Hypoglycemic peptide preparation from Bacillus subtilis fermented with Pyropia: Identification, molecular docking, and in vivo confirmation.

Hypoglycemic foods have attracted increasing research interest. This study prepared a hypoglycemic product from Bacillus subtilis fermented with Pyropia (PBP), which has promising industrial potential, and elucidated its hypoglycemic mechanism. The aqueous PBP solution was orange, with protein as the main functional component. In vivo experiments demonstrated that PBP could increase insulin secretion and inhibit α-glucosidase activity, resulting in a hypoglycemic effect superior to that of acarbose at the same dose. Molecular docking revealed that the peptides APPVDID, GPPDSPY, PPSSPRP, and SPPPPPA from PBP could inhibit both α-glucosidase and dipeptidyl peptidase-IV (DPP-IV) activities. Pro residues promoted PBP peptide binding to the hydrophobic pocket S1 of DPP-IV. Additionally, PBP reduced inflammation and promoted the growth of beneficial gut bacteria (Prevotellaceae_UCG_003, Lachnospiraceae_UCG_001). This study presents a novel approach for the high-value utilization of Pyropia and a new option for the production of hypoglycemic functional foods and medicines.

Purification and Identification of Novel Dipeptidyl Peptidase IV Inhibitory Peptides Derived from Bighead Carp (Hypophthalmichthys nobilis).

Dipeptidyl peptidase IV (DPP-IV) inhibitors are widely used in treating type 2 diabetes due to their ability to lower blood glucose levels. However, synthetic versions often lead to gastrointestinal side effects. This study explores DPP-IV inhibitory properties in peptides from bighead carp skin. Collagen was prepared, hydrolyzed into collagen peptides, and then fractionated for DPP-IV inhibitory activity examination. The most effective fractions were identified, and their peptide sequences were determined. Molecular docking analysis identified nine peptides with potential inhibitory activity, four of which (VYP, FVA, PPGF, PGLVG) were synthesized and tested in vitro. PPGF exhibited the highest potency with an IC50 of 4.63 nM, competitively binding to key DPP-IV sites, including ARG125, VAL711, TYR666, and TYR662. Other peptides showed varying effectiveness, with IC50 values of 398.87 nM (VYP), 402.02 nM (FVA), and 110.20 nM (PGLVG). These findings highlight bighead carp skin peptides as potent DPP-IV inhibitors with hypoglycemic potential, suggesting a novel avenue for diabetes management using natural peptides. Moreover, this research underscores the utilization of bighead carp by-products, contributing to environmental sustainability.

Influence of InVitro Digestion on Dipeptidyl Peptidase-IV (DPP-IV) Inhibitory Activity of Plant-Protein Hydrolysates Obtained from Agro-Industrial By-Products.

This study investigates the production of protein hydrolysates with dipeptidyl peptidase-IV (DPP-IV) inhibitory activity from agro-industrial by-products, namely olive seed, sunflower seed, rapeseed, and lupin meals, as well as from two plant protein isolates such as pea and potato. Furthermore, the effect of simulated gastrointestinal digestion on the DPP-IV inhibitory activity of all the hydrolysates was evaluated. Overall, the lowest values of IC50 (1.02 ± 0.09 - 1.24 ± 0.19 mg protein/mL) were observed for the hydrolysates with a high proportion of short-chain [< 1 kDa] peptides (i.e., olive seed, sunflower seed, and lupin) or high content of proline (i.e., rapeseed). Contrarily, the IC50 of the pea and potato hydrolysates was significantly higher (1.50 ± 0.13 - 1.93 ± 0.13 mg protein/mL). In vitro digestion led to an increase in peptides <1 kDa for almost all hydrolysates (except olive and sunflower seed meals), which was noticeable for rapeseed, pea, and potato hydrolysates. Digestion did not significantly modify the DPP-IV inhibitory activity of olive, sunflower, rapeseed, and potato hydrolysates, whereas a significant decrease in IC50 value was obtained for pea hydrolysate and a significant increase in IC50 was obtained for lupin hydrolysate. Thus, this work shows the potential of agro-industrial by-products for the production of protein hydrolysates exhibiting DPP-IV inhibition.

DPP-IV Inhibitory Peptide against In Vitro Gastrointestinal Digestion Derived from Goat's Milk Protein and Its Activity Enhancement via Amino Acid Substitution.

