Synthesis and In Silico Analysis of New Polyheterocyclic Molecules Derived from [1,4]-Benzoxazin-3-one and Their Inhibitory Effect against Pancreatic α-Amylase and Intestinal α-Glucosidase.

This study focuses on synthesizing a new series of isoxazolinyl-1,2,3-triazolyl-[1,4]-benzoxazin-3-one derivatives 5a-5o. The synthesis method involves a double 1,3-dipolar cycloaddition reaction following a "click chemistry" approach, starting from the respective [1,4]-benzoxazin-3-ones. Additionally, the study aims to evaluate the antidiabetic potential of these newly synthesized compounds through in silico methods. This synthesis approach allows for the combination of three heterocyclic components: [1,4]-benzoxazin-3-one, 1,2,3-triazole, and isoxazoline, known for their diverse biological activities. The synthesis procedure involved a two-step process. Firstly, a 1,3-dipolar cycloaddition reaction was performed involving the propargylic moiety linked to the [1,4]-benzoxazin-3-one and the allylic azide. Secondly, a second cycloaddition reaction was conducted using the product from the first step, containing the allylic part and an oxime. The synthesized compounds were thoroughly characterized using spectroscopic methods, including 1H NMR, 13C NMR, DEPT-135, and IR. This molecular docking method revealed a promising antidiabetic potential of the synthesized compounds, particularly against two key diabetes-related enzymes: pancreatic α-amylase, with the two synthetic molecules 5a and 5o showing the highest affinity values of 9.2 and 9.1 kcal/mol, respectively, and intestinal α-glucosidase, with the two synthetic molecules 5n and 5e showing the highest affinity values of -9.9 and -9.6 kcal/mol, respectively. Indeed, the synthesized compounds have shown significant potential as antidiabetic agents, as indicated by molecular docking studies against the enzymes α-amylase and α-glucosidase. Additionally, ADME analyses have revealed that all the synthetic compounds examined in our study demonstrate high intestinal absorption, meet Lipinski's criteria, and fall within the required range for oral bioavailability, indicating their potential suitability for oral drug development.

Structure-function relationships in (poly)phenol-enzyme binding: Direct inhibition of human salivary and pancreatic α-amylases.

(Poly)phenols inhibit α-amylase by directly binding to the enzyme and/or by forming starch-polyphenol complexes. Conventional methods using starch as the substrate measure inhibition from both mechanisms, whereas the use of shorter oligosaccharides as substrates exclusively measures the direct interaction of (poly)phenols with the enzyme. In this study, using a chromatography-based method and a short oligosaccharide as the substrate, we investigated the detailed structural prerequisites for the direct inhibition of human salivary and pancreatic α-amylases by over 50 (poly)phenols from the (poly)phenol groups: flavonols, flavones, flavanones, flavan-3-ols, polymethoxyflavones, isoflavones, anthocyanidins and phenolic acids. Despite being structurally very similar (97% sequence homology), human salivary and pancreatic α-amylases were inhibited to different extents by the tested (poly)phenols. The most potent human salivary α-amylase inhibitors were luteolin and pelargonidin, while the methoxylated anthocyanidins, peonidin and petunidin, significantly blocked pancreatic enzyme activity. B-ring methoxylation of anthocyanidins increased inhibition against both human α-amylases while hydroxyl groups at C3 and B3' acted antagonistically in human salivary inhibition. C4 carbonyl reduction, or the positive charge on the flavonoid structure, was the key structural feature for human pancreatic inhibition. B-ring glycosylation did not affect salivary enzyme inhibition, but increased pancreatic enzyme inhibition when compared to its corresponding aglycone. Overall, our findings indicate that the efficacy of interaction with human α-amylase is mainly influenced by the type and placement of functional groups rather than the number of hydroxyl groups and molecular weight.

Comparative analysis of pancreatic amylase activity in laboratory rodents.

