Identification, characterization, and insights into the mechanism of novel dipeptidyl peptidase-IV inhibitory peptides from yak hemoglobin by in silico exploration, molecular docking, and in vitro assessment.

Dipeptidyl peptidase IV (DPP-IV) inhibitory peptides were screened and identified from yak hemoglobin for the first time by in silico analysis, molecular docking, and in vitro evaluation. Results showed that yak hemoglobin had a high potential to produce DPP-IV inhibitory peptides based on the sequence alignment and bioactive potential evaluation. Furthermore, "pancreatic elastase + stem bromelain" was the optimal combined-enzymatic strategy by simulated proteolysis. Additionally, 25 novel peptides were found from its simulated hydrolysate, among which 10 peptides had high binding affinities with DPP-IV by molecular docking. Most of these peptides were also in silico characterized with favorable physicochemical properties and biological potentials, including relatively low molecular weight, high hydrophobicity, several net charges, good water solubility, nontoxicity, acceptable sensory quality, and good human intestinal absorption. Finally, six novel DPP-IV inhibitory peptides were identified via in vitro assessment, among which EEKA (IC50 = 235.26 µM), DEV (IC50 = 339.45 µM), and HCDKL (IC50 = 632.93 µM) showed the strongest capacities. The hydrogen bonds and electrostatic attractions formed with core residues within the S2 pocket of DPP-IV could be mainly responsible for their inhibition performances. This work provided a time-saving method and broadened application for yak by-products development as sources of functional foods.

Discovery of anthraquinones as DPP-IV inhibitors: Structure-activity relationships and inhibitory mechanism.

Dipeptidyl peptidase IV (DPP-IV) is an integrated type II transmembrane protein that reduces endogenous insulin contents and increases plasma glucose levels by hydrolyzing glucagon-like peptide-1 (GLP-1). Inhibition of DPP-IV regulates and maintains glucose homeostasis, making it an attractive drug target for the treatment of diabetes II. Natural compounds have tremendous potential to regulate glucose metabolism. In this study, we examined the DPP-IV inhibitory activity of a series of natural anthraquinones and synthetic structural analogues on DPP-IV using fluorescence-based biochemical assays. The inhibitory efficiency differed among anthraquinone compounds with different structures. Alizarin (7), aloe emodin (11), emodin (13) emerged the outstanding inhibitory potential for DPP-IV with IC50 values lower than 5 µM. To clarifying the inhibitory mechanism, inhibitory kinetics were performed, which showed that alizarin red S (8) and 13 were effective non-competitive inhibitors of DPP-IV, while alizarin complexone (9), rhein (12), and anthraquinone-2-carboxylic acid (23) were mixed inhibitors. Emodin was determined as inhibitor with the strongest DPP-IV-binding affinity determined via molecular docking. Structure-activity relationship (SAR) demonstrated that hydroxyl group at C-1 and C-8 sites and hydroxyl, hydroxymethyl or carboxyl group at the C-2 or C-3 site were very essential for DPP-IV inhibition, replacement of hydroxyl group with amino group at C-1 could led to an increase of the inhibitory potential. Further fluorescence imaging showed that both compounds 7 and 13 significantly inhibited DPP-IV activity in RTPEC cells. Overall, the results indicated that anthraquinones would be a natural functional ingredient for inhibiting DPP-IV and provided new ideas for searching and developing potential antidiabetic compounds.

Exploration of Dipeptidyl Peptidase-IV (DPP-IV) Inhibitory Peptides from Silkworm Pupae (Bombyx mori) Proteins Based on In Silico and In Vitro Assessments.