Goat milk protein can release a variety of bioactive peptides after digestion, while most of them are digested into free amino acids or dipeptides via the GI tract. We investigated the peptide profiles of goat milk protein following in vitro gastrointestinal digestion using LC-MS/MS and identified 683 bioactive peptides, including 105 DPP-IV inhibitory peptides. Among these peptides, ILDKVGINY (IL), derived from ß-lactoglobulin, was found to be high in content and resistance to digestion. Herein, we explore the effect of amino acid residue substitution at the second N-terminus on its DPP-IV inhibitory activity. Three 9 polypeptide fragments (peptide IL, IP, and II) were synthesized and subjected to molecular docking and activity analysis. The peptide IL demonstrated the highest affinity for DPP-IV with a binding energy of -8.4 kcal/mol and a moderate IC50 value of 1.431 mg/mL determined based on the Caco-2 cell model. The replacement of specific amino acid residues by Pro and Leu led to an increase in the hydrophobic force interaction between the inhibitor peptide and DPP-IV. The inhibition rates of the three peptides were significantly different (p < 0.05). Peptide II containing an Ile residue instead of Leu resulted in a significant enhancement of DPP-IV inhibitory activity, with an IC50 value of 0.577 mg/mL. The GRAVY changes in the three peptides were consistent with the trend of the inhibitory rates. Therefore, the GRAVY of peptides and branch-chain amino acids should be considered in its activity improvement. The present study revealed the presence and activity of DPP-IV inhibitory peptides in goat milk, providing important insights for further investigation of their potential food functionality and health benefits.

LIRAGLUTIDE PREVENTS BODY AND FAT MASS GAIN IN OVARIECTOMIZED WISTAR RATS.

Estrogens exert beneficial metabolic effects by reducing food intake and enhancing energy expenditure through both central and peripheral mechanisms. The decrease of estrogen, as occurs in ovariectomy (OVX), leads to metabolic disturbances, such as increased body weight, adipose tissue mass, basal blood glucose, and impaired glucose tolerance. These effects can be reversed by reintroducing estrogen. GLP-1 and its receptor agonists, known for their antihyperglycemic properties, also exhibit anorexigenic effects. Besides that, research indicates that GLP-1 analogs can induce metabolic changes peripherally, such as increased fatty acid oxidation and inhibited lipogenesis. Given the shared metabolic actions of GLP-1 and estrogens, we explored whether liraglutide, a GLP-1 agonist, could mitigate the metabolic effects of estrogen deficiency. We tested this hypothesis using ovariectomized rats, a model that simulates menopausal estrogen deficiency, and treated them with either liraglutide or 17ß-Estradiol benzoate for 21 days. Ovariectomy resulted in elevated DPP-IV activity in both plasma and inguinal white adipose tissue (iWAT). While estrogen replacement effectively countered the DPP-IV increase in both plasma and iWAT, liraglutide only prevented the rise in iWAT DPP-IV activity. Liraglutide prevented body weight and fat mass gain after ovariectomy to the same extent as estradiol treatment. This can be explained by the lower food intake and food efficiency caused by estradiol and liraglutide. However, liraglutide was associated with increased pro-inflammatory cytokines and inflammatory cells in white adipose tissue. Further research is crucial to fully understand the potential benefits and risks of using GLP-1 receptor agonists in the context of menopause.

Defatted Wheat Germ Protein-Derived Peptides Showed Multiple Biological Activities from the Stomach to Small Intestine: In Silico and In Vitro Approaches.

This study aimed to test the hypothesis that bioactive peptides can exert multiple bioactivities at different sites in the gastrointestinal tract. Our previous research identified 33 gastric-resistant peptides derived from wheat germ with potential antiadhesive activity against Helicobacter pylori in the stomach. In this work, in silico digestion of these peptides with trypsin, thermolysin, and chymotrypsin produced 67 peptide fragments. Molecular docking was conducted to predict their ACE and DPP-IV inhibitory activities in the small intestine. Three peptides (VPIPNPSGDR, VPY, and AR) were selected and synthesized for in vitro validation. Their generation in the gastrointestinal tract was verified via in vitro digestion, followed by mass spectrometry analysis. The IC50 values for ACE inhibition were 199.5 µM (VPIPNPSGDR), 316.3 µM (VPY), and 446.7 µM (AR). For DPP-IV inhibition, their IC50 values were 0.5, 1.6, and 4.0 mM, respectively. This research pioneers new directions in the emerging field of multifunctional peptides, providing scientific evidence to support the utilization of wheat germ as value-added food ingredients.

