Investigation of anti-Alzheimer's activity of aqueous extract of areca nuts (Areca catechu L): in vitro and in vivo studies / Investigación de la actividad anti-Alzheimer del extracto acuoso de nueces de areca (Areca catechu L): estudios in vitro e in vivo

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Sever cognitive and memory impairments, huge increase in the prevalence of the disease, and lacking definite cure have absorbed worldwide efforts to develop therapeutic approaches. Since many drugs have failed in the clinical trials due to multifactorial nature of AD, symptomatic treatments are still in the center attention and now, nootropic medicinal plants have been found as versatile ameliorators to reverse memory disorders. In this work, anti-Alzheimer's activity of aqueous extract of areca nuts (Areca catechu L.) was investigated via in vitro and in vivo studies. It depicted good amyloid ß (Aß) aggregation inhibitory activity, 82% at 100 µg/mL. In addition, it inhibited beta-secretase 1 (BACE1) with IC50 value of 19.03 µg/mL. Evaluation of neuroprotectivity of the aqueous extract of the plant against H2O2-induced cell death in PC12 neurons revealed 84.5% protection at 1 µg/mL. It should be noted that according to our results obtained from Morris Water Maze (MWM) test, the extract reversed scopolamine-induced memory deficit in rats at concentrations of 1.5 and 3 mg/kg.

La enfermedad de Alzheimer (EA) es un trastorno neurodegenerativo relacionado con la edad. Los severos deterioros cognitivos y de la memoria, el enorme aumento de la prevalencia de la enfermedad y la falta de una cura definitiva han absorbido los esfuerzos mundiales para desarrollar enfoques terapéuticos. Dado que muchos fármacos han fallado en los ensayos clínicos debido a la naturaleza multifactorial de la EA, los tratamientos sintomáticos siguen siendo el centro de atención y ahora, las plantas medicinales nootrópicas se han encontrado como mejoradores versátiles para revertir los trastornos de la memoria. En este trabajo, se investigó la actividad anti-Alzheimer del extracto acuoso de nueces de areca (Areca catechu L.) mediante estudios in vitro e in vivo. Representaba una buena actividad inhibidora de la agregación de amiloide ß (Aß), 82% a 100 µg/mL. Además, inhibió la beta-secretasa 1 (BACE1) con un valor de CI50 de 19,03 µg/mL. La evaluación de la neuroprotección del extracto acuoso de la planta contra la muerte celular inducida por H2O2 en neuronas PC12 reveló una protección del 84,5% a 1 µg/mL. Cabe señalar que, de acuerdo con nuestros resultados obtenidos de la prueba Morris Water Maze (MWM), el extracto revirtió el déficit de memoria inducido por escopolamina en ratas a concentraciones de 1,5 y 3 mg/kg.

Characterization and thermal inactivation kinetics of highly thermostable ramie leaf ß-amylase.

We characterized ramie leaf ß-amylase, and determined its thermostability and kinetic parameters. The enzyme was purified 53-fold using ammonium sulfate fractionation (40-60% saturation), anion exchange chromatography on DEAE-cellulose and gel permeation chromatography on Superdex-200. The purified enzyme was identified as ß-amylase with molecular mass of 42kD. The enzyme displayed Km and kcat values for soluble potato starch of 1.1mg/mL and 7.8s-1, respectively. The enzyme had a temperature optimum of 65°C, and its activity at 70°C was 92% of that at the optimal temperature after a 15-min incubation. Furthermore, enzyme activity was stable during treatment at 55°C for 60min but was inactivated rapidly at >75°C. This thermal behavior indicates that ramie leaf ß-amylase has excellent intermediate temperature-stable enzyme properties for the baking and bio-industries. Inactivation of the enzyme followed first-order kinetics in the range of 55-80°C. The enthalpy change of thermal inactivation (ΔH‡), ΔG‡, and ΔS‡ were 237.2kJ/mol, 107.7kJ/mol, and 0.39kJ/molK at 333K, respectively. The D-value at 65°C (=110min) and the z-value (=9.4°C) are given for food processing.

CuAAC click chemistry with N-propargyl 1,5-dideoxy-1,5-imino-D-gulitol and N-propargyl 1,6-dideoxy-1,6-imino-D-mannitol provides access to triazole-linked piperidine and azepane pseudo-disaccharide iminosugars displaying glycosidase inhibitory properties.