This study aimed at exploring dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides from silkworm pupae proteins by in silico analysis and in vitro assessments. In silico analysis of 274 silkworm pupae proteomes indicated that DPP-IV inhibitory peptides can be released from silkworm pupae proteins. In vitro assessments revealed that pepsin and bromelain led to better production of DPP-IV inhibitory peptides from silkworm pupae protein. Notably, peptide fractions (<1 kDa) from pepsin- and bromelain-treated hydrolysates exhibited more potent DPP-IV inhibitory activities. Two novel DPP-IV inhibitory peptides (Leu-Pro-Pro-Glu-His-Asp-Trp-Arg and Leu-Pro-Ala-Val-Thr-Ile-Arg) were identified by LC-MS/MS with IC50 values of 261.17 and 192.47 µM, respectively. Enzyme kinetics data demonstrated that these two peptides displayed a mixed-type DPP-IV inhibition mode, which was further validated by molecular docking data. Overall, in silico analysis combined with in vitro assessments can serve as an effective and rapid approach for discovery of DPP-IV peptides from silkworm pupae proteins.

Hypoglycemic peptide-enriched hydrolysates of Corbicula fluminea and Chlorella sorokiniana possess synergistic hypoglycemic activity through inhibiting α-glucosidase and dipeptidyl peptidase-4 activity.

BACKGROUND: The prevalence of diabetes mellitus worldwide has increased in recent decades. Maintaining the level of blood glucose is the most basic and important issue for diabetics. This study aimed to investigate the hypoglycemic activity of a combination of hypoglycemic peptide-enriched hydrolysates of Corbicula fluminea (ACH) and Chlorella sorokiniana (PCH). RESULTS: Combined supplementation of ACH and PCH synergistically inhibited α-glucosidase and DPP4 activities in vitro. After 4 weeks of treatment with ACH and/or PCH, the plasma glucose concentration and insulin, homeostasis model assessment-estimated insulin resistance (HOMA-IR), total cholesterol (TC) and triglyceride (TG) levels significantly decreased. The hypoglycemic peptides in ACH and PCH were purified and assayed for α-glucosidase and DPP4 activity. The hypoglycemic peptides in ACH and PCH effectively decreased α-glucosidase and DPP4 activities. In silico assays showed that these two peptide types have different docking poses, which determined their inhibitory effect against α-glucosidase and DPP4 activity. CONCLUSION: Combined treatment with hypoglycemic peptide-enriched ACH and PCH could modulate blood glucose by synergistically inhibiting α-glucosidase and DPP4 activities. © 2021 Society of Chemical Industry.

Identification of in vitro angiotensin-converting enzyme and dipeptidyl peptidase IV inhibitory peptides from draft beer by virtual screening and molecular docking.

BACKGROUND: Hypertension and diabetes are two kinds of senile diseases which often occur simultaneously. The commonly used drugs in clinic may produce certain side effects. Food-derived polypeptide is a kind of polypeptide with great development potential, which has many functions of regulating human physiological function. Beer is rich in nutrition but there are few researches on bioactive peptides in beer. RESULTS: In this study, a rapid virtual screening method was established to obtain bioactive peptides from Tsingtao draft beer. The peptide sequence was analyzed by ultra-performance liquid chromatography-quadrupole-Orbitrap-tandem mass spectrometry (UPLC-Q-Orbitrap-MS2 ), and 50 peptides were identified. Eight peptides with potential biological activities were screened by using Peptide Ranker software and previous literature references. On the basis of absorption prediction, toxicity prediction, and molecular docking analysis, LNFDPNR and LPQQQAQFK were finally confirmed. The molecular docking results showed that two peptides could bind angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) tightly by hydrogen bonding and hydrophobic interaction. The in vitro activity evaluation results showed that two peptides had obvious ACE and DPP-IV inhibitory activity. CONCLUSION: This study established a method for rapidly screening bioactive peptides from Tsingtao draft beer, screened two ACE and DPP-IV inhibitory peptides in beer and analyzed their active action mechanism. This article may have great theoretical significance and practical value to further explore the health function of beer. © 2021 Society of Chemical Industry.