Multiple roles of food-derived bioactive peptides in the management of T2DM and commercial solutions: A review.

Type 2 diabetes mellitus (T2DM), a disease that threatens public health worldwide and can cause a series of irreversible complications, has been a major concern. Although the treatment based on hypoglycemic drugs is effective, its side effects should not be ignored, which has led to an urgent need for developing new hypoglycemic drugs. Bioactive peptides with antidiabetic effects obtained from food proteins have become a research hotspot as they are safer and with higher specificity than traditional hypoglycemic drugs. Here, we reviewed antidiabetic peptides that have the ability to inhibit key enzymes (α-glucosidase, α-amylase, and DPP-IV) in T2DM, the hypoglycemic mechanisms and structure-activity relationships were summarized, some antidiabetic peptides that improve insulin resistance and reverse gut microbiota and their metabolites were overviewed, the bitterness of antidiabetic peptides was predicted in silico, proposed solutions to the current challenges encountered in the development of antidiabetic peptide drugs, and provided an outlook on the future focus of commercial production. It provides a reference for the application of food-derived antidiabetic peptides.

Exploring the dipeptidyl peptidase IV inhibitory potential of probiotic-fermented milk: An in vitro and in silico comprehensive investigation into peptides from milk of different farm animals.

Bioactive peptides produced via enzymatic hydrolysis have been widely investigated for their dipeptidyl peptidase-IV (DPP-IV) inhibitory properties. However, deficit of studies on fermentation as a mean to produce DPP-IV inhibitory peptides prompted us to draw a comparative study on DPP-IV inhibitory peptides generated from cow, camel, goat, and sheep milk using probiotic fermentation. Further, peptide identification, in silico molecular interactions with DPP-IV, and ensemble docking were performed. Results obtained suggested that goat milk consistently exhibited higher hydrolysis than other milk types. Further, Pediococcus pentosaceus (PP-957) emerged as a potent probiotic, with significantly lower DPP-IV-IC50 values 0.17, 0.12, and 0.25 µg/mL protein equivalent in fermented cow, camel, and goat milk, respectively. Overall, peptides (RPPPPVAM, CHNLDELKDTR, and VLSLSQPK) exhibited strong binding affinity with binding energies of -9.31, -9.18 and -8.9 Kcal·mol-1, respectively, suggesting their potential role as DPP-IV inhibitors. Overall, this study, offers valuable information toward antidiabetic benefits of fermented milk products via inhibition of DPP-IV.

The Discovery and Characterization of a Potent DPP-IV Inhibitory Peptide from Oysters for the Treatment of Type 2 Diabetes Based on Computational and Experimental Studies.

The inhibition of dipeptidyl peptidase-IV (DPP-IV) is a promising approach for regulating the blood glucose levels in patients with type 2 diabetes (T2D). Oysters, rich in functional peptides, contain peptides capable of inhibiting DPP-IV activity. This study aims to identify the hypoglycemic peptides from oysters and investigate their potential anti-T2D targets and mechanisms. This research utilized virtual screening for the peptide selection, followed by in vitro DPP-IV activity assays to validate the chosen peptide. Network pharmacology was employed to identify the potential targets, GO terms, and KEGG pathways. Molecular docking and molecular dynamics simulations were used to provide virtual confirmation. The virtual screening identified LRGFGNPPT as the most promising peptide among the screened oyster peptides. The in vitro studies confirmed its inhibitory effect on DPP-IV activity. Network pharmacology revealed that LRGFGNPPT exerts an anti-T2D effect through multiple targets and signaling pathways. The key hub targets are AKT1, ACE, and REN. Additionally, the molecular docking results showed that LRGFGNPPT exhibited a strong binding affinity with targets like AKT1, ACE, and REN, which was further confirmed by the molecular dynamics simulations showcasing a stable peptide-target interaction. This study highlights the potential of LRGFGNPPT as a natural anti-T2D peptide, providing valuable insights for potential future pharmaceutical or dietary interventions in T2D management.

