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.

Inhibition mechanism of human salivary α-amylase by lipid transfer protein from Vigna unguiculata.

VuLTP1.1, a LTP1 from Vigna unguiculata, inhibits 78.1 % of the human salivary α-amylase (HSA) activity at 20 µM. We had performed a correlation study between VuLTP1.1 structure and HSA inhibitory activity and showed that two VuLTP1.1 regions are responsible for HSA inhibition. In one of them we had characterized the crucial importance of an Arg39 for inhibition. In this work, we analyzed the VuLTP1.1-HSA interaction by protein-protein docking to understand the most probable interaction model and the mechanism of HSA inhibition by VuLTP1.1. The VuLTP1.1 tertiary structure quality and refinement as well as the docking assay between VuLTP1.1 and HSA were done by bioinformatic programs. HSA inhibition occurs by direct interaction of the VuLTP1.1 with the HSA causing the obstruction of the carbohydrate biding cleft with Gibbs free energy of -18.5 Kcal/mol and the dissociation constant of 2.6E-14 M. The previously identified Arg39 of VuLTP1.1 is burrowed into the active site of the HSA and there it interacts with the Asp300 of HSA catalytic site by a hydrogen bond. We had confirmed the importance of the Arg39 of VuLTP1.1 for the HSA inhibition which interacts with the Asp300 at the HSA active site. I-2, a LTP-like peptide, presents the same HSA inhibition pattern that VuLTP1.1, which indicates that the inhibition mechanism of the LTPs towards α-amylase is very similar. For the best of our knowledge, it is the first time that the HSA inhibition mechanism was understood and described for the LTP1s using VuLTP1.1 and I-2 as prototype inhibitors.

Colorimetric Detection of Salivary α-Amylase Using Maltose as a Noncompetitive Inhibitor for Polysaccharide Cleavage.

This paper describes an approach for colorimetric detection of salivary α-amylase, one of the potential biomarkers of autonomic nervous system (ANS) activity, for enabling assessment of fatigue. The ability of α-amylase to cleave α-bonds of polysaccharides is utilized for developing a colorimetric assay. In the proposed approach, 2-chloro-4-nitrophenyl-α-d-maltotrioside as substrate releases a colored byproduct upon cleavage by salivary α-amylase. Introduction of maltose as a noncompetitive inhibitor yields desirable linear responses in the physiologically relevant concentration range (20-500 µg/mL) with a limit of detection (LOD) of 8 µg/mL (in aqueous solution). The concentrations of substrate and noncompetitive inhibitor are subsequently optimized for colorimetric detection of salivary α-amylase. A facile paper-based "strip" assay is proposed for analysis of human saliva samples with marginal interference from saliva components. The proposed assay is rapid, specific, and easy-to-implement for colorimetric detection of salivary α-amylase between 20 and 500 µg/mL. Complementary RGB (red, green, blue components) analysis offers quantitative detection with a LOD of 11 µg/mL. The two assay formats are benchmarked against the Phadebas test, a state of the art method for spectrophotometric detection of α-amylase. The reported paper-based methodology possesses a high potential for estimation of altered ANS responses toward stressors that possibly could find applications in assessment of fatigue and for monitoring onset of fatigue.

Acid induced reduction of the glycaemic response to starch-rich foods: the salivary α-amylase inhibition hypothesis.

Numerous studies have reported that the glycaemic response to starch-rich meals can be reduced by 20-50% with acidic drinks or foods. A number of candidate explanations have been put forward, but this phenomenon still remains vaguely understood. This study intends to demonstrate the remarkable effect of acid inhibition of salivary α-amylase during oro-gastric hydrolysis of starch, shedding light on this often overlooked mechanism. Oro-gastric digestions of bread, wheat and gluten-free pastas, combined with either water or lemon juice were performed using a dynamic in vitro system that reproduces gastric acidification kinetics observed in humans. In the presence of water, large proportions of starch (25-85%) and oligosaccharides (15-50%) were released from all foods within the first hour of gastric digestion (pH > 3.5). In the presence of lemon juice (pH < 3.5 at all time), starch release was about twice as low, and amylolysis into oligosaccharides was completely interrupted. Acid-inhibition of salivary α-amylase may explain, at least in part, the reduction of the blood glucose response through acidification of starch-rich foods/meals. This offers new perspectives for the development of strategies to improve the glycaemic response elicited by starch-rich diets.

Chlorogenic and phenolic acids are only very weak inhibitors of human salivary α-amylase and rat intestinal maltase activities.

