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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.

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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.

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Our previous study has demonstrated that both the amino acid at N3 position and peptide length affected the DPP-IV inhibitory activity of Gly-Pro-type peptides. To further elucidate their molecular mechanism, a combined approach of QSAR modeling, enzymatic kinetics and molecular docking was used. Results showed that the QSAR models of Gly-Pro-type tripeptides and Gly-Pro-type peptides containing 3-12 residues were successfully constructed by 5z-scale descriptor with R2 of 0.830 and 0.797, respectively. The lower values of electrophilicity, polarity, and side-chain bulk of amino acid at N3 position caused higher DPP-IV inhibitory activity of Gly-Pro-type peptides. Moreover, an appropriate increase in the length of Gly-Pro-type peptides did not change their competitive inhibition mode, but decreased their inhibition constants (Ki values) and increased interactions with DPP-IV. More importantly, the interactions between the residues at C-terminal of Gly-Pro-type peptides containing 5 âˆ¼ 6 residues with S2 extensive subsites (Ser209, Phe357, Arg358) of DPP-IV increased the interactions of Gly residue at N1 position with the S2 subsites (Glu205, Glu206, Asn710, Arg125, Tyr662) and decreased the acylation level of DPP-IV-peptide complex, and thereby increasing peptides' DPP-IV inhibitory activity.

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Flixweed (sophia) seed meal and camelina, both by-products of oil processing, were employed to generate protein hydrolysates by applying Flavourzyme and Alcalase. This study aimed to integrate in vitro and in silico methods to analyze sophia and camelina protein hydrolysates for releasing potent antioxidative, dipeptidyl peptidase IV (DPP IV) inhibitors and angiotensin-converting enzyme (ACE) inhibitory peptides. In vitro methods were used to investigate the antioxidant potential of sophia/camelina protein hydrolysates. Bioinformatics techniques, including Peptideranker, BIOPEP, Toxinpred, AlgPred, and SwissADME, were employed to obtain the identification of bioactive peptides produced during the hydrolysis process. Protein hydrolysates produced from sophia and camelina seed meal exhibited higher ABTS and DPPH radical scavenging activities Ithan their protein isolates. Among the produced protein hydrolysates, Alcalase-treated samples showed the highest oxygen radical absorbance capacity and hydroxyl radical scavenging activity. In addition, sophia/camelina hydrolysates prevented hydroxyl and peroxyl radical-induced DNA scission and LDL cholesterol oxidation. In silico proteolysis was conducted on Alcalase-treated samples, and resultant peptides showed potential DPP IV and ACE-inhibitory activities. Identified peptides were further assessed for their toxicity and medicinal properties. Results indicate that all digestive-resistant peptides were non-toxic and had desirable drug-like properties. The findings of this study suggest that sophia/camelina protein hydrolysates are promising candidates for functional foods, nutraceuticals, and natural therapeutics.

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The ability of peptides from an aqueous and salt-soluble protein extract of dry-cured pork loins to inhibit the action of dipeptidyl peptidase IV was determined. This activity was assessed at different times of the production process, i.e., 28, 90, 180, 270 and 360 days. The resistance of the biological property during the simulated digestive process was also assessed. For this, the extracts were hydrolyzed with pepsin and pancreatin as a simulated digestion step of the gastrointestinal tract and fractionated (>7 kDa) as an intestinal absorption step. The results indicate that dried-pork-loin peptides may have potential as functional food ingredients in the prevention and treatment of type 2 diabetes mellitus. In particular, the APPPPAEV, APPPPAEVH, KLPPLPL, RLPLLP, VATPPPPPPK, VPIPVPLPM and VPLPVPVPI sequences show promise as natural food compounds helpful in maintaining good health.

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Swertiamarin is a lead, biologically active compound obtained from Enicostemma littorale Blume and known to be identified for the anti-diabetic activity. Present work comprises the synthesis and structural optimization of seven novel swertiamarin analogues and those were not being reported elsewhere till date. Swertiamarin was isolated, followed by modifications that have been accomplished amidst fluorinating, acetylating and oxidizing agents and also performed chromatographic purity and characterization of analogues. Furthermore, the swertiamarin analogues were screened for dipeptidyl peptidase IV (DPP-IV) enzyme inhibition with in silico studies. Besides, the pharmacokinetics and toxicity of analogues were predicted using ADMET software. In a nutshell, the compounds such as SNIPERSV-4 and SNIPERSV-7 have to pose good initial activity (∼48%) in comparison to standard DPP-IV inhibitor (Sitagliptin). The identified analogues were active against DPP-IV enzyme in preliminary screenings, and these findings would be beneficial for the new age researchers also for the therapy of diabetes.