Emerging production techniques and potential health promoting properties of plant and animal protein-derived bioactive peptides.

Bioactive peptides (BPs) are short amino acid sequences that that are known to exhibit physiological characteristics such as antioxidant, antimicrobial, antihypertensive and antidiabetic properties, suggesting that they could be exploited as functional foods in the nutraceutical industry. These BPs can be derived from a variety of food sources, including milk, meat, marine, and plant proteins. In the past decade, various methods including in silico, in vitro, and in vivo techniques have been explored to unravel underlying mechanisms of BPs. To forecast interactions between peptides and their targets, in silico methods such as BIOPEP, molecular docking and Quantitative Structure-Activity Relationship modeling have been employed. Additionally, in vitro research has examined how BPs affect enzyme activities, protein expressions, and cell cultures. In vivo studies on the contrary have appraised the impact of BPs on animal models and human subjects. Hence, in the light of recent literature, this review examines the multifaceted aspects of BPs production from milk, meat, marine, and plant proteins and their potential bioactivities. We envisage that the various concepts discussed will contribute to a better understanding of the food derived BP production, which could pave a way for their potential applications in the nutraceutical industry.

Preparation, characterization, and mechanism of DPP-IV inhibitory peptides derived from Bactrian camel milk.

In this study, double enzyme hydrolysis significantly enhanced the DPP-IV inhibition rate compared to single enzyme. The α + K enzymes exhibited the highest inhibition rate. Ultrasonic pretreatment for 30 min improved the hydrolysis efficiency and DPP-IV inhibition rate, potentially due to the structural changes in hydrolysates, such as the increased surface hydrophobicity, and reduced particle size, α-helix and ß-turn. Six peptides were screened and verified in vitro. QPY, WPEYL, and YPPQVM displayed competitive inhibition, while LPAAP and IPAPSFPRL displayed mixed competitive/non-competitive inhibition. The interactions between these six peptides and DPP-IV primarily occurred through hydrogen bonds, electrostatic and hydrophobic interactions. Network pharmacological analysis indicated that LPAAP might inhibit DPP-IV activity trough interactions with diabetes-related targets such as CASP3, HSP90AA1, MMP9, and MMP9. These results uncover the potential mechanism of regulating blood glucose by camel milk hydrolysates, establishing camel milk peptide as a source of DPP-IV inhibitory peptide.

Peptide array screening with anti-GLP-1 monoclonal antibody: Discovery of cysteine-containing DPP-IV inhibitory peptides.

Inhibition of dipeptidyl peptidase IV (DPP-IV) is an effective pharmacotherapy for the management of type 2 diabetes. Recent findings have suggested that various dietary proteins can serve as precursors to peptides that inhibit DPP-IV. Although several DPP-IV inhibitory peptides derived from food materials have been reported, more effective inhibitory peptides remain to be discovered. This study aimed to identify potent DPP-IV inhibitory peptides that earlier approaches had overlooked by employing a screening method that combined peptide arrays and neutralizing antibodies. Octa-peptides covering the complete amino acid sequences of four casein proteins and two whey proteins were synthesized on arrays via a solid-phase method. These peptides were then reacted with a monoclonal antibody specifically engineered to recognize glucagon-like peptide 1 (GLP-1), a substrate of DPP-IV. The variable region of the anti-GLP-1 monoclonal antibody is utilized to mimic the substrate-binding region of DPP-IV, enabling the antibody to bind to peptides that interact with DPP-IV. Based on this feature, 26 peptides were selected as DPP-IV inhibitory peptide candidates, 11 of which showed strong DPP-IV inhibitory activity. Five of these peptides consistently contained cysteines positioned two to four residues from the N-terminus. Treatment with disulfide formation decreased the DPP-IV inhibitory activity of these cysteine-containing peptides, while the inhibitory activity of α-lactalbumin hydrolysates increased with reducing treatment. These results revealed that the thiol group is important for DPP-IV inhibitory activity. This study provides a useful screen for DPP-IV inhibitory peptides and indicates the importance of reductive cysteine residues within DPP-IV inhibitory peptides.

Effect of thermal pretreatment and gastrointestinal digestion on the bioactivity of dry-cured ham bone enzymatic hydrolyzates.

