Glucoregulatory Properties of a Protein Hydrolysate from Atlantic Salmon (Salmo salar): Preliminary Characterization and Evaluation of DPP-IV Inhibition and Direct Glucose Uptake In Vitro.

Metabolic disorders are increasingly prevalent conditions that manifest pathophysiologically along a continuum. Among reported metabolic risk factors, elevated fasting serum glucose (FSG) levels have shown the most substantial increase in risk exposure. Ultimately leading to insulin resistance (IR), this condition is associated with notable deteriorations in the prognostic outlook for major diseases, including neurodegenerative diseases, cancer risk, and mortality related to cardiovascular disease. Tackling metabolic dysfunction, with a focus on prevention, is a critically important aspect for human health. In this study, an investigation into the potential antidiabetic properties of a salmon protein hydrolysate (SPH) was conducted, focusing on its potential dipeptidyl peptidase-IV (DPP-IV) inhibition and direct glucose uptake in vitro. Characterization of the SPH utilized a bioassay-guided fractionation approach to identify potent glucoregulatory peptide fractions. Low-molecular-weight (MW) fractions prepared by membrane filtration (MWCO = 3 kDa) showed significant DPP-IV inhibition (IC50 = 1.01 ± 0.12 mg/mL) and glucose uptake in vitro (p ≤ 0.0001 at 1 mg/mL). Further fractionation of the lowest MW fractions (<3 kDa) derived from the permeate resulted in three peptide subfractions. The subfraction with the lowest molecular weight demonstrated the most significant glucose uptake activity (p ≤ 0.0001), maintaining its potency even at a dilution of 1:500 (p ≤ 0.01).

Purification, identification and molecular mechanism of dipeptidyl peptidase IV inhibitory peptides from discarded shrimp (Penaeus vannamei) head.

DPP-IV plays a key role for regulation of glucose metabolism in the body. The object of this study was to obtain DPP-IV inhibitors from discarded but protein-rich Penaeus vannamei (P. vannamei) head, and to explore the potential mechanism between DPP-IV and its inhibitors. P. vannamei head protein was hydrolyzed by five food grade proteases, respectively. The animal protease hydrolysate showed the highest inhibitory active. Then the hydrolysate was sequentially separated by ultrafiltration, gel filtration chromatography and reversed phase high-performance liquid chromatography (RP-HPLC), the peptides sequences were identified by LC-MS/MS and four potential peptides YPGE, VPW, HPLY, YATP showed superior DPP-IV inhibitory activity. Meanwhile, molecular docking effectively explored their mechanism through formed hydrogen bonds and hydrophobic regions. The four peptides showed better DPP-IV inhibitory activity stability with heating treatment, pH (1-10) treatment, and in vitro gastrointestinal digestion. Our results demonstrated that the protein hydrolysate from discarded P. vannamei head can be considered as a promising natural source of DPP-IV inhibitor for helping to improve glycaemic control in Type 2 diabetes.

Potential DPP IV Inhibitory Peptides from Dry-Cured Pork Loins after Hydrolysis: An In Vitro and In Silico Study.

Peptidyl peptidase IV (DPP-IV) is a pharmacotherapeutic target in type 2 diabetes, and inhibitors of this enzyme are an important class of drugs for the treatment of type 2 diabetes. In the present study, peptides (<7 kDa) isolated from dry-cured pork loins after pepsin and pancreatin hydrolysis were identified by mass spectrometry and tested as potential inhibitors of DPP-IV by the in silico method. Two peptides, namely WTIAVPGPPHS from myomesin (water-soluble fraction, A = 0.9091) and FKRPPL from troponin (salt-soluble fraction, A = 0.8333), were selected as the most promising inhibitors of DPP-IV. Both peptides were subjected to ADMET analysis. Fragments of these peptides showed promising drug-likeness properties as well as favorable absorption, distribution, metabolism, excretion, and toxicity functions, suggesting that they are novel leads in the development of DPP-IV inhibitors from food.

Application of experimental design in HPLC method optimization and robustness for the simultaneous determination of canagliflozin, empagliflozin, linagliptin, and metformin in tablet.