Protecting group-free synthesis of 1,2:5,6-di-anhydro-D-mannitol, followed by ring opening with propargylamine and subsequent ring closure produced a separable mix of piperidine N-propargyl 1,5-dideoxy-1,5-imino-D-gulitol and azepane N-propargyl 1,6-dideoxy-1,6-imino-D-mannitol. In O-acetylated form, these two building blocks were subjected to CuAAC click chemistry with a panel of three differently azide-substituted glucose building blocks, producing iminosugar pseudo-disaccharides in good yield. The overall panel of eight compounds, plus 1-deoxynojirimycin (DNJ) as a benchmark, was evaluated as prospective inhibitors of almond ß-glucosidase, yeast α-glucosidase and barley ß-amylase. The iminosugar pseudo-disaccharides showed no inhibitory activity against almond ß-glucosidase, while the parent N-propargyl 1,5-dideoxy-1,5-imino-D-gulitol and N-propargyl 1,6-dideoxy-1,6-imino-D-mannitol likewise proved to be inactive against yeast α-glucosidase. Inhibitory activity could be reinstated in the former series by appropriate substitution on nitrogen. The greater activity of the piperidine could be rationalized based on docking studies. Further, potent inhibition of ß-amylase was observed with compounds from both the piperidine and azepane series.

Chemical modification of wheat ß-amylase by trinitrobenzenesulfonic acid, methoxypolyethylene glycol, and glutaraldehyde to improve its thermal stability and activity.

The amino groups of wheat ß-amylase (WBA) were modified by 2,4,6-trinitrobenzenesulfonic acid (TNBS), 2,4-bis (O-methoxypolyethylene glycol)-6-chloro-s-triazine (mPEG), and glutaraldehyde (GA) to improve its thermal stability and activity. Modification of WBA by 5mM TNBS, 4.8µM mPEG and 11 mM GA improved its T50 (the temperature at which 50% of its activity is lost after 30 min of incubation) from 47 ± 1°C to 48 ± 2, 55 ± 2, and 54 ± 2°C, respectively. The catalytic activity of WBA was reduced by 15% and 59% with modification by 5mM TNBS and 11mM GA, respectively. In all cases, the enhancement of thermostability of modified WBA was entropically driven. The activity of WBA modified by 4.8µM mPEG was enhanced by 39% at 25°C. Therefore, the thermal stability of WBA was significantly improved by modification with mPEG, GA and slightly by TNBS and its catalytic activity was enhanced by mPEG.

Kinetic and thermodynamic analysis of the inhibitory effects of maltose, glucose, and related carbohydrates on wheat ß-amylase.

Inhibition of wheat ß-amylase (WBA) by glucose and maltose was studied by kinetics and thermodynamics. The inhibitory effects of fructose, difructose, sucrose, trehalose, cellobiose, acarbose, and 1-deoxynojirimycin on WBA were also evaluated. The half maximal inhibitory concentrations (IC50) of acarbose, maltose and glucose were 0.06±0.01M, 0.22±0.09M, and 1.41±0.17M, respectively. The inhibitor constant (Ki) and the thermodynamic parameters such as changes in Gibbs energy (ΔG), enthalpy (ΔH), and entropy (ΔS) of the dissociation reactions of the WBA-glucose and WBA-maltose complexes were temperature and pH-dependent. The dissociation reactions were endothermic and enthalpy-driven. Both glucose and maltose behaved as competitive inhibitors at pH 3.0 and 5.4 at a temperature of 25°C with respective Ki values of 0.33±0.02M and 0.12±0.03M. In contrast, both sugars exhibited uncompetitive inhibition at pH 9 at a temperature of 25°C with Ki values of 0.21±0.03M for glucose and 0.11±0.04M for maltose. The pH-dependence of the inhibition type and Ki values indicate that the ionizing groups of WBA influence drastically the interaction with these carbohydrates. This evidence enables us to consider temperature and pH in the WBA-catalyzed hydrolysis to manipulate the inhibition by end-product, maltose, and even by glucose.