Alpha-amylase is the main enzyme for starch digestion in the mammalian gastrointestinal tract. There are species differences in the enzymatic activity of pancreatic amylase that are related to the digestive strategy and natural diet of a species. This aspect is well investigated in pet and farm animals, while in common laboratory animal rodents, information is scarce. In the context of the 3R concept, detailed knowledge of the digestive physiology should be the basis of adequate nutrition, experimental planning and data interpretation. The present study aimed to obtain reference data on amylase activity in pancreatic tissue and duodenal digesta in laboratory mice, rats and hamsters. In addition, digesta was stained with Lugol's iodine to visualize starch in the process of degradation throughout the gastrointestinal tract. Amylase activity in pancreatic tissue and duodenal digesta was significantly lower in hamsters than rats and mice. The Lugol staining showed intense starch degradation in the hamsters' forestomachs, presumably by microbial fermentation. A possible explanation is that the prae-duodenal microbial starch fermentation enhances digestibility and reduces the need for pancreatic amylase in hamsters. Rats and mice may rely more on pancreatic amylase for prae-caecal starch digestion, while the microbial fermentation is mainly located in the caecum. The results clearly show species differences in the digestive capacity for starch in mice, rats and hamsters that need to be considered in the feeding of these species in the laboratory setting as well as in the use of rodents as translational animal models.

The genome of Anoplarchus purpurescens (Stichaeidae) reflects its carnivorous diet.

Digestion is driven by digestive enzymes and digestive enzyme gene copy number can provide insights on the genomic underpinnings of dietary specialization. The "Adaptive Modulation Hypothesis" (AMH) proposes that digestive enzyme activity, which increases with increased gene copy number, should correlate with substrate quantity in the diet. To test the AMH and reveal some of the genetics of herbivory vs carnivory, we sequenced, assembled, and annotated the genome of Anoplarchus purpurescens, a carnivorous prickleback fish in the family Stichaeidae, and compared the gene copy number for key digestive enzymes to that of Cebidichthys violaceus, a herbivorous fish from the same family. A highly contiguous genome assembly of high quality (N50 = 10.6 Mb) was produced for A. purpurescens, using combined long-read and short-read technology, with an estimated 33,842 protein-coding genes. The digestive enzymes that we examined include pancreatic α-amylase, carboxyl ester lipase, alanyl aminopeptidase, trypsin, and chymotrypsin. Anoplarchus purpurescens had fewer copies of pancreatic α-amylase (carbohydrate digestion) than C. violaceus (1 vs. 3 copies). Moreover, A. purpurescens had one fewer copy of carboxyl ester lipase (plant lipid digestion) than C. violaceus (4 vs. 5). We observed an expansion in copy number for several protein digestion genes in A. purpurescens compared to C. violaceus, including trypsin (5 vs. 3) and total aminopeptidases (6 vs. 5). Collectively, these genomic differences coincide with measured digestive enzyme activities (phenotypes) in the two species and they support the AMH. Moreover, this genomic resource is now available to better understand fish biology and dietary specialization.

Insights on the Hypoglycemic Potential of Crocus sativus Tepal Polyphenols: An In Vitro and In Silico Study.

Post-prandial hyperglycemia typical of diabetes mellitus could be alleviated using plant-derived compounds such as polyphenols, which could influence the activities of enzymes involved in carbohydrate digestion and of intestinal glucose transporters. Here, we report on the potential anti-hyperglycemic effect of Crocus sativus tepals compared to stigmas, within the framework of valorizing these by-products of the saffron industry, since the anti-diabetic properties of saffron are well-known, but not those of its tepals. In vitro assays showed that tepal extracts (TE) had a greater inhibitory action than stigma extracts (SE) on α-amylase activity (IC50: TE = 0.60 ± 0.09 mg/mL; SE = 1.10 ± 0.08 mg/mL; acarbose = 0.051 ± 0.07) and on glucose absorption in Caco-2 differentiated cells (TE = 1.20 ± 0.02 mg/mL; SE = 2.30 ± 0.02 mg/mL; phlorizin = 0.23 ± 0.01). Virtual screening performed with principal compounds from stigma and tepals of C. sativus and human pancreatic α-amylase, glucose transporter 2 (GLUT2) and sodium glucose co-transporter-1 (SGLT1) were validated via molecular docking, e.g., for human pancreatic α-amylase, epicatechin 3-o-gallate and catechin-3-o-gallate were the best scored ligands from tepals (-9.5 kcal/mol and -9.4 kcal/mol, respectively), while sesamin and episesamin were the best scored ones from stigmas (-10.1 kcal/mol). Overall, the results point to the potential of C. sativus tepal extracts in the prevention/management of diabetes, likely due to the rich pool of phytocompounds characterized using high-resolution mass spectrometry, some of which are capable of binding and interacting with proteins involved in starch digestion and intestinal glucose transport.