There is increasing evidence that consumption of polyphenol and phenolic-rich foods and beverages have the potential to reduce the risk of developing diabetes type 2, with coffee a dominant example according to epidemiological evidence. One of the proposed mechanisms of action is the inhibition of carbohydrate-digesting enzymes leading to attenuated post-prandial blood glucose concentrations, as exemplified by the anti-diabetic drug, acarbose. We determined if the phenolic, 5-caffeoylquinic acid, present in coffee, apples, potatoes, artichokes and prunes, for example, and also selected free phenolic acids (ferulic acid, caffeic acid and 3,4-dimethoxycinnamic acid), could inhibit human salivary α-amylase and rat intestinal maltase activities, digestive enzymes involved in the degradation of starch and malto-oligosaccharides. Using validated assays, we show that phenolic acids, both free and linked to quinic acid, are poor inhibitors of these enzymes, despite several publications that claim otherwise. 5-CQA inhibited human α-amylase only by <20% at 5 mM, with even less inhibition of rat intestinal maltase. The most effective inhibition was with 3,4-dimethoxycinnamic acid (plateau at maximum 32% inhibition of human α-amylase at 0.6 mM), but this compound is found in coffee in the free form only at very low concentrations. Espresso coffee contains the highest levels of 5-CQA among all commonly consumed foods and beverages with a typical concentration of ~5 mM, and much lower levels of free phenolic acids. We therefore conclude that inhibition of carbohydrate-digesting enzymes by chlorogenic or phenolic acids from any food or beverage is unlikely to be sufficient to modify post-prandial glycaemia, and so is unlikely to be the mechanism by which chlorogenic acid-rich foods and beverages such as coffee can reduce the risk of developing type 2 diabetes.

Sour cherry extract inhibits human salivary α-amylase and growth of Streptococcus mutans (a pilot clinical study).

The purpose of this study was to determine whether cherry extract has any effect on salivary α-amylase activity (sAA) or on the level of Streptococcus mutans in human saliva. 70 patients (45 females and 25 males) in three age groups (22 children, 25 young adults, and 23 adults) were examined. All participants completed a questionnaire to obtain information on their oral health behaviour and life style. Clinical examination was performed to record the number of decayed, missing and filled teeth (DMF-T). Saliva samples were collected for the measurement of sAA and the salivary S. mutans level before and after chewing a gum with or without cherry extract. Statistical evaluation of data was performed. S. mutans and the sAA level of unstimulated saliva samples did not depend on either age or gender. The basal sAA value of adult patients was in linear correlation with the dental caries status. Habitual chewing-gum use decreased the resting sAA and the mean of DMF-T. The number of S. mutans cells was significantly lower in the resting saliva of allergic patients. The applied mechanical and gustatory stimuli by chewing gum resulted in higher sAA and S. mutans levels and a slow decrease of values was observed in the control group for the next 30 min. Thereafter, sAA and S. mutans levels decreased earlier in the presence of sour cherry extract than those of control cases. Chewing gum with sour cherry extract may be useful for the prevention of dental caries.

Oleanolic acid and ursolic acid as potential inhibitors of human salivary α-amylase: insights from in vitro assays and in silico simulations.

It is known that inhibiting α-amylase, an important enzyme in digestion of starch and glycogen, is a useful strategy for treating disorders in carbohydrate uptake. Two natural components distributed in many fruits and plants, oleanolic acid and ursolic acid, are endowed with important pharmacological activities and wide therapeutic possibilities. Until now, only a tiny fraction of their applications have been identified and exploited. Our in vitro inhibition studies demonstrated that oleanolic acid and ursolic acid non-competitively inhibit the activity and function of human salivary α-amylase. The molecular simulations revealed that oleanolic acid and ursolic acid interact with amino acid residues within the binding pocket of human salivary α-amylase, among which the side chain of Arg195 and Asp 197 was supposed to be important in imparting the inhibitory activity of triterpenoids. The present work will provide meaningful information for future development of functional drugs for the treatment of disorders in carbohydrate metabolism. Graphical abstract This work is valuable for providing a deeper insight into the interaction mechanism of oleanolic acid and ursolic acid with α-amylase.

Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-ß-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.

Pouteria ramiflora extract inhibits salivary amylolytic activity and decreases glycemic level in mice.