Characterization of peptides with antioxidant activity and antidiabetic potential obtained from chickpea (Cicer arietinum L.) protein hydrolyzates.

Alcalase hydrolyzates were prepared from the albumin (AH) and globulin (GH) fractions of eight chickpea (Cicer arietinum L.) genotypes from Mexico and 10 from other countries. Protein content, antioxidant activity (AA) (ABTS, DPPH), and degree of hydrolysis were evaluated and the best genotype was selected by principal component analysis. The hydrolyzates of the chosen genotype were analyzed for its antidiabetic potential measured as inhibition of α-amylase, α-glucosidase, and dipeptidyl peptidase-4 (DPP4). Peptide profiles were obtained by liquid chromatography-mass spectrometry (UPLC-DAD-MS), and the most active peptides were analyzed by molecular docking. The average antioxidant activity of albumin hydrolyzates was higher than that of globulin hydrolyzates. ICC3761 was the selected genotype and peptides purified from the albumin hydrolyzate showed the best antioxidant activity and antidiabetic potential (FEI, FEL, FIE, FKN, FGKG, and MEE). FEI, FEL, and FIE were in the same chromatographic peak and this mixture showed the best ABTS scavenging (78.25%) and DPP4 inhibition (IC50  = 4.20 µg/ml). MEE showed the best DPPH scavenging (47%). FGKG showed the best inhibition of α-amylase (54%) and α-glucosidase (56%) and may be a competitive inhibitor based on in silico-predicted interactions with catalytic amino acids in the active site of both enzymes. These peptides could be used as nutraceutical supplements against diseases related to oxidative stress and diabetes. PRACTICAL APPLICATION: This study showed that chickpea protein hydrolyzates are good sources of peptides with antidiabetic potential, showing high antioxidant activity and inhibition of enzymes related to carbohydrate metabolism and type 2 diabetes. These hydrolyzates could be formulated in functional foods for diabetes.

Protease Inhibitors Purified from the Canola Meal Extracts of Two Genetically Diverse Genotypes Exhibit Antidiabetic and Antihypertension Properties.

Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse Brassica napus genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (pI) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of pI 8.65 and 9.9, and one acidic of pI 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC50 values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.

Synthesis, Biological Evaluation, and QPLD Studies of Piperazine Derivatives as Potential DPP-IV Inhibitors.

BACKGROUND: Diabetes mellitus is a serious global health issue, currently affecting 425 million people and is set to affect over 690 million people by 2045. It is a chronic disease characterized by hyperglycemia due to relative or absolute insulin hormone deficiency. Dipeptidyl peptidase- IV (DPP-IV) inhibitors are hypoglycemic agents augmenting the action of the incretin hormones that stimulate insulin secretion from the pancreatic beta cells. OBJECTIVE: In this study, synthesis and biological evaluation of seven piperazine derivatives 3a-g was carried out. METHODS: The synthesized molecules were characterized using proton-nuclear magnetic resonance, carbon-nuclear magnetic resonance, infrared spectroscopy and mass spectrometry. RESULTS: In vitro biological evaluation study showed comparable DPP-IV inhibitory activity for the targeted compounds ranging from 19%-30% at 100 µM concentration. Furthermore, the in vivo hypoglycemic activity of 3d was evaluated using streptozotocin-induced diabetic mice. It was found that compound 3d significantly decreased the blood glucose level when the diabetic group treated with 3d was compared to the control diabetic group. Quantum-Polarized Ligand Docking (QPLD) studies demonstrate that 3a-g fit the binding site of DPP-IV enzyme and form H-bonding with the backbones of R125, E205, E206, K554, W629, Y631, Y662, R669, and Y752. CONCLUSION: Piperazine derivatives were successfully found to be new scaffolds as potential DPP-IV inhibitors.

Flavonol Glycosides: In Vitro Inhibition of DPPIV, Aldose Reductase and Combating Oxidative Stress are Potential Mechanisms for Mediating the Antidiabetic Activity of Cleome droserifolia.