This study reports the effect of thermal pretreatment and the use of different commercial proteolytic enzymes (Protamex, Flavourzyme, Protana prime, and Alcalase) on the free amino acid content (FAA), peptide profile, and antioxidant, antidiabetic, antihypertensive, and anti-inflammatory potential (DPPH, FRAP, and ABTS assay, DPP-IV, ACE-I, and NEP inhibitory activities) of dry-cured ham bone hydrolyzates. The effect of in vitro digestion was also determined. Thermal pretreatment significantly increased the degree of hydrolysis, the FAA, and the DPP-IV and ACE-I inhibitory activities. The type of peptidase used was the most significant factor influencing antioxidant activity and neprilysin inhibitory activity. Protana prime hydrolyzates failed to inhibit DPP-IV and neprilysin enzymes and had low values of ACE-I inhibitory activity. After in vitro digestion, bioactivities kept constant in most cases or even increased in ACE-I inhibitory activity. Therefore, hydrolyzates from dry-cured ham bones could serve as a potential source of functional food ingredients for health benefits.

Identification of DPP-IV inhibitory peptides derived from buffalo colostrum: Mining through bioinformatics, in silico and in vitro approaches.

Bioactive peptides derived from foods provide physiological health benefits beyond nutrition. This study focused on profiling small peptide inhibitors against two key serine proteases, dipeptidyl peptidase-IV (DPP-IV) and prolyl oligopeptidase (POP). DPP-IV is a well-known protein involved in diverse pathways regulating inflammation, renal, cardiovascular physiology, and glucose homeostasis. POP is yet another key target protein for neurodegenerative disorders. The study evaluated peptide libraries of buffalo colostrum whey and fat globule membrane proteins derived from pepsin and pepsin-pancreatin digestion through in silico web tools and structure-based analysis by molecular docking and binding free-energy estimation, followed by in vitro assay for DPP-IV inhibition for the lead peptides. The bioinformatic study indicated 49 peptides presented motifs with DPP-IV inhibition while 5 peptides with sequences for POP inhibition. In the molecular docking interactions study, 22 peptides interacted with active site residues of DPP-IV and 3 peptides with that of POP. The synthesized peptides, SFVSEVPEL and LTFQHNF inhibited DPP-IV in vitro with an IC50 of 193.5 µM and 1.782 mM, respectively. The study revealed the key residues for inhibition of DPP-IV and POP thus affirming the DPP-IV inhibitory potential of milk-derived peptides.

Molecular Mechanistic Insights into Dipeptidyl Peptidase-IV Inhibitory Peptides to Decipher the Structural Basis of Activity.

Dipeptidyl peptidase-IV (DPP-IV) inhibiting peptides have attracted increased attention because of their possible beneficial effects on glycemic homeostasis. However, the structural basis underpinning their activities has not been well understood. This study combined computational and in vitro investigations to explore the structural basis of DPP-IV inhibitory peptides. We first superimposed the Xaa-Pro-type peptide-like structures from several crystal structures of DPP-IV ligand-protein complexes to analyze the recognition interactions of DPP-IV to peptides. Thereafter, a small set of Xaa-Pro-type peptides was designed to explore the effect of key interactions on inhibitory activity. The intramolecular interaction of Xaa-Pro-type peptides at the first and third positions from the N-terminus was pivotal to their inhibitory activities. Residue interactions between DPP-IV and residues of the peptides at the fourth and fifth positions of the N-terminus contributed significantly to the inhibitory effect of Xaa-Pro-type tetrapeptides and pentapeptides. Based on the interaction descriptors, quantitative structure-activity relationship (QSAR) studies with the DPP-IV inhibitory peptides resulted in valid models with high R2 values (0.90 for tripeptides; 0.91 for tetrapeptides and pentapeptides) and Q2 values (0.33 for tripeptides; 0.68 for tetrapeptides and pentapeptides). Taken together, the structural information on DPP-IV and peptides in this study facilitated the development of novel DPP-IV inhibitory peptides.

Tracking bioactive peptides and their origin proteins during the in vitro digestion of meat and meat products.