Synthesis, α-glucosidase and α-amylase inhibitory activities, acute toxicity and molecular docking studies of thiazolidine-2,4-diones derivatives.

In the present study, a series of thiazolidine-2,4-diones derivatives (3a-3e) and (4a-4e) were synthesized and characterized by 1H NMR, 13C NMR and ESI-MS spectrometry. All compounds were screened for their α-glucosidase and α-amylase inhibitory activities. In vitro biological investigations revealed that most of compounds were active against α-glucosidase with IC50 values in the range of 43.85 ± 1.06 to 380.10 ± 1.02 µM, and α-amylase with IC50 in the range of 18.19 ± 0.11 to 208.10 ± 1.80 µM. Some of the tested compounds were found to be more potent inhibitors than the clinical drug Acarbose (IC50glucosidase = 97.12 ± 0.35 µM and IC50amylase = 2.97 ± 0.004 µM). The lead compounds were evaluated for their acute toxicity on Swiss mice and found to be completely non-toxic with LD > 2000 mg/kg BW. Furthermore, the Structure-activity relationship (SAR) and the binding interactions of all compounds with the active site of α-glucosidase and α-amylase were confirmed through molecular docking and stabilizing energy calculations. This study has identified the inhibitory potential a new class of synthesized thiazolidine-2,4-diones in controlling both hyperglycemia and type 2 diabetes mellitus. Furthermore, the theoretical binding mode of the target molecules was evaluated by molecular docking studies against the 3D Crystal Structure of human pancreatic α-amylase (PDB ID: 1B2Y) and α-glucosidase (PDB ID: 3W37)Communicated by Ramaswamy H. Sarma.

Slowly digestible property of highly branched α-limit dextrins produced by 4,6-α-glucanotransferase from Streptococcus thermophilus evaluated in vitro and in vivo.

Starch molecules are first degraded to slowly digestible α-limit dextrins (α-LDx) and rapidly hydrolyzable linear malto-oligosaccharides (LMOs) by salivary and pancreatic α-amylases. In this study, we designed a slowly digestible highly branched α-LDx with maximized α-1,6 linkages using 4,6-α-glucanotransferase (4,6-αGT), which creates a short length of α-1,4 side chains with increasing branching points. The results showed that a short length of external chains mainly composed of 1-8 glucosyl units was newly synthesized in different amylose contents of corn starches, and the α-1,6 linkage ratio of branched α-LDx after the chromatographical purification was significantly increased from 4.6% to 22.1%. Both in vitro and in vivo studies confirmed that enzymatically modified α-LDx had improved slowly digestible properties and extended glycemic responses. Therefore, 4,6-αGT treatment enhanced the slowly digestible properties of highly branched α-LDx and promises usefulness as a functional ingredient to attenuate postprandial glucose homeostasis.

Opuntia dillenii (Ker Gawl.) Haw., Seeds Oil Antidiabetic Potential Using In Vivo, In Vitro, In Situ, and Ex Vivo Approaches to Reveal Its Underlying Mechanism of Action.

Opuntia dillenii Ker Gawl. is one of the medicinal plants used for the prevention and treatment of diabetes mellitus (DM) in Morocco. This study aims to investigate the antihyperglycemic effect of Opuntia dillenii seed oil (ODSO), its mechanism of action, and any hypoglycemic risk and toxic effects. The antihyperglycemic effect was assessed using the OGTT test in normal and streptozotocin (STZ)-diabetic rats. The mechanisms of action were explored by studying the effect of ODSO on the intestinal absorption of d-glucose using the intestinal in situ single-pass perfusion technique. An Ussing chamber was used to explore the effects of ODSO on intestinal sodium-glucose cotransporter 1 (SGLT1). Additionally, ODSO's effect on carbohydrate degrading enzymes, pancreatic α-amylase, and intestinal α-glucosidase was evaluated in vitro and in vivo using STZ-diabetic rats. The acute toxicity test on mice was performed, along with a single-dose hypoglycemic effect test. The results showed that ODSO significantly attenuated the postprandial hyperglycemia in normal and STZ-diabetic rats. Indeed, ODSO significantly decreased the intestinal d-glucose absorption in situ. The ex vivo test (Ussing chamber) showed that the ODSO significantly blocks the SGLT1 (IC50 = 60.24 µg/mL). Moreover, ODSO indu\ced a significant inhibition of intestinal α-glucosidase (IC50 = 278 ± 0.01 µg/mL) and pancreatic α-amylase (IC50 = 0.81 ± 0.09 mg/mL) in vitro. A significant decrease of postprandial hyperglycemia was observed in sucrose/starch-loaded normal and STZ-diabetic ODSO-treated rats. On the other hand, ODSO had no risk of hypoglycemia on the basal glucose levels in normal rats. Therefore, no toxic effect was observed in ODSO-treated mice up to 7 mL/kg. The results of this study suggest that ODSO could be suitable as an antidiabetic functional food.