Diabetes is a major health problem that is associated with high risk of various complications. Medicinal plants hold great promise against diabetes. The traditional use of Cleome droserifolia as an antidiabetic agent was correlated to its flavonol glycosides content. In the current study, five major flavonol glycosides appeared on the RP-HPLC chromatogram of the aqueous extract namely; quercetin-3-O-ß-d-glucosyl-7-O-α-rhamnoside (1), isorhamnetin-7-O-ß-neohesperidoside (2), isorhamnetin-3-O-ß-d-glucoside (3) kaempferol-4'-methoxy-3,7-O-α-dirhamnoside (4), and isorhamnetin-3-O-α-(4″-acetylrhamnoside)-7-O-α-rhamnoside (5). The inhibitory activities of these compounds were tested in vitro against several enzymes involved in diabetes management. Only the relatively less polar methoxylated flavonol glycosides (4, 5) showed mild to moderate α-amylase and α-glucosidase inhibitory activities. Compounds 1-4 displayed remarkable inhibition of dipeptidyl peptidase IV (DPPIV) enzyme (IC50 0.194 ± 0.06, 0.573 ± 0.03, 0.345 ± 0.02 and 0.281 ± 0.05 µg/mL, respectively) comparable to vildagliptin (IC50 0.154 ± 0.02 µg/mL). Moreover, these compounds showed high potential in preventing diabetes complications through inhibiting aldose reductase enzyme and combating oxidative stress. Both isorhamnetin glycoside derivatives (2, 3) exhibited the highest activities in aldose reductase inhibition and compound 2 (IC50 5.45 ± 0.26 µg/mL) was even more potent than standard quercetin (IC50 7.77 ± 0.43 µg/mL). Additionally, these flavonols exerted excellent antioxidant capacities through 2, 2-diphenyl-1-picrylhydrazil (DPPH) and ferric reducing antioxidant (FRAP) assays.

Identification of Dipeptidyl Peptidase-4 and α-Amylase Inhibitors from Melicope glabra (Blume) T. G. Hartley (Rutaceae) Using Liquid Chromatography Tandem Mass Spectrometry, In Vitro and In Silico Methods.

The present study investigated the antidiabetic properties of the extracts and fractions from leaves and stem bark of M. glabra based on dipeptidyl peptidase-4 (DPP-4) and α-Amylase inhibitory activity assays. The chloroform extract of the leaves was found to be most active towards inhibition of DPP-4 and α-Amylase with IC50 of 169.40 µg/mL and 303.64 µg/mL, respectively. Bioassay-guided fractionation of the leaves' chloroform extract revealed fraction 4 (CF4) as the most active fraction (DPP-4 IC50: 128.35 µg/mL; α-Amylase IC50: 170.19 µg/mL). LC-MS/MS investigation of CF4 led to the identification of trans-decursidinol (1), swermirin (2), methyl 3,4,5-trimethoxycinnamate (3), renifolin (4), 4',5,6,7-tetramethoxy-flavone (5), isorhamnetin (6), quercetagetin-3,4'-dimethyl ether (7), 5,3',4'-trihydroxy-6,7-dimethoxy-flavone (8), and 2-methoxy-5-acetoxy-fruranogermacr-1(10)-en-6-one (9) as the major components. The computational study suggested that (8) and (7) were the most potent DPP-4 and α-Amylase inhibitors based on their lower binding affinities and extensive interactions with critical amino acid residues of the respective enzymes. The binding affinity of (8) with DPP-4 (-8.1 kcal/mol) was comparable to that of sitagliptin (-8.6 kcal/mol) while the binding affinity of (7) with α-Amylase (-8.6 kcal/mol) was better than acarbose (-6.9 kcal/mol). These findings highlight the phytochemical profile and potential antidiabetic compounds from M. glabra that may work as an alternative treatment for diabetes.