A novel RING finger gene, SbRFP1, increases resistance to cold-induced sweetening of potato tubers.

The modulation of the activity of enzymes associated with carbohydrate metabolism is important for potato cold-induced sweetening (CIS). A novel RING finger gene SbRFP1 was cloned and its expression was found to be cold-inducible in potato tubers of the CIS-resistant genotypes. Transformation of SbRFP1 in potatoes confirmed its role in inhibiting ß-amylase and invertase activity, which consequently slowed down starch and sucrose degradation and the accumulation of reducing sugars in cold stored tubers. These findings strongly suggest that SbRFP1 may function as a negative regulator of BAM1 and StvacINV1 to decelerate the accumulation of reducing sugars in the process of potato CIS.

Chemical genetics and cereal starch metabolism: structural basis of the non-covalent and covalent inhibition of barley ß-amylase.

There are major issues regarding the proposed pathway for starch degradation in germinating cereal grain. Given the commercial importance but genetic intractability of the problem, we have embarked on a program of chemical genetics studies to identify and dissect the pathway and regulation of starch degradation in germinating barley grains. As a precursor to in vivo studies, here we report systematic analysis of the reversible and irreversible inhibition of the major ß-amylase of the grain endosperm (BMY1). The molecular basis of inhibitor action was defined through high resolution X-ray crystallography studies of unliganded barley ß-amylase, as well as its complexes with glycone site binder disaccharide iminosugar G1M, irreversible inhibitors α-epoxypropyl and α-epoxybutyl glucosides, which target the enzyme's catalytic residues, and the aglycone site binders acarbose and α-cyclodextrin.

Deactivation of isoamylase and ß-amylase in the agitated reactor under supercritical carbon dioxide.

The current research examines the impact of agitation on deactivation of isoamylase and ß-amylase under supercritical carbon dioxide (SC-CO2). Our experimental results showed that the activity of either enzyme decreased with increasing pressure or speed of agitation. The degree of enzymatic deactivation caused by pressure became more prominent in the presence of agitation, suggesting that the agitation plays an important role in enzymatic deactivation in SC-CO2 environment. Moreover, the enzymatic deactivation behavior associated with agitation and pressure was further quantitatively analyzed using a proposed inactivation kinetic model. Our analysis indicated that isoamylase and ß-amylase exhibited significantly different relationships between the inverse of percentage residual activity and the product of number of revolution per time and time elapsed under pressurized carbon dioxide. We believe that the outcome from this work may provide a better understanding of the effects of agitation and pressure in enzyme deactivation behavior under SC-CO2.

Purification and characterization of camel (Camelus dromedarius) milk amylase.

Skimmed camel milk contains 59,900 U/L amylase, which is 39,363 times less than serum and plasma amylase. Camel milk beta-amylase was purified as a 61 KDa band using DEAE-Sepharose and Sephadex G-100 and yielded 561 U/mg. The optimum working pH, Km and temperature were 7.0, 13.6 mg/Lstarch, 30-40 degrees C, respectively. The enzyme has been shown higher affinity toward amylose and soluble starch than glycogen, amylopectin, dextrin, or pullulan. Magnesium chloride, CaCl(2) and NaCl activated the amylase, while EDTA and EGTA decreased its activity. While its activity was increased in the presence of Triton X-100 and Triton X-114. Phenylmethanesulfonyl fluoride did not show any effect on enzyme activity. However, the enzyme activity was inhibited by urea, SDS, DTNB, iodoacetamide, N-ethylmalimide, aprotinin, and trypsin inhibitor. It worked on starch to yield a maltose. Scanning electron microscope images demonstrated a nano-degrading ability on starch granules from various sources (potato, corn, cassava, and rice).

Catalytic activity of beta-amylase from barley in different pressure/temperature domains.

The depolymerization of starch by beta-amylase during exposure to hydrostatic pressure up to 700 MPa and within a temperature range from 20 to 70 degrees C has been investigated. Inactivation of the enzyme as well as alterations in conversion speed in response to combined pressure-temperature treatments were assessed by analyzing the kinetic rate constants. At 200 MPa a significant stabilization of the enzyme against heat inactivation was observed. However, high pressure also impedes the catalytic reaction and a progressive reduction of the conversion rate constants with increasing pressure was found at all temperatures investigated. For the overall reaction of maltose liberation from soluble starch in ACES buffer at pH 5.6 an optimum was identified at 106 MPa and at 63 degrees C, which is approximately 7 degrees C above the local maximum at ambient pressure (0.1 MPa). Gelatinization of nonsoluble starch granules in response to pressure-temperature (p-T) treatment has been inspected by phase-contrast microscopy and yielded circular curves of identical effect in the p-T plane.