Gliflozins and gliptins represent two different pharmacological drug classes that exert different and potentially complementary glucose-lowering effect in patients with type II diabetes mellitus. A novel, selective, and sensitive HPLC method was developed for the determination of canagliflozin, empaglifozin, linagliptin, and metformin in pure form, in laboratory prepared mixtures, and in pharmaceutical dosage form. Experimental design optimization was applied by using Plackett-Burman and face-centered composite designs to achieve the best resolution with minimum experimental trials. Three significant variables affecting optimization, namely buffer pH, percentage of methanol, and percentage of acetonitrile, were studied. Chromatographic separation was achieved using an Agilent Eclipse C8 column, and column temperature was kept at 45°C. The mobile phase was composed of dipotassium hydrogen phosphate buffer (0.05 M, adjusted to pH 6 using o-phosphoric acid):acetonitrile:methanol (50:25:25, v/v/v) at a flow rate of 1.5 mL/min. Sharp and well-resolved peaks of the cited drugs were obtained. The method was fully validated in terms of linearity, accuracy, precision, selectivity and robustness in agreement with the International Council of Harmonization (ICH) guidelines Q2 (R1). Satisfactory results were obtained by the analysis of tablets through applying the developed method. Therefore, it could be performed for the analysis of the cited drugs in quality control laboratories.

Gouda Cheese with Modified Content of ß-Casein as a Source of Peptides with ACE- and DPP-IV-Inhibiting Bioactivity: A Study Based on In Silico and In Vitro Protocol.

In silico and in vitro methods were used to analyze ACE- and DPP-IV-inhibiting potential of Gouda cheese with a modified content of ß-casein. Firstly, the BIOPEP-UWM database was used to predict the presence of ACE and DPP-IV inhibitors in casein sequences. Then, the following Gouda cheeses were produced: with decreased, increased, and normative content of ß-casein after 1 and 60 days of ripening each (six variants in total). Finally, determination of the ACE/DPP-IV-inhibitory activity and the identification of peptides in respective Gouda-derived water-soluble extracts were carried out. The identification analyses were supported with in silico calculations, i.e., heatmaps and quantitative parameters. All Gouda variants exhibited comparable ACE inhibition, whereas DPP-IV inhibition was more diversified among the samples. The samples derived from Gouda with the increased content of ß-casein (both stages of ripening) had the highest DPP-IV-inhibiting potency compared to the same samples measured for ACE inhibition. Regardless of the results concerning ACE and DPP-IV inhibition among the cheese samples, the heatmap showed that the latter bioactivity was predominant in all Gouda variants, presumably because it was based on the qualitative approach (i.e., peptide presence in the sample). Our heatmap did not include the bioactivity of a single peptide as well as its quantity in the sample. In turn, the quantitative parameters showed that the best sources of ACE/DPP-IV inhibitors were all Gouda-derived extracts obtained after 60 days of the ripening. Although our protocol was efficient in showing some regularities among Gouda cheese variants, in vivo studies are recommended for more extensive investigations of this subject.

Separation, identification and molecular binding mechanism of dipeptidyl peptidase IV inhibitory peptides derived from walnut (Juglans regia L.) protein.

Walnut protein was hydrolyzed with different proteases to evaluate the hydrolytic efficiency and dipeptidyl peptidase IV (DPP-IV) inhibitory activity in vitro. All of walnut protein hydrolysates (WPHs) exhibited DPP-IV inhibitory activity and Alcalase-derived hydrolysate (WPH-Alc) with better DPP-IV inhibitory activity of 33.90% (at 0.50 mg/mL) was subsequently separated by ultrafiltration and cation exchange chromatography on a SP Sephadex C-25 column. The results showed that fractions with lower molecular weight and higher basic amino acid residues possessed stronger DPP-IV inhibitory activity. Comparably, the obtained fraction B with the yield of 19.80% had the highest DPP-IV inhibitory activity of 76.19% at 0.25 mg/mL. Moreover, nine novel DPP-IV inhibitory peptides were identified using MALDI-TOF/TOF-MS. Molecular docking revealed the peptides could interact with DPP-IV through hydrogen bonds, salt bridges, hydrophobic interactions, π-cation bonds and π-π bonds. The walnut DPP-IV inhibitory peptides showed better stability with heating treatment, pH treatment, or in vitro gastrointestinal digestion.

Development and validation of an ultrasensitive LC-MS/MS method for the quantification of cetagliptin in human plasma and its application in a microdose clinical trial.

