Synephrine and phenylephrine act as α‐amylase, α‐glycosidase,acetylcholinesterase, butyrylcholinesterase,and carbonic anhydrase enzymes inhibitors

In this paper, synephrine and phenylephrine compounds showed excellent inhibitory effects against human carbonic anhydrase (hCA) isoforms I and II, α‐amylase, α‐glycosidase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). Synephrine and phenylephrine had K_i values of 199.02 ± 16.01 and 65.01 ± 5.00 μM against hCA I and 336.02 ± 74.01 and 92.04  ±  18.03 μM against hCA II, respectively. On the other hand, their K_i values were found to be 169.10  ±  80.03 and 88.03  ±  5.01 nM against AChE and 177.06  ±  6.01 and 78.03  ±  3.05 nM against BChE, respectively. α‐Amylase and α‐glycosidase enzymes were easily inhibited by these compounds. α‐Glycosidase inhibitors, generally defined to as starch blockers, are anti‐diabetic drugs that help to decrease post comestible blood glucose levels.     

The impact of some natural phenolic compounds on carbonic anhydrase,acetylcholinesterase, butyrylcholinesterase,and α‐glycosidase enzymes: An antidiabetic,anticholinergic, and antiepileptic study

Natural products from food and plant sources have been used for medicinal usage for ages. Also, natural products with therapeutic significance are compounds derived from animals, plants, or any microorganism. In this study, chrysin, carvacrol, hesperidin, zingerone, and naringin as natural phenols showed excellent inhibitory effects against human (h) carbonic anhydrase (CA) isoforms I and II (hCA I and II), α‐glucosidase (α‐Gly), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). These phenolic compounds were tested for the inhibition of α‐glycosidase, hCA I, hCA II, AChE, and BChE enzymes and demonstrated efficient inhibition profiles with K_i values in the range of 3.70 ± 0.92–79.66 ± 20.81 nM against hCA I, 2.98 ± 0.33–84.88 ± 40.32 nM against hCA II, 4.93 ± 2.01–593.60 ± 134.74 nM against α‐Gly, 0.52 ± 0.18–46.80 ± 17.15 nM against AChE, and 1.25 ± 0.22–32.08 ± 2.68 against BChE.     

Inhibition profiles of Voriconazole against acetylcholinesterase, α‐glycosidase,and human carbonic anhydrase I and II isoenzymes

In this work, the inhibitory activity of Voriconazole was measured against some metabolic enzymes, including human carbonic anhydrase (hCA) I and II isoenzymes, acetylcholinesterase (AChE), and α‐glycosidase; the results were compared with standard compounds including acetazolamide, tacrine, and acarbose. Half maximal inhibition concentration (IC₅₀) values were obtained from the enzyme activity (%)‐[Voriconazole] graphs, whereas K_i values were calculated from the Lineweaver‐Burk graphs. According to the results, the IC₅₀ value of Voriconazole was 40.77 nM for α‐glycosidase, while the mean inhibition constant (K_i) value was 17.47 ± 1.51 nM for α‐glycosidase. The results make an important contribution to drug design and have pharmacological applications. In addition, the Voriconazole compound demonstrated excellent inhibitory effects against AChE and hCA isoforms I and II. Voriconazole had K_i values of 29.13 ± 3.57 nM against hCA I, 15.92 ± 1.90 nM against hCA II, and 10.50 ± 2.46 nM against AChE.     