Inhibition of beta-amylase activity by calcium, magnesium and zinc ions determined by spectrophotometry and isothermal titration calorimetry.

The inhibition effect of metal ions on beta amylase activity was studied. The inhibitor-binding constant (Ki) was determined by spectrophotometric and isothermal titration calorimetric (ITC) methods. The binding of calcium, magnesium and zinc ion as inhibitors at the active site of barley beta amylase was studied at pH = 4.8 (sodium acetate 16 mM) and T = 300K. The Ki and enthalpy of binding for calcium (13.4, 13.1 mM and -14.3 kJ/mol), magnesium (18.6, 17.8mM and -17.7 kJ/mol) and zinc (17.5, 17.7 mM and -20.0 kJ/mol) were found by spectrophotometric and ITC methods respectively.

Two additional carbohydrate-binding sites of beta-amylase from Bacillus cereus var. mycoides are involved in hydrolysis and raw starch-binding.

In the previous X-ray crystallographic study, it was found that beta-amylase from Bacillus cereus var. mycoides has three carbohydrate-binding sites aside from the active site: two (Site2 and Site3) in domain B and one (Site1) in domain C. To investigate the roles of these sites in the catalytic reaction and raw starch-binding, Site1 and Site2 were mutated. From analyses of the raw starch-binding of wild-type and mutant enzymes, it was found that Site1 contributes to the binding affinity to raw-starch more than Site2, and that the binding capacity is maintained when either Site1 or Site2 exists. The raw starch-digesting ability of this enzyme was poor. From inhibition studies by maltitol, GGX and alpha-CD for hydrolyses of maltopentaose (G5) and amylose ( (n) = 16) catalyzed by wild-type and mutant enzymes, it was found that alpha-CD is a competitive inhibitor, while, maltitol behaves as a mixed-type or competitive inhibitor depending on the chain length of the substrate and the mutant enzyme. From the analysis of the inhibition mechanism, we conclude that the bindings of maltitol and GGX to Site2 in domain B form an abortive ESI complex when amylose ( (n) = 16) is used as a substrate.

Production and partial characterization of a beta-amylase by Xanthophyllomyces dendrorhous.

AIMS: The characterization of a beta-amylase produced by Xanthophyllomyces dendrorhous. METHODS AND RESULTS: Growth in different culture media showed that X. dendrorhous produces an amylase whose synthesis is repressed by the carbon source and induced by starch and maltose. Enzymatic assays using substrates with different degrees of polymerization together with viscosity experiments revealed that the enzyme was beta-amylase. According to the biochemical characterization, the enzyme has a molecular weight of 240 kDa and a Km of 1.35 mg ml-1. The optimum pH and temperature were 5.5 and 50 degrees C, respectively. Using different inhibitors of the enzymatic activity it was shown that cysteine, tryptophan and serine are essential amino acids for catalysis. CONCLUSIONS: Xanthophyllomyces dendrorhous CECT1690 synthesizes and secretes beta-amylase that could be a by-product, in addition to carotenoid pigments, in the fermentation downstream. SIGNIFICANCE AND IMPACT OF THE STUDY: The beta-amylase produced by X. dendrorhous may have certain industrial applications.

Effects of guanidine hydrochloride and high pressure on subsite flexibility of beta-amylase.