In this study, extracts of plant species from the Cerrado biome were assessed in order to find potential inhibitors of human salivary alpha-amylase. The plants were collected and extracts were obtained from leaves, bark, and roots. We performed a preliminary phytochemical analysis and a screening for salivar alpha-amylase inhibitory activity. Only three botanical families (Sapotaceae, Sapindaceae and Flacourtiaceae) and 16 extracts showed a substantial inhibition (>75%) of alpha-amylase. The ethanolic extracts of Pouteria ramiflora obtained from stem barks and root barks decreased amylolytic activity above 95% at a final concentration of 20 µg/mL. Thus, adult male Swiss mice were treated orally with P. ramiflora in acute toxicity and glycemic control studies. Daily administration with 25, 50 and 100 mg/kg of aqueous extract of P. ramiflora for eight days can reduce significantly body weight and blood glucose level in mice. These data suggest that the crude polar extract of P. ramiflora decreases salivary amylolytic activity while lowering the blood levels of glucose.

Oral Fusobacterium nucleatum subsp. polymorphum binds to human salivary α-amylase.

Fusobacterium nucleatum acts as an intermediate between early and late colonizers in the oral cavity. In this study, we showed that F. nucleatum subsp. polymorphum can bind to a salivary component with a molecular weight of approximately 110 kDa and identified the protein and another major factor of 55 kDa, as salivary α-amylase by time-of-flight mass spectrometry and immuno-reactions. Salivary α-amylase is present in both monomeric and dimeric forms and we found that formation of the dimer depends on copper ions. The F. nucleatum adhered to both monomeric and dimeric salivary α-amylases, but the numbers of bacteria bound to the dimeric form were more than those bound to the monomeric form. The degree of adherence of F. nucleatum to four α-amylases from different sources was almost the same, however its binding to ß-amylase was considerably decreased. Among four α-amylase inhibitors tested, acarbose and type 1 and 3 inhibitors derived from wheat flour showed significant activity against the adhesion of F.nucleatum to monomeric and dimeric amylases, however voglibose had little effect. Moreover F. nucleatum cells inhibited the enzymatic activity of salivary α-amylase in a dose-dependent manner. These results suggest that F. nucleatum plays more important and positive role as an early colonizer for maturation of oral microbial colonization.

Selective inhibition of CD4+ T-cell cytokine production and autoimmunity by BET protein and c-Myc inhibitors.

Bromodomain-containing proteins bind acetylated lysine residues on histone tails and are involved in the recruitment of additional factors that mediate histone modifications and enable transcription. A compound, I-BET-762, that inhibits binding of an acetylated histone peptide to proteins of the bromodomain and extra-terminal domain (BET) family, was previously shown to suppress the production of proinflammatory proteins by macrophages and block acute inflammation in mice. Here, we investigated the effect of short-term treatment with I-BET-762 on T-cell function. Treatment of naïve CD4(+) T cells with I-BET-762 during the first 2 d of differentiation had long-lasting effects on subsequent gene expression and cytokine production. Gene expression analysis revealed up-regulated expression of several antiinflammatory gene products, including IL-10, Lag3, and Egr2, and down-regulated expression of several proinflammatory cytokines including GM-CSF and IL-17. The short 2-d treatment with I-BET-762 inhibited the ability of antigen-specific T cells, differentiated under Th1 but not Th17 conditions in vitro, to induce pathogenesis in an adoptive transfer model of experimental autoimmune encephalomyelitis. The suppressive effects of I-BET-762 on T-cell mediated inflammation in vivo were accompanied by decreased recruitment of macrophages, consistent with decreased GM-CSF production by CNS-infiltrating T cells. These effects were mimicked by an inhibitor of c-myc function, implicating reduced expression of c-myc and GM-CSF as one avenue by which I-BET-762 suppresses the inflammatory functions of T cells. Our study demonstrates that inhibiting the functions of BET-family proteins during early T-cell differentiation causes long-lasting suppression of the proinflammatory functions of Th1 cells.

Isolation of a new phlorotannin, a potent inhibitor of carbohydrate-hydrolyzing enzymes, from the brown alga Sargassum patens.

Ethanol extracts from 15 kinds of marine algae collected from the coast of the Noto Peninsula in Japan were examined for their inhibitory effects on human salivary α-amylase. Four extracts significantly suppressed the enzyme activity. An inhibitor was purified from the extract of Sargassum patens . The compound was a new phloroglucinol derivative, 2-(4-(3,5-dihydroxyphenoxy)-3,5-dihydroxyphenoxy) benzene-1,3,5-triol (DDBT), which strongly suppressed the hydrolysis of amylopectin by human salivary and pancreatic α-amylases. The 50% inhibitory activity (IC(50)) for α-amylase inhibition of DDBT (3.2 µg/mL) was much lower than that of commercially available α-amylase inhibitors, acarbose (26.3 µg/mL), quercetagetin (764 µg/mL), and α-amylase inhibitor from Triticum aestivum (88.3 µg/mL). A kinetic study indicated that DDBT was a competitive α-amylase inhibitor with a K(i) of 1.8 µg/mL. DDBT also inhibited rat intestinal α-glucosidase with an IC(50) value of 25.4 µg/mL for sucrase activity and 114 µg/mL for maltase activity. These results suggest that DDBT, a potent inhibitor of carbohydrate-hydrolyzing enzymes, may be useful as a natural nutraceutical to prevent diabetes.