Synthesis and discovery of potent carbonic anhydrase,acetylcholinesterase, butyrylcholinesterase,and α‐glycosidase enzymes inhibitors: The novel N,N′‐bis‐cyanomethylamine and alkoxymethylamine derivatives

During this investigation, N,N′‐bis‐azidomethylamines, N,N′‐bis‐cyanomethylamine, new alkoxymethylamine and chiral derivatives, which are considered to be a new generation of multifunctional compounds, were synthesized, functional properties were investigated, and anticholinergic and antidiabetic properties of those compounds were studied through the laboratory tests, and it was approved that they contain physiologically active compounds rather than analogues. Novel N‐bis‐cyanomethylamine and alkoxymethylamine derivatives were effective inhibitors of the α‐glycosidase, cytosolic carbonic anhydrase I and II isoforms, butyrylcholinesterase (BChE), and acetylcholinesterase (AChE) with K_i values in the range of 0.15–13.31 nM for α‐glycosidase, 2.77–15.30 nM for human carbonic anhydrase isoenzymes I (hCA I), 3.12–21.90 nM for human carbonic anhydrase isoenzymes II (hCA II), 23.33–73.23 nM for AChE, and 3.84–48.41 nM for BChE, respectively. Indeed, the inhibition of these metabolic enzymes has been considered as a promising factor for pharmacologic intervention in a diversity of disturbances.     

Interaction of antidiabetic α‐glucosidase inhibitors and gut bacteria α‐glucosidase

Carbohydrate hydrolyzing α‐glucosidases are commonly found in microorganisms present in the human intestine microbiome. We have previously reported crystal structures of an α‐glucosidase from the human gut bacterium Blaubia (Ruminococcus) obeum (Ro‐αG1) and its substrate preference/specificity switch. This novel member of the GH31 family is a structural homolog of human intestinal maltase‐glucoamylase (MGAM) and sucrase–isomaltase (SI) with a highly conserved active site that is predicted to be common in Ro‐αG1 homologs among other species that colonize the human gut. In this report, we present structures of Ro‐αG1 in complex with the antidiabetic α‐glucosidase inhibitors voglibose, miglitol, and acarbose and supporting binding data. The in vitro binding of these antidiabetic drugs to Ro‐αG1 suggests the potential for unintended in vivo crossreaction of the α‐glucosidase inhibitors to bacterial α‐glucosidases that are present in gut microorganism communities. Moreover, analysis of these drug‐bound enzyme structures could benefit further antidiabetic drug development.     

Evaluation of In Vitro α‐Amylase and α‐Glucosidase Inhibitory Potentials of 14 Medicinal Plants Constituted in Thai Folk Antidiabetic Formularies

The sporadic increase in the occurrence and prevalence of diabetes mellitus have compelled and vigorous search for alternative anti‐diabetic therapeutic approach from medicinal plants and its bioactive. One of the major approach employed is the reduction of gastrointestinal glucose levels through the inhibition of carbohydrate digesting enzymes notably α‐amylase and α‐glucosidase. In this study, the ethanol extracts of 14 selected plants from Mor Porn's recipe were screened for their α‐amylase and α‐glucosidase inhibitory activity. The ethanolic extract from the stem of Vitex glabrata displayed the highest percentage inhibitory activity of 84.98 ± 0.59 and 84.71 ± 1.51 against α‐glucosidase and α‐amylase enzymes, respectively. Chemical investigation of the active extract of V. glabrata indicated that pentacyclic triterpenes were the major compounds responsible for the activity. The result obtained from this study suggests the potential use of V. glabrata as an alternative natural source for the treatment of diabetes mellitus.     

Potential of the bean α‐amylase inhibitor αAI‐1 to inhibit α‐amylase activity in true bugs (Hemiptera)

True bugs (Hemiptera) are an important pest complex not controlled by Bt‐transgenic crops. An alternative source of resistance includes inhibitors of digestive enzymes, such as protease or amylase inhibitors. αAI‐1, an α‐amylase inhibitor from the common bean, inhibits gut‐associated α‐amylases of bruchid pests of grain legumes. Here we quantify the in vitro activity of α‐amylases of 12 hemipteran species from different taxonomic and functional groups and the in vitro inhibition of those α‐amylases by αAI‐1. α‐Amylase activity was detected in all species tested. However, susceptibility to αAI‐1 varied among the different groups. α‐Amylases of species in the Lygaeidae, Miridae and Nabidae were highly susceptible, whereas those in the Auchenorrhyncha (Cicadellidae, Membracidae) had a moderate susceptibility, and those in the Pentatomidae seemed to be tolerant to αAI‐1. The species with αAI‐1 susceptible α‐amylases represented families which include both important pest species but also predatory species. These findings suggest that αAI‐1‐expressing crops have potential to control true bugs in vivo.     