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Inhibition of dipeptidyl peptidase-IV (DPP-IV) has been identified as a promising approach for the treatment of type 2 diabetes mellitus (T2DM). Therefore, development of DPP-IV inhibitors with new chemical scaffold is of utmost importance to medicinal chemistry. In the present study, we identified benzophenone thio- and semicarbazone scaffolds as novel DPP-IV inhibitors. For that purpose, benzophenone thio- and semicarbazone were synthesized through a 2-step reaction. These newly synthetic derivatives were characterized by different spectroscopic techniques, including HREI-MS and NMR. whereas stereochemistry of the iminic bond was predicted by NOESY experiments. Thio- and semicarbazones derivatives were evaluated for their DPP-IV inhibitory potential and found to exhibit a good to moderate enzyme inhibitory activity. Most active and non-cytotoxic derivatives were further evaluated for their DPP-IV inhibitory potential in in cellulo model. The binding sites as well as affinity of active compounds for DPP- IV enzyme were predicted by in silico studies, and compared to a standard drug, sitagliptin. Pharmacophore studies of thio- and semicarbazones derivatives 1-29 suggest that substitution of aryl group, particularly a lipophilic substituents at C-4″ of benzene ring, and a hydroxyl at C-4' strongly influenced the DPP-IV inhibitory activity. Compound 9 showed the highest inhibitory activity (IC50 = 15.0 ± 0.6 µM), whereas compounds 10, 17, 12, 14 and 23 showed a moderate activity with IC50 values in the range of 28.9-39.2 µM. This study identifies thio- and semicarbazones as new classes of DPP-IV inhibitors which may translate into safe and effective therapeutics for a better management of type 2 diabetes.Communicated by Ramaswamy H. Sarma.

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INTRODUCTION: Radical resection is the only curative treatment for pancreatic cancer, which is a life-threatening disease. However, it is often not easy to accurately identify the extent of the tumor before and during surgery. Here we describe the development of a novel method to detect pancreatic tumors using a tumor-specific enzyme-activatable fluorescence probe. METHODS: Tumor and non-tumor lysate or small specimen collected from the resected specimen were selected to serve as the most appropriate fluorescence probe to distinguish cancer tissues from noncancerous tissues. The selected probe was sprayed onto the cut surface of the resected specimen of cancer tissue to acquire a fluorescence image. Next, we evaluated the ability of the probe to detect the tumor and calculated the tumor-to-background ratio (TBR) by comparing the fluorescence image with the pathological extent of the tumor. Finally, we searched for a tumor-specific enzyme that optimally activates the selected probe. RESULTS: Using a library comprising 309 unique fluorescence probes, we selected GP-HMRG as the most appropriate activatable fluorescence probe. We obtained eight fluorescence images of resected specimens, among which four approximated the pathological findings of the tumor, which achieved the highest TBR. Finally, dipeptidyl-peptidase IV (DPP-IV) or a DPP-IV-like enzyme was identified as the target enzyme. CONCLUSION: This novel method may enable rapid and real-time visualization of pancreatic cancer through the enzymatic activities of cancer tissues.

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Camel milk proteins are an important substrate for bioactive peptides generation. This study investigates in-vitro antidiabetic effect (via inhibition of α-amylase (AA), α-glucosidase (AG) and dipeptidyl peptidase IV (DPP-IV)) of bovine (BC) and camel casein (CC) hydrolysates. Further, effect of simulated gastrointestinal digestion (SGID) on inhibitory potential of generated hydrolysates was also explored. Both BC and CC hydrolysates displayed potent inhibitory properties against AA (IC50 value- 0.58 & 0.59 mg/mL), AG (IC50 value- 1.04 & 0.59 mg/mL) and DPP-IV (IC50 value- 0.62 & 0.66 mg/mL), respectively. Among different peptides identified in BC and CC hydrolysates, it was observed that FLWPEYGAL was predicted to be most potent inhibitory peptide against AA. While LPTGWLM, MFE and GPAHCLL as most active inhibitor of AG and HLPGRG, QNVLPLH and PLMLP were predicted to be active against DPP-IV. Overall, BC and CC hydrolysates can be proposed to be used in different food formulations as functional antidiabetic agents.