We investigated the effects of guanidine hydrochloride (GuHCl) and high pressure on the conformational flexibility of the active site of sweet potato beta-amylase by monitoring the sulfhydryl reaction and the enzymatic activity. The reactivity of Cys345 at the active site, one of six inert half cystine residues of this enzyme, was enhanced by GuHCl at concentrations below 0.5 M. A GuHCl-induced change of the active site was also observed through an intensity change in the near-UV circular dichroism (CD) spectrum. On the other hand, the native conformation of sweet potato beta-amylase observed through fluorescence polarization, far-UV CD spectrum and intrinsic fluorescence was not influenced by GuHCl at concentrations below 0.5 M. Therefore, Cys345 reaction caused by GuHCl was due to an alteration of the local conformation of the active site. GuHCl-induced reaction of Cys345, located in the vicinity of subsites 3 and 4, is attributed to enhanced subsite flexibility, which is responsible for substrate slipping in a single-chain attack mechanism. Due to the flexible conformation, the local region of the subsite is more susceptible to GuHCl perturbation than the molecule overall. The enzymatic activity of sweet potato beta-amylase was reversibly inhibited by GuHCl at concentrations below 0.5 M, and kinetic analysis of the enzymatic mechanism showed that GuHCl decreases the kcat value. High pressure below 400 MPa also inactivated sweet potato beta-amylase with an increase in Cys345 reactivity. These findings indicated that excessively enhanced subsite flexibility reduced the enzymatic activity of sweet potato beta-amylase.

Mutations of barley beta-amylase that improve substrate-binding affinity and thermostability.

Three allelic forms of barley beta-amylase (Sd1, Sd2H and Sd2L) exhibit different thermostability and kinetic properties. These differences critically influence the malting quality of barley varieties. To understand the molecular basis for the different properties of these three allelic forms, Sd1 and Sd2L beta-amylase cDNAs were cloned, and the effects of the amino acid substitutions between them were evaluated by site-directed mutagenesis. The results showed that an R115C mutation is responsible for the difference in kinetic properties. This substitution resulted in an additional hydrogen bond which may create a more favourable environment for substrate-binding. The different thermostabilities of the beta-amylase forms are due to two amino acid substitutions (V233A and L347S), which increased the enzyme's thermostability index T50 by 1.9 degrees C and 2.1 degrees C, respectively. The increased thermostability associated with these two mutations may be due to relief of steric strain and the interaction of the protein surface with solvent water. Although both V233A and L347S mutations increased thermostability, they affected the thermostability in different ways. The replacement of L347 by serine seems to increase the thermostability by slowing thermal unfolding of the protein during heating, while the replacement of V233 by alanine appears to cause an acceleration of the refolding after heating. Because the different beta-amylase properties determined by the three mutations (R115C, V233A and L347S) are associated with malting quality of barley variety, a mutant with high thermostability and substrate-binding affinity was generated by combining the three preferred amino acid residues C115, A233 and S347 together. A possible approach to producing barley varieties with better malting quality by genetic engineering is discussed.

Inhibition of beta-amylase activity, starch degradation and sucrose formation by indole-3-acetic acid during banana ripening.

In order to observe the effect of indole-3-acetic acid (IAA) on carbohydrate metabolism, unripe banana (Musa acuminata AAA, cv. Nanicão) slices were infiltrated with the hormone and left to ripen under controlled conditions. The climacteric respiration burst was reduced by the action of IAA, and starch degradation and sucrose formation were delayed. Sucrose synthase (SuSy; EC 2.4.1.13) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) activities and transcript levels were not affected, indicating that prevention of sucrose accumulation was not related to sucrose-metabolizing enzymes. Impairment of sucrose synthesis could be a consequence of lack of substrate, since starch degradation was inhibited. The increase in activity and transcript level of beta-amylase was delayed, indicating that this enzyme could be important in starch-to-sucrose metabolism in bananas and that it might be, at least partially, controlled at the transcriptional level. This is the first report showing that IAA can delay starch degradation, possibly affecting the activity of hydrolytic enzymes such as beta-amylase (EC 3.2.1.2).

Removal of the four C-terminal glycine-rich repeats enhances the thermostability and substrate binding affinity of barley beta-amylase.

Barley beta-amylase undergoes proteolytic cleavage in the C-terminal region after germination. The implication of the cleavage in the enzyme's characteristics is unclear. With purified native beta-amylases from both mature barley grain and germinated barley, we found that the beta-amylase from germinated barley had significantly higher thermostability and substrate binding affinity for starch than that from mature barley grain. To better understand the effect of the proteolytic cleavage on the enzyme's thermostability and substrate binding affinity for starch, recombinant barley beta-amylases with specific deletions at the C-terminal tail were generated. The complete deletion of the four C-terminal glycine-rich repeats significantly increased the enzyme's thermostability, but an incomplete deletion with one repeat remaining did not change the thermostability. Although different C-terminal deletions affect the thermostability differently, they all increased the enzyme's affinity for starch. The possible reasons for the increased thermostability and substrate binding affinity, due to the removal of the four C-terminal glycine-rich repeats, are discussed in terms of the three-dimensional structure of beta-amylase.