This study established and validated an LC-MS/MS method for the ultrasensitive determination of cetagliptin in human plasma. Sample pretreatment was achieved by liquid-liquid extraction with ethyl acetate, and chromatographic separation was performed on an XB-C18 analytical column (50 × 2.1 mm, 5 µm) with gradient elution (0.1% formic acid in acetonitrile and 0.1% formic acid) at a flow rate of 1.0 mL/min. For mass spectrometric detection, multiple reaction monitoring was used, and the ion transitions monitored were m/z 421.2-86.0 for cetagliptin and m/z 424.2-88.0 for cetagliptin-d3. Method validation was performed according to the U.S. Food and Drug Administration Bioanalytical Method Validation Guidance, for which the calibration curve was linear in the range of 50.0-2000 pg/mL. All of the other results, such as selectivity, lower limit of quantitation, precision, accuracy, matrix effect, recovery, and stability, met the acceptance criteria. The validated method was successfully applied in a microdose clinical trial to systematically investigate the pharmacokinetic profile of cetagliptin in healthy subjects. Both rapid absorption and prolonged duration demonstrate the potential value of cetagliptin for diabetes treatment.

Inhibitory evaluation of Curculigo latifolia on α-glucosidase, DPP (IV) and in vitro studies in antidiabetic with molecular docking relevance to type 2 diabetes mellitus.

The inhibition of α-glucosidase and DPP enzymes capable of effectively reducing blood glucose level in the management of type 2 diabetes. The purpose of the present study is to evaluate the inhibitory potential of α-glucosidase and DPP (IV) activity including with the 2-NBDG uptake assay and insulin secretion activities through in vitro studies. The selected of active compounds obtained from the screening of compounds by LC-MS were docked with the targeted enzyme that involved in the mechanism of T2DM. From the results, root extracts displayed a better promising outcome in α-glucosidase (IC50 2.72 ± 0.32) as compared with the fruit extracts (IC50 3.87 ± 0.32). Besides, root extracts also displayed a better activity in the inhibition of DPP (IV), enhance insulin secretion and glucose uptake activity. Molecular docking results revealing that phlorizin binds strongly with α-glucosidase, DPP (IV) and Insulin receptor (IR) enzymes with achieving the lowest binding energy value. The present work suggests several of the compounds have the potential that contribute towards inhibiting α-glucosidase and DPP (IV) and thus effective in lowering post-prandial hyperglycaemia.

Simultaneous Natural Deep Eutectic Solvent-Based Ultrasonic-Assisted Extraction of Bioactive Compounds of Cinnamon Bark and Sappan Wood as a Dipeptidyl Peptidase IV Inhibitor.

Cinnamon bark (Cinnamomum burmannii) and sappan wood (Caesalpinia sappan) have been reported to be beneficial for Type-2 Diabetes Mellitus (T2DM) and the combination is commonly used by Indonesian herbal industries. In the present study, the simultaneous extraction of bioactive compounds from both plants was conducted using natural deep eutectic solvent (NADES), their content analyzed using high-performance liquid chromatography (HPLC), and their dipeptidyl peptidase IV (DPP IV) inhibitory activity evaluated. An additional in silico molecular docking analysis was conducted to ensure their activity. The results showed that NADES (with a composition of choline chloride-glycerol) extraction from cinnamon and sappan wood had DPP IV inhibitory activity of 205.0 and 1254.0 µg/mL, respectively. Brazilin as a marker substance from sappan wood was responsible for the DPP IV inhibitory activity, while none of the marker substances chosen for cinnamon bark (trans-cinnamaldehyde, coumarin, and trans-cinnamic acid) were found to have significant DPP IV inhibitory activity. These results were confirmed by molecular docking conducted in brazilin, trans-cinnamaldehyde, coumarin, and trans-cinnamic acid.

Exploration of dipeptidyl-peptidase IV (DPP IV) inhibitors in a low-molecular mass extract of the earthworm Eisenia fetida and identification of the inhibitors as amino acids like methionine, leucine, histidine, and isoleucine.