Thermally processed lignin reduces the apparent hydrolysis rate of pancreatic α-amylase in starchy foods.

Lignin, despite being the second most abundant constituent of plant cell walls, is thought to be chemically inert during gastrointestinal digestion and therefore attracts little attention for its role in the human diet. This study explores the heat modifications of lignin to derive species capable of slowing starch digestion in vitro. We applied various advanced biochemical (e.g. enzymic digestion, solubility) and physio-chemical (e.g. scanning electron microscopy, Fourier-Transform-Infrared Spectroscopy, 13C-NMR) analyses to characterize the structure-function of lignin induced by heat treatment. It was found that lignin thermally processed above 300 °C reduced the apparent hydrolysis rate of pancreatic α-amylase, which is ascribed mainly to the insoluble lignin with a modified particle surface morphology. Further kinetic experiments showed that lignin species derived by thermal processing slowed in vitro digestion rates of potato starch and pasta. These findings highlight the potential for utilizing thermally processed lignin in slowing digestion of starchy foods.

Diet-ruminal microbiome-host crosstalk contributes to differential effects of calf starter and alfalfa hay on rumen epithelial development and pancreatic α-amylase activity in yak calves.

Dietary supplementation of alfalfa hay or calf starter during the preweaning period was beneficial to the gastrointestinal development in dairy calves and lambs. In the present study, we designed 2 experiments using weaning with calf starter and alfalfa hay to investigate the diet-ruminal microbiome-host crosstalk in yak calves by analyzing the ruminal microbiota and rumen epithelial transcriptome. During the preweaning period, supplementation with either alfalfa hay or the starter significantly promoted animal growth and organ development in yak calves, including increases in body weight, body height, body length, chest girth, and development of liver, spleen, and thymus. These improvements could be attributed to increased dry matter intake, rumen fermentation, and development. Butyrate concentration increased in yak calves fed alfalfa hay or the starter, which could further promote ruminal epithelium development. Using 16S rRNA gene amplicon sequencing, we determined that butyrate-producing genera were increased by the supplementation with alfalfa hay or the starter. Transcriptomic analysis of the rumen epithelia revealed that the PI3K-Akt signaling pathway, which is critical in mediating many aspects of cellular function such as cell growth, was upregulated in response to alfalfa hay or the starter supplementation. The starter supplementation also increased the jejunal α-amylase activity, whereas alfalfa hay supplementation reduced the ileal α-amylase activity. Furthermore, the co-supplementation of both the starter and alfalfa hay reduced intestinal α-amylase activity. The starter increased ruminal propionate concentration, whereas alfalfa hay exhibited the opposite trend. The observed opposite effects of the starter and alfalfa hay on rumen propionate concentration corresponded with up- and downregulation, respectively, of the ruminal cholecystokinin involved in pancreatic secretion pathway, and thereby increased and decreased pancreatic α-amylase activity. In conclusion, both alfalfa hay and the starter could promote the growth and ruminal epithelial development of yak calves. The starter and alfalfa hay also differentially affected the intestinal α-amylase activities due to their different chemical components and different effects on ruminal fermentation, especially the ruminal propionate production.

Extending the investigation of 4-thiazolidinone derivatives as potential multi-target ligands of enzymes involved in diabetes mellitus and its long-term complications: A study with pancreatic α-amylase.