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Asymmetric Allylation of α‐Ketoester‐Derived N‐Benzoylhydrazones Promoted by Chiral Sulfoxides/N‐Oxides Lewis Bases: Highly Enantioselective Synthesis of Quaternary α‐Substituted α‐Allyl‐α‐Amino Acids

Chiral sulfoxides/N‐oxides (R)‐ 1 and (R,R)‐ 2 are effective chiral promoters in the enantioselective allylation of α‐keto ester N‐benzoylhydrazone derivatives 3a , 3b , 3c , 3d , 3e , 3f , 3g to generate the corresponding N‐benzoylhydrazine derivatives 4a , 4b , 4c , 4d , 4e , 4f , 4g , with enantiomeric excesses as high as 98%. Representative hydrazine derivatives 4a , 4b were subsequently treated with SmI₂, and the resulting amino esters 5a , 5b with LiOH to obtain quaternary α‐substituted α‐allyl α‐amino acids 6a , 6b , whose absolute configuration was assigned as (S), with fundament on chemical correlation and electronic circular dichroism (ECD) data. Chirality 25:529–540, 2013. © 2013 Wiley Periodicals, Inc.     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Raw starch–degrading α‐amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene,bioinformatics and biochemical characterization of the recombinant enzyme

Aims

The aims were to isolate a raw starch–degrading α‐amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli.

Methods and Results

A 1539 complete open reading frame of a starch–degrading α‐amylase gene baqA from B. aquimaris MKSC 6·2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B. aquimaris MKSC 6.2 α‐amylase (BaqA) has no starch‐binding domain, and together with a few putative α‐amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch.

Conclusions

A novel raw starch–degrading B. aquimaris MKSC 6.2 α‐amylase BaqA is proposed to be a member of new GH13 subfamily.

Significance and Impact of the Study

This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly.     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

Binding of synthetic fragments of β‐endorphin to nonopioid β‐endorphin receptor

Selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin (SLTCLVKGFY) was labeled with tritium (the specific activity of 24 Ci/mmol). [³H]Immunorphin was found to bind to nonopioid β‐endorphin receptor of mouse peritoneal macrophages (K_d = 2.0 ± 0.1 nM ). The [³H]immunorphin specific binding with macrophages was inhibited by unlabeled β‐endorphin (K_i = 2.9 ± 0.2 nM ) and was not inhibited by unlabeled naloxone, α‐endorphin, γ‐endorphin and [Met⁵]enkephalin (K_i > 10 µM ). Thirty fragments of β‐endorphin have been synthesized and their ability to inhibit the [³H]immunorphin specific binding to macrophages was studied. Unlabeled fragment 12–19 (TPLVTLFK, the author's name of the peptide octarphin) was found to be the shortest peptide possessing practically the same inhibitory activity as β‐endorphin (K_i = 3.1 ± 0.3 nM ). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol). [³H]Octarphin was found to bind to macrophages with high affinity (K_d = 2.3 ± 0.2 nM ). The specific binding of [³H]octarphin was inhibited by unlabeled immunorphin and β‐endorphin (K_i = 2.4 ± 0.2 and 2.7 ± 0.2 nM , respectively). Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Phenolic compounds prevent the oligomerization of α‐synuclein and reduce synaptic toxicity

Lewy bodies, mainly composed of α‐synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low‐order αS oligomers are neurotoxic and critical species in the pathogenesis of α‐synucleinopathies. To develop disease modifying therapies for α‐synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo‐induced cross‐linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N‐terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long‐term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers.