Phytosterol supplements do not inhibit dipeptidyl peptidase-4.

BACKGROUND AND AIMS: Several commercially available phytosterol supplements are promoted for their cholesterol-lowering effects. However, limited information is available about their potential anti-hyperglycaemic effects. This study aimed to evaluate the dipeptidyl peptidase-4 (DPP-4) inhibitory effects of phytosterol supplements in silico and in vitro to determine their potential for anti-diabetic activity. METHODS: Docking studies were carried out in silico to evaluate the potential for interactions between three major phytosterol compounds (stigmasterol, ß-sitosterol, campesterol) and the DPP-4 enzyme, the enzyme that is inhibited by the anti-diabetic gliptins. Gas chromatography-tandem mass spectrometry (GC-MS/MS) was used to analyse three different supplements for phytosterol content. DPP-4 inhibitory activity was tested in vitro for these phytosterol supplements and two major phytosterol standards. RESULTS: In silico calculations predicted free binding energies for DPP-4 with the phytosterols to be: stigmasterol -8.78 kcal/mol; ß-sitosterol -8.70 kcal/mol; campesterol -8.40 kcal/mol. These binding energies indicated a potential for significant DPP-4 inhibition. However, these results were not supported by the in vitro studies. Stigmasterol and ß-sitosterol had an IC50 > 50 mg/ml (maximum tested concentration) and the Thompson's Cholesterol Manager® and Mega Strength Beta Sitosterol® supplements gave an IC50 > 100 mg/ml (maximum tested concentration). Blackmores Cholesterol Health® gave an IC50 value of 40 mg/ml which was attributed to ß-carotene content. CONCLUSIONS: Phytosterol supplements do not appear to offer any anti-diabetic activity potential via pathways that involve the inhibition of DPP-4.

Purification and biochemical characterization of a novel secretory dipeptidyl peptidase IV from porcine serum.

Purification of DPP-IV enzyme from porcine serum, is presented in this study for the first time. The high molecular weight DPP-IV from porcine serum was fractioned using Sephadex G-75 gel filtration followed by DEAE Sephadex anion exchange and Sephadex G-100 gel filtration chromatography columns with a final yield of 11.25%. The SDS-PAGE of the purified sample showed a single band of molecular mass nearing 160 kDa. Distinct single band was observed after PAS staining confirmed it to be a glycoprotein. The purified enzyme showed an optimum pH and temperature of 8 and 37 °C, respectively. The enzyme effectively cleaved fluorogenic substrate Gly-Pro-AMC with Km and Vmax of 4.578 µM and 90.84 nmoles/min, respectively. Purified DPP-IV activity was inhibited by Diprotin A with an IC50 value of 8.473 µM. Among the three plant extracts used to study DPP-IV inhibition, the aqueous hot extract of Terminalia chebula showed the highest inhibition of 87.19%, followed by the aqueous cold extract of Momordica carantia, ( 31.6%) and Azadirachta indica (34.16%) at the concentration of 25 µg.

Assessment of the Multifunctional Behavior of Lupin Peptide P7 and Its Metabolite Using an Integrated Strategy.

LTFPGSAED (P7) is a multifunctional hypocholesterolemic and hypoglycemic lupin peptide. While assessing its angiotensin-converting enzyme (ACE) inhibitory activity, it was more effective in intestinal Caco-2 cells (IC50 of 13.7 µM) than in renal HK-2 cells (IC50 of 79.6 µM). This discrepancy was explained by the metabolic transformation mediated by intestinal peptidases, which produced two main detected peptides, TFPGSAED and LTFPG. Indeed LTFPG, dynamically generated by intestinal dipeptidyl peptidase IV as well as its parent peptide P7 were linearly absorbed by mature Caco-2 cells. An in silico study demonstrated that the metabolite was a better ligand of the ACE enzyme than P7. These results are in agreement with an in vivo study, previously performed by Aluko et al., which has shown that LTFPG is an effective hypotensive peptide. Our work highlights the dynamic nature of bioactive food peptides that may be modulated by the metabolic activity of intestinal cells.

