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.

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.

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.