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Food derived bioactive peptides can be generated from various protein sources and usually consist of between 2-30 amino acids with bulky, side-chain aromatic amino acids preferred in the ultimate and penultimate positions at the C-terminal end of the amino acid chain. They are reported to impart a myriad of preventative health beneficial effects to the consumer once ingested and these include heart health benefits through inhibition of enzymes including renin (EC 3.4.23.15) and angiotensin- I-converting enzyme (ACE-1; EC 3.4.15.1) within the renin angiotensin aldosterone system (RAAS) anti-inflammatory (due to inhibition of ACE-I and other enzymes) and anti-cancer benefits, prevention of type-2 diabetes through inhibition of dipeptidyl peptidase IV (DPP-IV), bone and dental strength, antimicrobial and immunomodulatory effects and several others. Peptides have also reported health benefits in the treatment of asthma, neuropathic pain, HIV and wound healing. However, the structure, amino acid composition and length of these peptides, along with the quantity of peptide that can pass through the gastrointestinal tract and often the blood-brain barrier (BBB), intact and reach the target organ, are important for the realisation of these health effects in an in vivo setting. This paper aims to collate recent important research concerning the generation and detection of peptides in the laboratory. It discusses products currently available as preventative healthcare peptide options and relevant legislation barriers to place a food peptide product on the market. The review also highlights useful in silico computer- based methods and analysis that may be used to generate specific peptide sequences from proteins whose amino acid sequences are known and also to determine if the peptides generated are unique and bioactive. The topic of food-derived bioactive peptides for health is of great interest to scientific research and industry due to evolving drivers in food product innovation, including health and wellness for the elderly, infant nutrition and optimum nutrition for sports athletes and the humanisation of pets. This paper provides an overview of what is required to generate bioactive peptide containing hydrolysates, what methods should be used in order to characterise the beneficial health effects of these hydrolysates and the active peptide sequences, potential applications of bioactive peptides and legislative requirements in Europe and the United States. It also highlights success stories and barriers to the development of peptide-containing food products that currently exist.

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Dipeptidyl peptidase-IV (DPP-IV) is an enzyme that break down the antidiabetic hormone glucagon-like peptide-1. Therefore, inhibition of DPP-IV could be an effective strategy to treat Type 2 diabetes (T2D). The α-lactalbumin-rich whey protein concentrate was hydrolyzed by trypsin, and the hydrolysates were then fractionated at a semi-preparative scale using a Superdex Gel filtration Chromatography. The peptides were analyzed by using HPLC coupled with tandem mass spectrometry (RP-HPLC-MS/MS), and their Dipeptidyl peptidase-IV inhibitory activity was determined by the enzymatic assay. Among tested fragments, a potent fragment (LDQWLCEKL), with the half-maximal inhibitory concentration (IC50) of 131 µM was obtained. Further analysis shows that the LDQWLCEKL peptide corresponds to the amino acid sequence of f(115-123) in α-lactalbumin. Furthermore, LDQWLCEKL exhibited a typical non-competitive mode of inhibition. The results indicate that α-lactalbumin contains active peptides with DPP-IV inhibitory activity that may be used to prevent and treat T2D.

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BACKGROUND: Lately, diabetes has become the main health concern for millions of people around the world. Dipeptidyl peptidase-IV (DPP-IV) inhibitors have emerged as a new class of oral antidiabetic agents. Formerly, acridines, N4-sulfonamido-succinamic, phthalamic, acrylic and benzoyl acetic acid derivatives, and sulfamoyl-phenyl acid esters were designed and developed as new DPP-IV inhibitors. OBJECTIVE: This study aims to develop a pharmacophore model of DPP-IV inhibitors and to evaluate phenanthridines as a novel scaffold for inhibiting DPP-IV enzyme. In addition, to assess their binding interactions with the enzyme through docking in the binding site of 4A5S (PDB). METHODS: Herein, Quantum-Polarized Ligand Docking (QPLD) and ligand-based pharmacophore modeling investigations were performed. Three novel 3,8-disubstituted-6-phenyl phenanthridine derivatives 3-5 have been designed, synthesized and characterized. In vitro biological testing against DPP-IV was carried out using fluorometric assay kit. RESULTS: QPLD study demonstrates that compounds 3-5 forms H-bond with Lys554, Trp629, and Tyr631, besides charge transfer interaction between their aromatic rings and the aromatic rings of Tyr547 and Tyr666. Moreover, they fit the three pharmacophoric point features of DPP-IV inhibitors and were proven to have in vitro DPP-IV inhibitory activity where compound 5 displayed a % inhibition of 45.4 at 100 µM concentration. CONCLUSION: Phenanthridines may serve as a potential lead compound for developing new DPP-IV inhibitors as a promising antidiabetic agent. Computational results suggest future structural simplification.