Diabetes mellitus is a multifactorial disease, which is frequently complicated by the development of hyperglycaemia-induced chronic complications. The therapy of diabetes mellitus often requires combinations of two or more drugs in order both to control glycaemic levels and to prevent hyperglycaemia-induced dangerous affairs. The application of multi-target agents, which are able to control simultaneously several pathogenic mechanisms, represents a useful alternative and, in fact, their discovery is a pursued aim of the research. Some (5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)acetic acids, which we had previously reported as inhibitors of selected enzymes critically implicated in diabetes mellitus, were tested against pancreatic α-amylase and intestinal α-glucosidase. These enzymes catalyse the hydrolysis of dietary oligo- and polysaccharides into monosaccharides and, consequently, are responsible for postprandial hyperglycaemia; therefore, their inhibition is one of the possible strategies to control glycaemic levels in diabetes mellitus. In addition, we investigated the aggregation tendency of the tested compounds, through direct and indirect methods, in order to evaluate the mechanism of their multiple action and discover if aggregation may contribute to the inhibition of the target enzymes. Overall, compounds 1, 3 and 4 exhibited the most favourable profile since they were shown to act as multi-target inhibitors of enzymes involved in pathways related to diabetes mellitus, without producing aggregates even at high micromolar concentrations and, therefore, can be promising agents for further developments.

Mechanistic study on inhibition of porcine pancreatic α-amylase using the flavonoids from dandelion.

This study was designed to investigate quantitatively the inhibition and molecular mechanism of pancreatic α-amylase exhibited by flavonoids from dandelion to reveal its potential use in relieving postprandial hyperglycemia. The results show that the flavonoids reversibly inhibited the α-amylase in a non-competitive manner with Michaelis-Menten constant (Km) and half-inhibitory concentration (IC50) value of 10.51 and 0.0067 mg/mL, respectively. The flavonoids present a strong ability to quench the intrinsic fluorescence of α-amylase through static quenching by forming a complex. The values of the binding site (n) at different temperatures were found to be approximately the unity, indicating the presence of a single class of molecular binding of the dandelion flavonoids on α-amylase. The positive values of enthalpy and entropy change reveal that the binding was predominately driven by hydrophobic interactions. This study suggests a benefit of incorporating the dandelion flavonoids in making functional foods in managing the diet of the diabetes.

Digestion kinetics of low, intermediate and highly branched maltodextrins produced from gelatinized starches with various microbial glycogen branching enzymes.

Twenty-four branched maltodextrins were synthesized from eight starches using three thermostable microbial glycogen branching enzymes. The maltodextrins have a degree of branching (DB) ranging from 5 % to 13 %. This range of products allows us to explore the effect of DB on the digestibility, which was quantified under conditions that mimic the digestion process in the small intestine. The rate and extent of digestibility were analyzed using the logarithm of the slope method, revealing that the branched maltodextrins consist of a rapidly and slowly digestible fraction. The amount of slowly digestible maltodextrin increases with an increasing DB. Surprisingly, above 10 % branching the fraction of slowly digestible maltodextrin remains constant. Nevertheless, the rate of digestion of the slowly digestible fraction was found to decline with increasing DB and shorter average internal chain length. These observations increase the understanding of the structural factors important for the digestion rate of branched maltodextrins.

Computer simulations explain the anomalous temperature optimum in a cold-adapted enzyme.

Cold-adapted enzymes from psychrophilic species show the general characteristics of being more heat labile, and having a different balance between enthalpic and entropic contributions to free energy barrier of the catalyzed reaction compared to mesophilic orthologs. Among cold-adapted enzymes, there are also examples that show an enigmatic inactivation at higher temperatures before unfolding of the protein occurs. Here, we analyze these phenomena by extensive computer simulations of the catalytic reactions of psychrophilic and mesophilic α-amylases. The calculations yield temperature dependent reaction rates in good agreement with experiment, and also elicit the anomalous rate optimum for the cold-adapted enzyme, which occurs about 15 °C below the melting point. This result allows us to examine the structural basis of thermal inactivation, which turns out to be caused by breaking of a specific enzyme-substrate interaction. This type of behaviour is also likely to be relevant for other enzymes displaying such anomalous temperature optima.

Inhibitory effect of a weight-loss Chinese herbal formula RCM-107 on pancreatic α-amylase activity: Enzymatic and in silico approaches.