     

Characterization of α‐rhamnosidase activity from a Patagonian Pichia guilliermondii wine strain

Aims: The purpose of this study was to characterize the α‐l ‐rhamnosidase of Pichia guilliermondii NPCC1053 indigenous wine strain from North‐Patagonian region. Methods and Results: The optimization of yeast culture conditions was carried out and the effects of oenological parameters on α‐l ‐rhamnosidase activity were evaluated. Additionally, the effect of direct contact with must and wine on α‐l ‐rhamnosidase activity was assayed. This strain showed an intracellular inducible α‐l ‐rhamnosidase activity. This enzyme was active at pH, glucose and SO₂ concentrations usually found at the beginning of the fermentation as well as retained high levels of activity after 24 h of incubation in must. Furthermore, P. guilliermondiiα‐l ‐rhamnosidase was able to release monoterpenols and alcohols from grape glycosidic extracts. Conclusions: The α‐l ‐rhamnosidase belonging to P. guilliermondii indigenous wine yeast strain showed mainly an intracellular location and evidenced interesting oenological characteristics. Significance and Impact of the Study: This study contributes to the knowledge of α‐l ‐rhamnosidases from yeast origin because at present, there are few reports about this enzymatic activity in these micro‐organisms. In addition, this work is relevant to the regional wine industry considering that this enzyme could be used in the production of more aromatic young wines.     

Synthesis of β‐Ketoamide Curcumin Analogs for Anti‐Diabetic and AGEs Inhibitory Activities

Two different series of novel β‐ketoamide curcumin analogs enriched in biological activities have been synthesized. The synthesized compounds were screened for their in vitro anti‐diabetic and AGEs inhibitory activities and exhibited potent to good anti‐diabetic and AGEs inhibitory activities. The molecular docking study was also performed with the α‐amylase enzyme.     

Enzymatic Resolution of α‐Methyleneparaconic Acids and Evaluation of their Biological Activity

Both enantiomers of three biologically relevant paraconic acids—MB‐3, methylenolactocin, and C75—were obtained with enantioselectivities up to 99% by kinetic enzymatic resolutions. Good enantiomeric excesses were obtained for MB‐3 and methylenolactocin, using α‐chymotrypsin and aminoacylase as enantiocomplementary enzymes, while C75 was resolved with aminoacylase. They all were evaluated for their antiproliferative, antibacterial, and antifungal activities, showing weak effects and practically no difference between enantiomers in each case. At high concentrations (16–64 µg/mL), (–)‐ C75 acted as an antimicrobial agent against Gram‐positive bacteria. Chirality 27:239–246, 2015. © 2015 Wiley Periodicals, Inc.     

DIGESTION IN ADULT FEMALES OF THE LEAF‐FOOTED BUG Leptoglossus zonatus (HEMIPTERA: COREIDAE) WITH EMPHASIS ON THE GLYCOSIDE HYDROLASES α‐AMYLASE, α‐GALACTOSIDASE,AND α‐GLUCOSIDASE

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is an emerging pest of several crops around the World and up to now very little is known of its digestive system. In this article, glycoside hydrolase (carbohydrase) activities in the adult midgut cells and in the luminal contents of L. zonatus adult females were studied. The results showed the distribution of digestive carbohydrases in adults of this heteropteran species in the different intestinal compartments. Determination of the spatial distribution of α‐glucosidase activity in L. zonatus midgut showed only one major molecular form, which was not equally distributed between soluble and membrane‐bound isoforms, being more abundant as a membrane‐bound enzyme. The majority of digestive carbohydrases were found in the soluble fractions. Activities against starch, maltose and the synthetic substrate NPαGlu were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on ion‐exchange chromatography elution profiles and banding patterns in mildly denaturing electrophoresis, both midgut α‐amylases and α‐galactosidases showed at least two isoforms. The data suggested that the majority of carbohydrases involved in initial digestion were present in the midgut lumen, whereas final digestion of starch and of galactosyl oligosaccharides takes place partially within the lumen and partially at the cell surface. The complex of carbohydrases here described was qualitatively appropriate for the digestion of free oligosaccharides and oligomaltodextrins released by α‐amylases acting on maize seed starch granules.