Inhibitory effect of chestnut (Castanea mollissima Blume) inner skin extract on the activity of α-amylase, α-glucosidase, dipeptidyl peptidase IV and in vitro digestibility of starches.

This study aimed to investigate the inhibitory effect of chestnut inner skin extract (CISE) on the activity of postprandial blood sugar-related enzymes. In total, 12 flavonoids were identified by HPLC-TOF-MS. CISE showed strong and weak inhibition on α-amylase and α-glucosidase, with the IC50 of 27.2 and 2.3 µg/mL, respectively. The inhibition modes of CISE against α-amylase and α-glucosidase were mixed-type and non-competitive type, respectively. Epicatechin gallate noncompetitively inhibited α-amylase, α-glucosidase and dipeptidyl peptidase IV (DPP-IV). Analysis by ultraviolet-visible spectroscopy, fluorescence spectroscopy and circular dichroism suggested that flavonoids altered the hydrophobicity and microenvironment of these enzymes. CISE decreased the starch bioavailability by reducing the enzymatic hydrolysis rate and increasing the fraction of undigested starch. The extract reduced the rapidly digestible starch and increased the resistant starch after incorporation into A-, B- or C- crystallinity starch. Thus, the chestnut inner skin is a useful resource for regulating postprandial blood sugar level.

Phytochemicals in Garlic Extract Inhibit Therapeutic Enzyme DPP-4 and Induce Skeletal Muscle Cell Proliferation: A Possible Mechanism of Action to Benefit the Treatment of Diabetes Mellitus.

Diabetes mellitus is a severe health problem in Mexico, and its prevalence is increasing exponentially every year. Recently, DPP-4 (dipeptidyl peptidase-4) inhibitors have become attractive oral anti-hyperglycemic agents to reduce the pathology of diabetes. Gliptin's family, such as sitagliptin, vildagliptin, and alogliptin, are in clinical use to treat diabetes mellitus but possess side effects. Therefore, there is a specific need to look for new therapeutic scaffolds (biomolecules). Garlic bulb is widely used as a traditional remedy for the treatment of diabetes. The garlic extracts are scientifically proven to control glucose levels in patients with diabetes, despite the unknown mechanism of action. The aim of the study is to investigate the antidiabetic effects of ultrasonication assisted garlic bulb extract. To achieve this, in-vitro assays such as DPP-4 inhibitory and antioxidant activities were investigated. Further, functional group analysis using FTIR and identification of phytochemicals using mass spectrometry analysis was performed. The results showed that 70.9 µg/mL of garlic bulb extract inhibited 50% DPP-4 activity. On top of that, the garlic extract exhibited a 20% scavenging activity, equivalent to 10 µg/mL of ascorbic acid. Molecular docking simulations on identified phytochemicals using mass spectrometry revealed their potential binding at the DPP-4 druggable region, and therefore the possible DPP-4 inhibition mechanism. These results suggest that prepared garlic extract contains phytochemicals that inhibit DPP-4 and have antioxidant activity. Also, the prepared extract induces skeletal muscle cell proliferation that demonstrates the antidiabetic effect and its possible mechanism of action.

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.

Model Optimization and In Silico Analysis of Potential Dipeptidyl Peptidase IV Antagonists from GC-MS Identified Compounds in Nauclea latifolia Leaf Extracts.