Reducing carbohydrates digestion by having a low glycaemic index (GI) foods has been linked to weight loss. Inhibiting related enzymes is an alternative way to decrease carbohydrate digestion. RCM-107 (Slimming Plus), an eight-herb formula that is modified from RCM-104, indicated significant weight-loss action in clinical trials. However, no published research has studied its mechanism of action on reducing carbohydrate absorption via suppressing the activities of porcine pancreatic alpha-amylase (PPA). In this paper, we used fluorescence PPA inhibition assay to investigate the inhibitory effects of RCM-107 and the individual herbs present in this herbal mixture on amylase activity. Subsequently, molecular docking predicted the key active compounds that may be responsible for the enzyme inhibition. According to our results, both the RCM-107 formula and several individual herbs displayed α-amylase inhibitory effects. Also, marginal synergistic effects of RCM-107 were detected. In addition, alisol B, (-)-epigallocatechin-3-gallate (EGCG) and plantagoside have been predicted as the key active compounds that may be responsible for the α-amylase inhibition effect of RCM-107 according to inter-residue contact analysis. Finally, Glu233, Gln63, His305, Asp300 and Tyr151 are predicted to be markers of important areas with which potential amylase inhibitors would interact. Therefore, our data has provided new knowledge on the mechanisms of action of the RCM-107 formula and its individual herbal ingredients for weight loss, in terms of decreasing carbohydrate digestion via the inhibition of pancreatic alpha-amylase.

α-Amylase expressed in human small intestinal epithelial cells is essential for cell proliferation and differentiation.

α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other tissues, but it is unclear whether the α-amylase is endogenously expressed in each tissue or mixed exogenously with one expressed by the pancreas or salivary glands. Furthermore, the biological significance of these α-amylases detected in tissues other than the pancreas and salivary glands has not been elucidated. We discovered that human α-amylase is expressed in intestinal epithelial cells and analyzed the effects of suppressing α-amylase expression. α-Amylase was found to be expressed at the second-highest messenger RNA level in the duodenum in human normal tissues after the pancreas. α-Amylase was detected in the cell extract of Caco-2 intestinal epithelial cells but not secreted into the culture medium. The amount of α-amylase expressed increased depending on the length of the culture of Caco-2 cells, suggesting that α-amylase is expressed in small intestine epithelial cells rather than the colon because the cells differentiate spontaneously upon reaching confluence in culture to exhibit the characteristics of small intestinal epithelial cells rather than colon cells. The α-amylase expressed in Caco-2 cells had enzymatic activity and was identified as AMY2B, one of the two isoforms of pancreatic α-amylase. The suppression of α-amylase expression by small interfering RNA inhibited cell differentiation and proliferation. These results demonstrate for the first time that α-amylase is expressed in human intestinal epithelial cells and affects cell proliferation and differentiation. This α-amylase may induce the proliferation and differentiation of small intestine epithelial cells, supporting a rapid turnover of cells to maintain a healthy intestinal lumen.

In silico evaluation of condensed and hydrolysable tannins as inhibitors of pancreatic α-amylase.

Amylases are interesting targets for antidiabetic drugs because their inhibition is able to lower glycaemia without the need of hormonal control, as promoted by insulin or glibenclamide. In this context, the comparison between the binding features of α-amylases with their substrate and known inhibitors may provide insights aiming at the discovery of new antidiabetic drugs. In this work, the structure of the porcine pancreatic α-amylase was modelled with the acarbose pentasaccharide inhibitor, and used in structure-based virtual screening simulations based on a library containing the structures of amylose (AMY), acarbose (ACA) and the more representative structures of condensed tannin (CTN) and hydrolysable tannin (HTN). After validation of the methodology by redocking (mean rmsd ~ 0.8 Å), the scores provided by programs AutoDock/Molegro were contradictory (- 1.5/- 23.3; - 3.5/- 24.6; - 4.3/- 14.6; -/- 19.5 for AMY, ACA, CTN and HTN respectively), indicating that a more sensitive methodology was necessary. The ΔGbinding was calculated by the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, which indicated that the HTN, ACA and CTN had higher affinities for the enzyme regarding the AMY substrate, with values of - 350.0, - 346.2, - 320.5 and - 209.2 kJ mol-1, respectively. The predicted relative affinities of HTN and CTN are in agreement with those obtained experimentally. The results provided useful information for the characterization of tannin binding to α-amylase, which can be applied in future studies aiming at finding new hypoglycaemic molecules among natural products.

A single-enzyme system for starch digestibility screening and its relevance to understanding and predicting the glycaemic index of food products.

