (TNFα)-stimulated EA.hy926 cells, LSW significantly inhibited oxidative stress, both reduced superoxide and malondialdehyde levels (p &lt; 0.001), owing to its free-radical-scavenging ability. LSW treatment also mitigated the elevated protein expression of vascular adhesion molecule-1 (p &lt; 0.001) and cyclooxygenase 2 (p &lt; 0.01) via inhibition of NF-κB and p38/JNK signaling, respectively. Additionally, LSW also inhibited the endogenous formation of TNFα and attenuated the expression of its two receptors in EA.hy926 cells. Furthermore, LSW upregulated sirtuin-1 level, which partially contributed to its anti-inflammatory activity. These results demonstrate the multiple roles of LSW in ameliorating vascular endothelial oxidative stress and inflammatory responses, which support its uses as a nutraceutical or functional food ingredient for combating endothelial dysfunction and cardiovascular diseases.

Molecular docking and antihypertensive effects of a novel angiotensin-I converting enzyme inhibitory peptide from yak bone.

A novel angiotensin-converting enzyme (ACE) inhibitory peptide ser-ala-ser-val-ile-pro-val-ser-ala-val-arg-ala (SASVIPVSAVRA) was purified and identified from yak bone by Electrospray Ionization-Time of Flight-Mass Spectrometry (ESI-TOF-MS). Results in vitro showed that the peptide exhibited strong ACE inhibition activities with an IC50 of 54.22 μM. Molecular docking results showed the binding between the peptide SASVIPVSAVRA and ACE mainly driven by van der Waals forces, hydrogen bonds and metal receptor. Interestingly, the ACE inhibition activities of the peptide increased about 19% after digestion, but none of its metabolites showed stronger activity than it. The in vivo experiment showed that the antihypertensive effect of peptide SASVIPVSAVRA at dose of 30 mg/kg is nearly equal to Captopril at dose of 10 mg/kg to spontaneously hypertensive rats (SHRs). The antihypertensive effect mechanism of SASVIPVSAVRA should be further studied through plasma metabolomics and bioanalysis. Structure analysis of amino acids and peptides produced during digestion may help better understand the antihypertensive effect of peptides.