Antidiabetic potential of α-amylase inhibitor from the seeds of Macrotyloma uniflorum in streptozotocin-nicotinamide-induced diabetic mice.

CONTEXT: Macrotyloma uniflorum (Lam.) Verdc. (Leguminosae) seeds, known as the poor man's pulse crop in India, have been used as a food and also used in the traditional method for treatment of kidney stones, diabetes, obesity, etc. OBJECTIVE: To investigate the antidiabetic effect of α-amylase inhibitor isolated from the seeds of Macrotyloma uniflorum seeds in streptozotocin-nicotinamide induced diabetic mice. MATERIALS AND METHOD: α-Amylase inhibitor was purified using a carboxymethyl cellulose (CMC) column. Kinetic studies were done using mouse pancreatic and human salivary α-amylase. Its antidiabetic effect was studied in streptozotocin-nicotinamide-induced diabetic mice. Biochemical parameters such as serum total cholesterol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined. Histopathological investigation was performed on the pancreas, kidney, and liver tissue samples. RESULTS: Macrotyloma uniflorum α-amylase inhibitor (MUAI) inhibited both the mouse pancreatic and human salivary α-amylase in a non-competitive manner with K(i) values of 11 and 8.8 µM and IC(50) value of 30 and 12.5 µg/mL, respectively. It decreased the serum glucose level in the treated diabetic mice. Histological findings suggested minimum pathological changes in the treated diabetic mice as compared to the diabetic control. DISCUSSION AND CONCLUSION: The results suggest that MUAI has an antihyperglycemic activity and therefore can be used in the dietary treatment of non-insulin dependent diabetes mellitus.

Effects of cigarette smoke borne reactive nitrogen species on salivary alpha-amylase activity and protein modifications.

Cigarette smoke (CS) is associated with a variety of human pathologies including cardiovascular disease and cancer. Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck. The major inducer of OSCC is exposure to tobacco. Recent studies demonstrated that oxidative and nitrosative stress contributes to the development of oral carcinogenesis through DNA damage. All salivary reactive nitrogen species (RNS) analyzed from OSCC patients are significantly higher in comparison with healthy subjects. Our findings show that CS and external RNS addition induced reduction in alpha-amylase activity and produced some excited carbonyl formation, but to a much less extant than CS. The addition of epigallocatechine-3-gallate (EGCG) to saliva produced no protective effect against damage to alpha-amylase activity. Our proposed mechanism for the decrease in alpha-amylase activity is the formation of adducts at SH groups of the alpha-amylase active site. In this case, EGCG was unable to counteract this phenomenon, as it does not reduce the concentration of disulfides, and does not alter the amount of protein-SH moieties. However, EGCG did reduce the levels of excited carbonyl formation. Our results indicate that although RNS are abundant in CS, a significant decrease in amylase activity is due to other components in CS, probably aldehydes, reacting with the thiol group of proteins by the Michael addition reaction.

Evidence for pentagalloyl glucose binding to human salivary alpha-amylase through aromatic amino acid residues.

We demonstrate here that pentagalloyl glucose (PGG), a main component of gallotannins, was an effective inhibitor of HSA and it exerted similar inhibitory potency to Aleppo tannin used in this study. The inhibition of HSA by PGG was found to be non-competitive and inhibitory constants of K(EI)=2.6 microM and K(ESI)=3.9 microM were determined from Lineweaver-Burk secondary plots. PGG as a model compound for gallotannins was selected to study the inhibitory mechanism and to characterize the interaction of HSA with this type of molecules. Surface plasmon resonance (SPR) binding experiments confirmed the direct interaction of HSA and PGG, and it also established similar binding of Aleppo tannin to HSA. Saturation transfer difference (STD) experiment by NMR clearly demonstrated the aromatic rings of PGG may be involved in the interaction suggesting a possible stacking with the aromatic side chains of HSA. The role of aromatic amino acids of HSA in PGG binding was reinforced by kinetic studies with the W58L and Y151M mutants of HSA: the replacement of the active site aromatic amino acids with aliphatic ones decreased the PGG inhibition dramatically, which justified the importance of these residues in the interaction.