There is currently great interest in increasing provisions of healthier carbohydrate foods, particularly those that possess a low Glycaemic Index (GI) when measured in vivo. The metabolic response to many starch-rich foods is driven largely by differences in the rate and extent of starch amylolysis. Enzyme-kinetic parameters obtained from high-throughput in vitro amylolysis assays therefore have potential for rapid prediction of GI for starch-rich foods. The aim of this study was to evaluate the usefulness of a starch digestibility screening method and resulting enzyme-kinetic parameters in comparing and predicting the GI of a range of carbohydrate-rich foods. Starch-rich foods (n = 20) with GI ranging from 36 to 81 were digested by porcine pancreatic α-amylase for 90 min under a fixed enzyme-substrate ratio (4 U/10 mg starch) at 37 °C on a rotary mixer. Starch digestion progress was determined by quantification of reducing sugar concentration in aliquots collected throughout the incubation. Indices of starch digestibility (C20, C60, C90, HI, C∞, and k) were obtained and compared with GI values. Digestibility curves revealed differences in the starch amylolysis for the broad range of foods tested. In vitro starch digestibility indices were significantly correlated (p < 0.01) with GI, with the exception of the rate constant, k. Out of all the indices tested, C90 and C∞ were the most strongly correlated with in vivo rankings for GI of matched food products (Tb = 0.596, p < 0.001 and Tb = 0.599, p < 0.01, respectively), however the digestibility plots obtained for some of the more slowly digested foods were linear over 90 min meaning that C∞ and k could not be obtained from first order kinetic analysis. C90 was most strongly correlated with the absolute GI values (r = 0.724, p < 0.001). Overall starch digestibility profiles reflected differences in starch amylolysis for food with varying GI, and C90 provided the best indication of absolute and relative GI values across all product categories. The in vitro starch digestibility screening method shows potential for rapid prediction of GI values and is recommended for early stage food product development and for mechanistic studies.

Folding Then Binding vs Folding Through Binding in Macrocyclic Peptide Inhibitors of Human Pancreatic α-Amylase.

De novo macrocyclic peptides, derived using selection technologies such as phage and mRNA display, present unique and unexpected solutions to challenging biological problems. This is due in part to their unusual folds, which are able to present side chains in ways not available to canonical structures such as α-helices and ß-sheets. Despite much recent interest in these molecules, their folding and binding behavior remains poorly characterized. In this work, we present cocrystallization, docking, and solution NMR structures of three de novo macrocyclic peptides that all bind as competitive inhibitors with single-digit nanomolar Ki to the active site of human pancreatic α-amylase. We show that a short stably folded motif in one of these is nucleated by internal hydrophobic interactions in an otherwise dynamic conformation in solution. Comparison of the solution structures with a target-bound structure from docking indicates that stabilization of the bound conformation is provided through interactions with the target protein after binding. These three structures also reveal a surprising functional convergence to present a motif of a single arginine sandwiched between two aromatic residues in the interactions of the peptide with the key catalytic residues of the enzyme, despite little to no other structural homology. Our results suggest that intramolecular hydrophobic interactions are important for priming binding of small macrocyclic peptides to their target and that high rigidity is not necessary for high affinity.

Water-soluble vitamins for controlling starch digestion: Conformational scrambling and inhibition mechanism of human pancreatic α-amylase by ascorbic acid and folic acid.

The inhibition of human pancreatic α-amylase (HPA) enzyme activity can offer facile routes to ameliorate postprandial hyperglycemia in diabetes via control of starch digestion. The present study utilizes complementary experimental (starch digestion kinetics, fluorescence quenching, Förster resonance energy transfer and X-ray diffraction) and computational (molecular docking and dynamics simulation) methods to evaluate the HPA inhibitory activity of eight water-soluble vitamins, for the first time. In particular, ascorbic acid inhibited HPA activity via non-competitive antagonism from two allosteric sites, by channeling the inhibition towards the active site cavity via the triose-phosphate isomerase (TIM) barrel. In contrast, folic acid inhibited HPA activity by binding competitively to the active site cavity and decreasing the disorder in the neighboring loops 3 and 7, which are important mobile loops in HPA for starch digestion. The infusion of such biocompatible and nutritional water-soluble vitamins alongside starch may offer new avenues for diabetes management.