We have reported previously that the water extract of the earthworm Eisenia fetida has inhibitory effect on human dipeptidyl-peptidase IV (DPP IV) in vitro. Here we studied to identify DPP IV inhibitors in a low-molecular mass extract (designated U3EE) under 3 kDa prepared from the water extract. U3EE showed 50 % inhibition (IC50) at the concentration of 5.3 ± 0.3 mg/mL. An inhibitory active fraction obtained by solid-phase extraction of U3EE was separated into three parts by reversed-phase HPLC. These parts were shown by GC/MS to be composed of ten (Ala, Gly, Thr, Ser, Asn, Asp, Lys, His, Orn, and cystine), two (Leu and Ile), and one (Met) amino acids, respectively. Among them, Met, Leu, and His showed strong inhibition with IC50 values of 3.4 ± 0.3, 6.1 ± 0.3 and 14.7 ± 1.2 mM, respectively; Ala, Lys, Orn, and Ile showed rather weaker inhibition than those, while the others showed no inhibition. Met, Leu, and Ile were competitive inhibitors and His was a mixed-type one. DPP IV inhibition by U3EE might be due to additive and/or synergistic effects of the inhibitory amino acids, suggesting that it could be useful as pharmaceutical and supplement for diabetes prevention.

Separation and identification of enzyme inhibition peptides from dark tea protein.

Two key enzymes like α-glucosidase and dipeptidyl peptidase-4 (DPP4) play an important role in the development of Type 2 diabetes (T2D), and inhibiting them is a common treatment strategy for T2D. In this study, an ultrafiltration- centrifugation approach was developed to isolate peptides from dark protein extract. Consequently, 6 peptides TAELLPR, CGKKFVR, AVPANLVDLNVPALLK, VVDLVFFAAAK, MSLYPR and QGQELLPSDFK were identified. The four peptides exhibited α-glucosidase inhibition activities with the IC50 values of 0.43 ± 0.03, 0.52 ± 0.09, 1.03 ± 0.13 and 0.04 ± 0.04 mg/mL, respectively, which were lower than the IC50 value of positive drug acarbose (1.51 ± 0.23 mg/mL); the last two peptides had DPP4 inhibition activities with the IC50 values of 1.35 ± 0.15 and 3.89 ± 0.22 mg/mL, respectively. This suggests that dark tea-derived peptides, for the first time, were found to be potential agents for α-glucosidase and DPP4 inhibition.

A dipyrrole derivative from Aloe vera inhibits an anti-diabetic drug target Dipeptidyl Peptidase (DPP)-IV in vitro.

Aloe vera, a succulent herb, has a long history of use in traditional medicine, including diabetes. Earlier studies from our laboratory demonstrated that the Aloe vera extract has the ability to inhibit the diabetic drug target dipeptidyl peptidase (DPP) IV in vitro. This current study focuses on the isolation of small water soluble active molecule(s) involved in DPP-IV inhibition from Aloe vera extract, and further to characterize its structure and to elucidate the mode of inhibition of the DPP-IV enzyme. Aloe vera gel ethanolic extract was subjected to preparative reverse-phase high-pressure liquid chromatography (RP-HPLC), LH-20 Sephadex gel filtration chromatography, followed by analytical RP-HPLC, to isolate the active molecule involved in DPP-IV inhibition. Based on the spectroscopic studies, the structure of the isolated DPP-IV inhibitor was predicted to be 3, 6-dioxo-3, 3a, 6, 6 a-tetrahydropyrrolo [3, 4-c] pyrrole-1, 4-dicarboxamide with the chemical formula C8H6N4O4, having the molecular weight of 225.175 Da. This molecule inhibited the DPP-IV enzyme in a noncompetitive manner with an IC50 value of 8.59 ± 2.61 µM, with a Ki of 4.7 ± 0.038 µM. Thus, the mechanism of DPP-IV inhibition and the inhibitory constants were determined. The results of our studies suggested that the inhibition of the DPP-IV enzyme as one of the pathways by which the Aloe vera extract may restore the pancreatic islets cell mass in diabetic animal model.

Standardized Emblica officinalis fruit extract inhibited the activities of α-amylase, α-glucosidase, and dipeptidyl peptidase-4 and displayed antioxidant potential.