Anatomy of a conformational transition of beta-strand 6 in soybean beta-amylase caused by substrate (or inhibitor) binding to the catalytical site.

A computational study of the five soybean beta-amylase X-ray structure reported so far revealed a peculiar conformational transition after substrate (or inhibitor) binding, which affects a segment of the beta-strand 6 (residues 341-343) in the (beta/alpha)8 molecular scaffold. Backbone distortions that involve considerable changes in the phi and psi angles were observed, as well as two sharp rotamer transitions for the Thr342 and Cys343 side chains. These changes caused the outermost CA-layer (at the C-terminal side of the barrel), which is involved in the catalysis, to shrink. Our observations strongly suggest that the 341FTC343 residue conformations in the free enzyme are not optimal for protein stability. Furthermore, as a result of conformational transitions in the ligand-binding process, there is a negative enthalpy change for these residues (-27 and -34 kcal/mol, after substrate or inhibitor binding, respectively). These findings support the proposed "stability-function" hypothesis for proteins that recognize a ligand (Shoichet BK, Baase WA, Kuroki R, Matthews BW. 1995. A relationship between protein stability and protein function. Proc Natl Acad Sci USA 92:452-456). They are also in good agreement with other experimental results in the literature that describe the role of the 341-343 segment in beta-amylase activity. Site-directed mutagenesis focused on these residues could be useful for undertaking functional studies of beta-amylase.

Construction of a plasmid used for the expression of a sevenfold-mutant barley beta-amylase with increased thermostability in Escherichia coli and properties of the sevenfold-mutant beta-amylase.

To increase the thermostability of beta-amylase, seven kinds of single-mutant plasmids were constructed with an expression vector of barley beta-amylase by mutagenesis. The remaining activity versus temperature curves were used to determine the temperatures (T50) at which 50% of the initial activity was lost during a 30-min heating period. These mutations increased the T50 values by amounts ranging from 0.8 to 3.2 degrees C. To express the sevenfold-mutant beta-amylase in Escherichia coli, plasmid pB927 was constructed. E. coli harboring plasmid pB927 produced sevenfold-mutant beta- amylase. The T50 value of purified sevenfold-mutant beta-amylase (69.0 degrees C) was higher than that of not only the original recombinant beta-amylase (57.4 degrees C) by 11.6 degrees C but also soybean beta-amylase (63.2 degrees C) by 5.8 degrees C. The intragenic amino acid replacements were found to have simple additive effects on the thermostability of beta-amylase. The sevenfold-mutant beta-amylase was found to be stable at pHs up to 12.5, while the original recombinant beta-amylase was unstable at pHs above 9.5. The data obtained from kinetics studies suggested that the sevenfold-mutant beta-amylase acquired enhanced thermostability, but its function as a beta-amylase remained unchanged.

Sweet potato beta-amylase. Primary structure and identification of the active-site glutamyl residue.

The complete amino acid sequence of a subunit of sweet potato beta-amylase, a homotetramer, was established by sequence analysis of peptides obtained by digestions with Achromobacter protease I and Staphylococcus aureus V8 protease and by cyanogen bromide cleavage of the S-carboxymethylated subunit. The subunit of the enzyme is a single polypeptide consisting of 498 amino acid residues. It showed 50-60% identity in the amino acid sequence with those of beta-amylases from soybean and barley, while it about 25% with those of three bacterial beta-amylases deduced from the cDNA sequences. Sweet potato beta-amylase was completely inactivated with 2,3-epoxypropyl alpha-D-[U-14C]glucopyranoside. Sequence analysis of the inactivated enzyme revealed that Glu187 was specifically esterified by the affinity labeling with the above reagent, proposing that Glu187 is a potent candidate involved directly in the catalysis with this plant beta-amylase.