Dipeptidyl peptidase IV (DPP-IV) is a pharmacotherapeutic target in type 2 diabetes. Inhibitors of this enzyme constitute a new class of drugs used in the treatment and management of type 2 diabetes. In this study, phytocompounds in Nauclea latifolia (NL) leaf extracts, identified using gas chromatography-mass spectroscopy (GC-MS), were tested for potential antagonists of DPP-IV via in silico techniques. Phytocompounds present in N. latifolia aqueous (NLA) and ethanol (NLE) leaf extracts were identified using GC-MS. DPP-IV model optimization and molecular docking of the identified compounds/standard inhibitors in the binding pocket was simulated. Drug-likeness, pharmacokinetic and pharmacodynamic properties of promising docked leads were also predicted. Results showed the presence of 50 phytocompounds in NL extracts of which only 2-O-p-methylphenyl-1-thio-ß-d-glucoside, 3-tosylsedoheptulose, 4-benzyloxy-6-hydroxymethyl-tetrahydropyran-2,3,5-triol and vitamin E exhibited comparable or better binding iGEMDOCK and AutoDock Vina scores than the clinically prescribed standards. These four compounds exhibited promising drug-likeness as well as absorption, distribution, metabolism, excretion and toxicity (ADMET) properties suggesting their candidature as novel leads for developing DPP-IV inhibitors.

In Silico and In Vitro Assessment of Portuguese Oyster (Crassostrea angulata) Proteins as Precursor of Bioactive Peptides.

In this study, the potential bioactivities of Portuguese oyster (Crassostrea angulata) proteins were predicted through in silico analyses and confirmed by in vitro tests. C. angulata proteins were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by proteomics techniques. Hydrolysis simulation by BIOPEP-UWM database revealed that pepsin (pH > 2) can theoretically release greatest amount of bioactive peptides from C. angulata proteins, predominantly angiotensin I-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory peptides, followed by stem bromelain and papain. Hydrolysates produced by pepsin, bromelain and papain have shown ACE and DPP-IV inhibitory activities in vitro, with pepsin hydrolysate (PEH) having the strongest activity of 78.18% and 44.34% at 2 mg/mL, respectively. Bioactivity assays of PEH fractions showed that low molecular weight (MW) fractions possessed stronger inhibitory activity than crude hydrolysate. Overall, in vitro analysis results corresponded with in silico predictions. Current findings suggest that in silico analysis is a rapid method to predict bioactive peptides in food proteins and determine suitable enzymes for hydrolysis. Moreover, C. angulata proteins can be a potential source of peptides with pharmaceutical and nutraceutical application.

Identification of a Novel Scaffold for Inhibition of Dipeptidyl Peptidase-4.

Dipeptidyl peptidase-4 (DPP-4) is considered a major drug target for type 2 diabetes mellitus (T2DM). In addition to T2DM, a regulatory role of DPP-4 was also found in cardiovascular diseases. Existing DPP-4 inhibitors have been reported to have several adverse effects. In this study, a computer-aided drug design approach and its use to detect a novel class of inhibitor for DPP-4 are reported. Through structure and pharmacophore-based screening, we identified 13 hit compounds from an ∼4-million-compound library. Physical interactions of these hits with DPP-4 were studied using docking and explicit solvent molecular dynamics (MD) simulations. Later, MMPBSA binding energy was calculated for the ligand/protein simulation trajectories to determine the stability of compounds in the binding cavity. These compounds have a novel scaffold and exhibited a stable binding mode. "Best-in-screen" compounds (or their closest available analogs) were resourced and their inhibition of DPP-4 activity was experimentally validated using an in vitro enzyme activity assay in the presence of 100 and 10 µM compounds. These assays identified a compound with a spirochromanone center with 53% inhibition activity at a 100 µM concentration. A further five spirochromanone compounds were synthesized and examined in silico and in vitro; again, one compound showed 53% inhibitory activity action at 100 µM. Overall, this study identified two novel "spirochromanone" compounds that lowered DPP-4 activity by more than ∼50% at 100 µM. This study also showed the impact of fast in silico drug design techniques utilizing virtual screening and MD to identify novel scaffolds to bind and inhibit DPP-4. Spirochromanone motif identified here may be used to design molecules to achieve drug-like inhibitory action against DPP-4.