BACKGROUND: Emblica officinalis, known as amla in Ayurveda, has been used as a folk medicine to treat numerous pathological conditions, including diabetes. However, the novel extract of E. officinalis fruit extract (amla fruit extract, AFE, Saberry®) containing 100 g kg-1 ß-glucogallin along with hydrolyzable tannins has not yet been extensively studied for its antidiabetic potential. OBJECTIVE: The aim of this study was to investigate the antidiabetic and antioxidant activities of AFE and its stability during gastric stress as well as its thermostability. METHODS: The effect of AFE on the inhibition of pancreatic α-amylase and salivary α-amylase enzymes was studied using starch and yeast α-glucosidase enzyme using 4-nitrophenyl α-d-glucopyranoside as substrate. Further, 2,2-diphenyl-1-picrylhydrazyl radical scavenging and reactive oxygen species inhibition assay was performed against AFE. RESULTS: AFE potently inhibited the activities of α-amylase and α-glucosidase in a concentration-dependent manner with half maximal inhibitory concentration (IC50 ) values of 135.70 µg mL-1 and 106.70 µg mL-1 respectively. Furthermore, it also showed inhibition of α-glucosidase (IC50 562.9 µg mL-1 ) and dipeptidyl peptidase-4 (DPP-4; IC50 3770 µg mL-1 ) enzyme activities. AFE is a potent antioxidant showing a free radical scavenging activity (IC50 2.37 µg mL-1 ) and protecting against cellular reactive oxygen species (IC50 1.77 µg mL-1 ), and the effects elicited could be attributed to its phytoconstituents. CONCLUSION: AFE showed significant gastric acid resistance and was also found to be thermostable against wet heat. Excellent α-amylase, α-glucosidase, and DPP-4 inhibitory activities of AFE, as well as antioxidant activities, strongly recommend its use for the management of type 2 diabetes mellitus. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Features of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from dietary proteins.

Dipeptidyl peptidase IV (DPP-IV) is involved in incretin hormone processing and therefore plays a key role in glycemic regulation. This review summarizes the latest developments in food protein-derived DPP-IV inhibitory peptides. The in silico approaches currently used to develop targeted strategies for the enzymatic release of DPP-IV inhibitory peptides from food proteins are outlined. The features within the primary sequences of potent DPP-IV inhibitory di-, tri-, and larger peptides, having half maximal inhibitory activity (IC50 ) < 100 µM, were evaluated and the outcomes are presented herein. It is proposed that detailed analysis of those food derived peptides identified in humans following ingestion may constitute a practical strategy for the targeted identification of novel bioavailable DPP-IV inhibitory peptides. Human intervention studies are required as the specific role of food protein-derived DPP-IV inhibitory peptides in the regulation of glycaemia in humans remains to be fully elucidated. PRACTICAL APPLICATIONS: This review provides recent information on dipeptidyl peptidase IV (DPP-IV) inhibitory peptides arising from food protein hydrolysates. Small animal studies have demonstrated that food protein hydrolysates with in vitro DPP-IV inhibitory properties also display antidiabetic activity. DPP-IV inhibitory peptides may be used as food ingredients to improve glycemic regulation in Type 2 diabetics. Therefore, the development of potent DPP-IV inhibitory hydrolysates containing bioavailable peptides in humans is of significant interest. This may help in the formulation of foods containing physiologically relevant doses of bioactive hydrolysates/peptides. Acquisition of detailed knowledge of DPP-IV inhibitory peptide features via the utilization of in silico tools may help to optimize the release of potent DPP-IV inhibitory peptides during enzymatic hydrolysis of food proteins. This review provides information on features within the primary sequences of potent DPP-IV inhibitory peptides and current in silico strategies which may be used to inform on the targeted enzymatic hydrolysis of food proteins.

First report of chromenyl derivatives from spineless marine cuttlefish Sepiella inermis: Prospective antihyperglycemic agents attenuate serine protease dipeptidyl peptidase-IV.