Existing reviews address bioactive peptides of meat proteins; however, comprehensive reviews summarizing the released sequences and their corresponding parent meat proteins in the digesta are limited. This review explores the bioactive peptides released during the in vitro gastrointestinal (GI) digestion of meat, connecting with parent proteins. The primary bioactivities of meat-derived peptides include angiotensin-converting enzyme (ACE) and dipeptidyl peptidase (DPP)-IV inhibition and antioxidant effects. Myofibrillar, sarcoplasmic, and stromal proteins play a significant role in peptide release during digestion. The release of bioactive peptides varies according to the parent protein and cryptides had short chains, non-toxicity, and great bioavailability and GI absorption scores. Moreover, the structural stability and bioactivities of peptides can be influenced by the digestive properties and amino acid composition of parent proteins. Investigating the properties and origins of bioactive peptides provides insights for enhancing the nutritional quality of meat and understanding its potential health benefits.

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).

Protein Hydrolysis as a Way to Valorise Squid-Processing Byproducts: Obtaining and Identification of ACE, DPP-IV and PEP Inhibitory Peptides.

The industrial processing of Argentine shortfin squid to obtain rings generates a significant amount of protein-rich waste, including the skin, which is rich in collagen and attached myofibrillar proteins. This waste is generally discarded. In this study, skin was used as a source of proteins that were hydrolysed using Trypsin, Esperase® or Alcalase®, which released peptides with antioxidant potential and, in particular, antihypertensive (ACE inhibition), hypoglycemic (DPP-IV inhibition) and/or nootropic (PEP inhibition) potential. Among the three enzymes tested, Esperase® and Alcalase produced hydrolysates with potent ACE-, DPP-IV- and PEP-inhibiting properties. These hydrolysates underwent chromatography fractionation, and the composition of the most bioactive fractions was analysed using HPLC-MS-MS. The fractions with the highest bioactivity exhibited very low IC50 values (16 and 66 µg/mL for ACE inhibition, 97 µg/mL for DPP-IV inhibition and 55 µg/mL for PEP inhibition) and were mainly derived from the hydrolysate obtained using Esperase®. The presence of Leu at the C-terminal appeared to be crucial for the ACE inhibitory activity of these fractions. The DPP-IV inhibitory activity of peptides seemed to be determined by the presence of Pro or Ala in the second position from the N-terminus, and Gly and/or Pro in the last C-terminal positions. Similarly, the presence of Pro in the peptides present in the best PEP inhibitory fraction seemed to be important in the inhibitory effect. These results demonstrate that the skin of the Argentine shortfin squid is a valuable source of bioactive peptides, suitable for incorporation into human nutrition as nutraceuticals and food supplements.

Antidiabetic effectiveness of Phyllanthus niruri bioactive compounds via targeting DPP-IV.

Phyllanthus niruri Linn. (Euphorbiaceae) is a small herb and is categorised as one of the rich medicinal plants throughout the world. This study aimed to evaluate the P. niruri L. whole plant extract (PNE) for secondary metabolite assay (total phenolic and terpenoid content) followed by the potential antioxidant activity (ABTS diammonium salt radical assay, DPPH· activity, superoxide anion (O2-) radicals' assay, and nitric oxide (NO) radical generation) and antidiabetic activity in vivo and in vitro in streptozotocin (STZ) induced albino mice. PNE showed good scavenging activity with a value of 286.45 ± 6.55 mg TE/g and 194.54 ± 4.64 mg TE/g in ABTS and DPPH assays respectively. In the superoxide anion assay, the PNE caused a dose-dependent inhibition at the lowest IC25 value of 0.17 ± 0.00 mg/mL compared to ascorbic acid (IC25 of 0.25 ± 0.02 mg/mL). The scavenging ability of PNE against nitric oxide showed an IC25 of 1.13 ± 0.04 mg/mL compared to ascorbic acid (IC25 4.78 ± 0.09 mg/mL). Unlike diabetic control mice, the PNE-treated diabetic mice presented significant amelioration of glycaemia and lipid dysmetabolism. Phytochemicals like Astragalin, Gallocatechin, Ellagic acid, Gallic acid, Brevifolin carboxylic acid, Phyllnirurin, and Hypophyllanthin showed significant docking score (> -4) of inhibitory potential with DPP-IV protein. Results indicated that PNE phytochemicals could be a promising antidiabetic agent by targeting DPP-IV.

Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles.

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100-1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20-70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.