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The aim of this study was to investigate the dipeptidyl peptidase-IV (DPP-IV) inhibition and metabolic stability of a casein-derived peptide Val-Pro-Tyr-Pro-Gln (VPYPQ) and its fragments as well as their release from casein following hydrolysis. Results showed that VPYPQ was the most potent DPP-IV inhibitory peptide among them with an IC50 value of 41.45 µM. This might be due to its two internal Pro residues at positions 2 and 4. Moreover, VPYPQ was resistant to hydrolysis by gastrointestinal enzymes and was relatively more stable to hydrolysis by DPP-IV and peptidases in plasma compared with its fragments. Additionally, oral administration of VPYPQ at a dose of 90 µmol/kg body weight could reduce the postprandial blood glucose levels in mice. More importantly, VPYPQ could be released efficiently from casein following hydrolysis by a combination of papain and in vitro digestion, reaching up to 3211.15 µg/g. Therefore, VPYPQ was a promising casein-derived DPP-IV inhibitor.

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Major proteins contained in dried giant grouper roe (GR) such as vitellogenin (from Epinephelus coioides; NCBI accession number: AAW29031.1), apolipoprotein A-1 precursor (from Epinephelus coioides; NCBI accession number: ACI01807.1) and apolipoprotein E (from Epinephelus bruneus; NCBI accession number: AEB31283.1) were characterized through compiled proteomics techniques (SDS-PAGE, in-gel digestion, mass spectrometry and on-line Mascot database analysis). These proteins were subjected to in silico analysis using BLAST and BIOPEP-UWM database. Sequence similarity search by BLAST revealed that the aligned vitellogenin sequences from Epinephelus coioides and Epinephelus lanceolatus share 70% identity, which indicates that the sequence sample has significant similarity with proteins in sequence databases. Moreover, prediction of potential bioactivities through BIOPEP-UWM database resulted in high numbers of peptides predominantly with dipeptidyl peptidase-IV (DPP-IV) and angiotensin-I-converting enzyme (ACE-I) inhibitory activities. Pepsin (pH > 2) was predicted to be the most promising enzyme for the production of bioactive peptides from GR protein, which theoretically released 82 DPP-IV inhibitory peptides and 47 ACE-I inhibitory peptides. Overall, this work highlighted the potentiality of giant grouper roe as raw material for the generation of pharmaceutical products. Furthermore, the application of proteomics and in silico techniques provided rapid identification of proteins and useful prediction of its potential bioactivities.

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Dipeptidyl peptidase IV (DPPIV) inhibition may be a promising therapeutic strategy for acute stroke treatment, given its potential to prolong the biological half-life of neuroprotective substrates. A related protease, fibroblast activation protein (FAP), was recently shown to inactivate the same substrates. Therefore, it should also be investigated as a potential target in stroke. The study aimed to investigate whether stroke severity and outcome correlate with DPPIV and FAP activities and their kinetics shortly after acute ischemic stroke. DPPIV and FAP activities were analyzed in the serum of 50 hyperacute stroke patients at admission, 1 day, 3 days, and 7 days after stroke onset and in 50 age-matched healthy controls. This was done as part of the Middelheim's Interdisciplinary Stroke Study. DPPIV activity tended to increase shortly after stroke compared to the control population. DPPIV and FAP activities steadily decreased in the first week after stroke onset. Higher infarct volumes (≥5 ml) and a more severe stroke (NIHSS >7) at admission were correlated with a stronger decrease in the activities of both enzymes. Moreover, these patients more often developed a progressive stroke, were more often institutionalized. Patients with a stronger increase in DPPIV activity at admission and decrease in the activity of both DPPIV and FAP during the first week after stroke onset had a more severe stroke and worse short-term outcomes.