Spineless marine cuttlefish Sepiella inermis has been considered as a popular dietary cephalopod species in Asian and Mediterranean coasts. Bioassay-directed purification of organic extract of S. inermis ensued in the characterization of two chromenyl derivatives. The studied compounds exhibited significantly greater antioxidant potencies (IC50  ≤ 0.5 mg/ml) when compared with α-tocopherol. The substituted 1H-isochromenyloxy-11-hydroxyethyl pentanoate isoform (compound 1) efficiently inhibited the carbolytic enzymes along with key regulator of insulin secretion dipeptidyl peptidase-IV (IC50 0.16 mg/ml). The molecular docking simulations displayed optimum binding affinity of the compound 1 (-10.01 kcal/mol) with dipeptidyl peptidase-IV and lesser inhibition constant (Ki 46.41 nM), which along with its permissible hydrophobic-hydrophilic balance (log Pow  ~ 2) appeared to play significant roles in its greater antihyperglycemic activity compared to other studied chromenyl isoform. The greater antioxidant and antidiabetic properties of compound 1 could be utilized as an important natural lead against hyperglycemic-related disorders. PRACTICAL APPLICATIONS: The edible spineless marine cuttlefish Sepiella inermis are ubiquitously available in Asian and Mediterranean coasts. The sequential chromatographic purification of the organic extract of S. inermis led to the identification of two pure chromenyl chemotypes. The metabolites with substituted 1H-isochromenyloxy-11-hydroxyethyl pentanoate isoform (compound 1) displayed potential antioxidative and antihyperglycemic activities compared to the chemotype (2) bearing 3H-isochromen-5-yl moiety. The attenuating potential of chromenyl chemotype 1 against carbohydrate hydrolyzing enzymes and insulin secretion regulator attributed towards its efficiency as an important natural lead against postprandial hyperglycemia and incretin hormone regulation to maintain glucose homeostasis in the biological system. The chromenyl metabolites isolated from S. inermis could be utilized as a functional food ingredient in the nutraceutical formulations against hyperglycemic-related disorders.

Antidiabetic Stilbenes from Peony Seeds with PTP1B, α-Glucosidase, and DPPIV Inhibitory Activities.

One unusual resveratrol tetramer, paeonilactiflorol (1), and 14 known compounds (2-15) were isolated from peony seeds ( Paeonia lactiflora) under the guidance of bioassay. Paeonilactiflorol (1) was determined by extensive HRESIMS, UV, IR, 1D and 2D NMR spectroscopic analyses. Most of the stilbenes showed obvious inhibition on PTP1B and α-glucosidase, superior to the monoterpene glycosides. Especially, the stilbene tetramer (1) and trimer (8) exhibited high activity inhibiting both PTP1B with IC50 values of 27.23 and 27.81 µM and α-glucosidase with IC50 values of 13.57 and 14.39 µM. Two trans-dimers (4 and 5) also showed dipeptidyl peptidase-4 (DPPIV) inhibitory activity (55.35% and 61.26%, 500 µM) in addition to PTP1B and α-glucosidase. Enzyme kinetic study indicated that the types of inhibition on PTP1B were noncompetitive for 3 and 5 and mixed for 8 and 10. Quantitative analysis suggested that the stilbene trimers 8 (23.17 ± 0.36 mg/g) and 10 (15.24 ± 0.25 mg/g) were the main contents in peony seeds and should be responsible for the antidiabetic effects. This investigation supports the therapeutic potential of peony seeds in the treatment of diabetes with stilbenes as the active constituents.

Peptide identification from a Porphyra dioica protein hydrolysate with antioxidant, angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory activities.

A Porphyra dioica protein extract was enzymatically hydrolysed and then fractionated using semi-preparative reverse-phase high performance chromatography. The hydrolysate and its fractions were tested for their oxygen radical absorbance capacity (ORAC) along with their angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. The most potent fraction was analysed by liquid chromatography mass spectrometry. Eight peptide sequences were selected for synthesis based on their structure-activity criteria for bioactivity. Asp-Tyr-Tyr-Lys-Arg showed the highest ORAC activity (4.27 ± 0.15 µmol Trolox equivalent per µM). Thr-Tyr-Ile-Ala had the highest ACE inhibitory activity (IC50: 89.7 ± 7.10 µM). Tyr-Leu-Val-Ala was the only peptide showing DPP-IV inhibitory activity (IC50: 439 ± 44 µM). Apart from Asp-Tyr-Tyr-Lys-Arg and Thr-Tyr-Ile-Ala, which displayed increased ORAC activity, the bioactivities of the peptides were either maintained or decreased following in vitro simulated gastrointestinal digestion. The results indicate that P. dioica-derived peptides may have potential applications as health enhancing ingredients.

Identification and Quantification of DPP-IV-Inhibitory Peptides from Hydrolyzed-Rapeseed-Protein-Derived Napin with Analysis of the Interactions between Key Residues and Protein Domains.

Previously reported peptides derived from napin of rapeseed ( Brassica napus) have been shown to inhibit DPP-IV in silico. In the present study, napin extracted from rapeseed was hydrolyzed by commercial enzymes and filtered by an ultrafiltration membrane. The napin hydrolysate was then purified by a Sephadex G-15 gel-filtration column and preparative RP-HPLC. A two-enzyme-combination approach with alcalase and trypsin was the most favorable in terms of the DPP-IV-inhibitory activity (IC50 = 0.68 mg/mL) of the napin hydrolysate. Three peptides and one modified peptide (pyroglutamate mutation at the N-terminus) were identified using HPLC-triple-TOF-MS/MS. DPP-IV-inhibitory activity and the types of enzyme inhibition were also determined. Meanwhile, key residues associated with the interactions between the selected peptides and DPP-IV were investigated by molecular docking. IPQVS has key amino acid residues (Tyr547, Glu205, and Glu206) that are consistent with Diprotin A. ELHQEEPL could form a better covalent bond with Arg358 in the S3 pocket of DPP-IV.

Identification of angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory peptides derived from oilseed proteins using two integrated bioinformatic approaches.

Angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) play critical roles in the development of hypertension and type 2 diabetes, respectively. Inhibiting ACE and DPP-IV activity using peptides has become part of new therapeutic strategies for supporting medicinal treatment of both diseases. In this study, oilseed proteins, including soybean, flaxseed, rapeseed, sunflower and sesame are evaluated for the possibility of generating ACE and DPP-IV inhibitory peptides using different integrated bioinformatic approaches (UniProt knowledgebase, ProtParam, BLAST, BIOPEP, PeptideRanker, Pepsite2 and ToxinPred), and three bovine proteins (ß-lactoglobulin, ß-casein and κ-casein) as comparisons. Compared with bovine proteins, the potency indices of ACE and DPP-IV inhibitory peptides, calculated using the BIOPEP database, suggest that oilseed proteins may be considered as good precursors of ACE inhibitory peptides but generate a relative lower yield of DPP-IV inhibitory peptides following subtilisin, pepsin (pH = 1.3) or pepsin (pH > 2) hydrolysis. Average scores aligned using PeptideRanker confirmed oilseed proteins as significant potential sources of bioactive peptides: over 105 peptides scored over 0.8. Pepsite2 predicted that these peptides would largely bind via Gln281, His353, Lys511, His513, Tyr520 and Tyr523 of ACE to inhibit the enzyme, while Trp629 would be the predominant binding site of peptides in reducing DPP-IV activity. All peptides were capable of inhibiting ACE and DPP-IV whilst 65 of these 105 peptides are not currently recorded in BIOPEP database. In conclusion, our in silico study demonstrates that oilseed proteins could be considered as good precursors of ACE and DPP-IV inhibitory peptides as well as so far unexplored peptides that potentially have roles in ACE and DPP-IV inhibition and beyond.

Dipeptidyl peptidase IV (DPP-IV) inhibitory properties of a camel whey protein enriched hydrolysate preparation.

Camel milk proteins contain dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. A camel whey protein concentrate (WPC, 44.7 ±â€¯3.4% (w/w) protein) was prepared and subsequently hydrolysed with trypsin at different temperatures, enzyme to substrate (E:S) ratios and hydrolysis times yielding fifteen hydrolysates (H1-H15). Their DPP-IV half maximal inhibitory concentrations (IC50) ranged from 0.55 ±â€¯0.05 to 1.52 ±â€¯0.16 mg L-1 for H8 and H6, respectively. E:S was the only factor having a significant effect on the DPP-IV IC50 value (p < 0.05). Relatively potent α-lactalbumin-derived DPP-IV inhibitory peptides (LAHKPL and ILDKEGIDY) were detected in selected hydrolysates. Additionally, three potent ß-CN-derived peptides, VPV, YPI and VPF having DPP-IV IC50 values of 6.6 ±â€¯0.5, 35.0 ±â€¯2.0 and 55.1 ±â€¯5.8 µM, respectively, were identified. After IPI, VPV is the second most potent DPP-IV inhibitory peptide identified to date, which supports the role of camel milk as an antidiabetic agent.