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RRDY, RL, and DPF were the top 3 of 21 peptides for inhibitions against dipeptidyl peptidase-IV (DPP-IV) from the pepsin hydrolysis of yam dioscorin in silico and were further investigated in a proof-of-concept study in normal ICR mice for regulating glucose metabolism by the oral glucose tolerance test (OGTT). The sample or sitagliptin (positive control) was orally administered by a feeding gauge; 30 min later, the glucose loads (2.5 g/kg) were performed. RRDY, yam dioscorin, or sitagliptin preload, but not DPF, lowered the area under the curve (AUC0-120) of blood glucose and DPP-IV activity and elevated the AUC0-120 of blood insulin, which showed significant differences compared to control (P < 0.05 or 0.001). These results suggested that RRDY and yam dioscorin might be beneficial in glycemic control in normal mice and need further investigations in diabetic animal models.

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Fibroblast growth factor 21 (FGF21) is an endocrine hormone that regulates carbohydrate and lipid metabolism. In humans, circulating FGF21 is inactivated by proteolytic cleavage of its C-terminus, thereby preventing signalling through a receptor complex. The mechanism for this cleavage event and the factors contributing to the post-translational regulation of FGF21 activity has previously been unknown. In a recent issue of the Biochemical Journal, Zhen et al. have identified fibroblast activation protein (FAP) as the endopeptidase responsible for this site-specific cleavage of human FGF21 (hFGF21), and propose that inhibition of FAP may be a therapeutic strategy to increase endogenous levels of active FGF21.

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Ethanol extracts (Et) from the stem (S) and leaf (L) of Vitis thunbergii var. taiwaniana (VTT) were used to investigate yeast α-glucosidase and porcine kidney dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. Both VTT-Et showed complete α-glucosidase inhibition at 0.1 mg/mL; VTT-S-Et and VTT-L-Et showed 26 and 11% DPP-IV inhibition, respectively, at 0.5 mg/mL. The VTT-Et interventions (20 and 50 mg/kg) resulted in improvements in impaired glucose tolerance of diet-induced obese rats. (+)-Hopeaphenol, (+)-vitisin A, and (-)-vitisin B were isolated from the ethyl acetate fractions of S-Et and showed yeast α-glucosidase inhibition (IC50 = 18.30, 1.22, and 1.02 µM) and porcine kidney DPP-IV inhibition (IC50 = 401, 90.75, and 15.3 µM) compared to acarbose (6.39 mM) and sitagliptin (47.35 nM), respectively. Both (+)-vitisin A and (-)-vitisin B showed mixed noncompetitive inhibition against yeast α-glucosidase and porcine kidney DPP-IV, respectively. These results proposed that VTT extracts might through inhibitions against α-glucosidase and DPP-IV improve the impaired glucose tolerance in diet-induced obese rats.

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BACKGROUND: The metabolic syndrome (MS) is termed a cluster of multiple metabolic risk criteria which is positively correlated with cardiovascular disease and type 2 diabetes mellitus (DM). Yam dioscorins have been reported to exhibit biological activities, however, little is known their preventive effects on the MS. Therefore, a high-fat (HF) diet was used to induce Wistar rat obesity and then yam dioscorin (50 mg/kg, dio50) was intervened daily concurrent HF diet (HF diet + dio50) for five weeks to check the changes of weights of body and tissues, blood pressures, and impaired glucose tolerances. The in vitro peptic hydrolysates of dioscorin with molecular mass between 3 kDa and 10 kDa and less than 3 kDa were used to determine dipeptidyl peptidase IV (DPP IV) inhibitory activities which DPP IV inhibitor has been reported to prevent and treat type 2 DM. RESULTS: There were no significant difference in body weights, feed intakes, feed conversion, and weights of adipose tissues of obese rats in groups of HF and (HF diet + dio50). However, the systolic blood pressures in obese rats of 2-, 3- and 4-week dioscorin interventions were showed significantly lower (P < 0.05) compared to the HF group. The dioscorin intervention (HF+ dio50) was showed significantly different (P < 0.05) and improved the impaired glucose tolerances compared to HF group in obese rats by the oral glucose tolerance tests. It was also found that the fraction with different molecular mass of dioscorin peptic hydrolysates (5 mg/ml) showed inhibitory activities against DPP IV using sitagliptin phosphate as positive controls. CONCLUSIONS: Yam dioscorins exhibit improved MS activities in obese rats which the related mechanisms may need further investigations.