Filipa V.M. Silva

Alicyclobacillus acidoterrestris spores in fruit products and design of pasteurization processes

Abstract Alicyclobacillus acidoterrestris is a thermoacidophilic, non-pathogenic and sporeforming bacterium which has been found in commercial pasteurized fruit juices in the past. Only few and recent studies were available in the literature, since only in 1984 Cerny et al. [Cerny, G., Hennlich, W., & Poralla, K. (1984). Fruchtsaftverderb durch bacillen: isolierung und charakterisierung des verderbserregers. Z. Lebensmitt. Unters. Forsch. 179, 224–227] reported a spoiled aseptically packaged apple juice with A. acidoterrestris and in 1987 Deinhard et al. [Deinhard, G., Blanz, P., Poralla, K., & Altan, E. (1987). Bacillus acidoterrestris sp. nov., a new thermotolerant acidophile isolated from different soils. Systematic and Applied Microbiology, 10, 47–53] named first this species. Detection and identification methods for A. acidoterrestris were reviewed and data regarding heat resistance of spores and growth in fruits were collected. Finally, a new methodology to design pasteurization processes for high acidic fruit products is presented.

Bioactivity studies and chemical profile of the antidiabetic plant Genista tenera

AIM OF THE STUDY Genista tenera is a plant endemic to the island of Madeira and is used in folk medicine to control diabetes. In the present work we evaluate the antihyperglycaemic activity of its n-butanol extract and determine its chromatographic profile. In addition, this extract, the ethyl acetate and diethyl ether plant extracts were studied in order to assess the plant antioxidant and acetylcholinesterase inhibitory activities, as well as its cyto- and genotoxicities. MATERIALS AND METHODS HPLC-DAD-ESI-MS was used to analyze the flavonoid profile of the n-butanol extract. The antihyperglycaemic activity of this extract was performed over streptozotocin induced diabetic Wistar rats (200 mg/kg, bw/day), for 15 days. Antioxidant activity (DPPH assay) and acetylcholinesterase inhibitory effect (Ellman method) were also performed. Acute cytotoxicity and genotoxicity were assessed by proliferative index quantification and the short-term chromosomal aberration technique, after exposure of lymphocytes to the extracts. RESULTS AND CONCLUSIONS The n-butanol extract, where 21 monoglycosyl and 12 diglycosyl flavonoids were detected, significantly lowered blood glucose levels, bringing them to normal values after 15 days of treatment. The best radical scavenging activity was observed for the ethyl acetate extract (48.7% at 139.1 microg/mL), which was also the most effective one at the minimal concentration tested. The highest acetylcholinesterase inhibitory activity (77.0% at 70.0 microg/mL) was also obtained with the ethyl acetate extract. In vitro toxicity studies showed no evidence for acute cytotoxicity or genotoxicity. This is the first report on antidiabetic activity of genus Genista.

Phytochemical Profile and Anticholinesterase and Antimicrobial Activities of Supercritical versus Conventional Extracts of Satureja montana

Winter savory Satureja montana is a medicinal herb used in traditional gastronomy for seasoning meats and salads. This study reports a comparison between conventional (hydrodistillation, HD, and Soxhlet extraction, SE) and alternative (supercritical fluid extraction, SFE) extraction methods to assess the best option to obtain bioactive compounds. Two different types of extracts were tested, the volatile (SFE-90 bar, second separator vs HD) and the nonvolatile fractions (SFE-250 bar, first and second separator vs SE). The inhibitory activity over acetyl- and butyrylcholinesterase by S. montana extracts was assessed as a potential indicator for the control of Alzheimers disease. The supercritical nonvolatile fractions, which showed the highest content of (+)-catechin, chlorogenic, vanillic, and protocatechuic acids, also inhibited selectively and significantly butyrylcholinesterase, whereas the nonvolatile conventional extract did not affect this enzyme. Microbial susceptibility tests revealed the great potential of S. montana volatile supercritical fluid extract for the growth control and inactivation of Bacillus subtilis and Bacillus cereus, showing some activity against Botrytis spp. and Pyricularia oryzae. Although some studies were carried out on S. montana, the phytochemical analysis together with the biological properties, namely, the anticholinesterase and antimicrobial activities of the plant nonvolatile and volatile supercritical fluid extracts, are described herein for the first time.

Synthesis of novel purine nucleosides towards a selective inhibition of human butyrylcholinesterase

The search for new and potent cholinesterase inhibitors is an ongoing quest mobilizing many organic chemistry groups around the world as these molecules have been shown to treat the late symptoms of Alzheimers disease as well as to act as neuroprotecting agents. In this work, we disclose the synthesis of novel 2-acetamidopurine nucleosides and, for the first time, regioselective N(7)-glycosylation with 2-acetamido-6-chloropurine, promoted by trimethylsilyl triflate, was accomplished by tuning the reaction conditions (acetonitrile as solvent, 65 degrees C, 5h) starting from 1-acetoxy bicyclic glycosyl donors, or by direct coupling of a methyl glucopyranoside with the nucleobase to obtain only N(7) nucleosides in reasonable yield (55-60%). The nucleosides as well as their sugar precursors were screened for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition. While none of the compounds tested inhibited AChE, remarkably, some of the N(7) nucleosides and sugar bicyclic derivatives showed potent inhibition towards BChE. Nanomolar inhibition was obtained for one compound competing well with rivastigmine, a drug currently in use for the treatment of Alzheimers disease. Experimental results showed that the presence of benzyl groups on the carbohydrate scaffold and the N(7)-linked purine nucleobase were necessary for strong BChE inactivation. A preliminary evaluation of the acute cytotoxicity of the elongated bicyclic sugar precursors and nucleosides was performed indicating low values, in the same order of magnitude as those of rivastigmine.

Alkyl deoxy-arabino-hexopyranosides: Synthesis, surface properties, and biological activities

Octyl and dodecyl glycosides possessing 2-deoxy-arabino-hexopyranoside moieties belonging to the D- and L-series in their alpha- and beta-forms were synthesized by reaction of an acetyl protected glycal with octanol or dodecanol, catalyzed by triphenylphosphine hydrobromide, followed by deprotection. Their surface properties were studied and discussed in terms of the adsorption and aggregation parameters, pC(20), CMC, and gamma(CMC). The antimicrobial activities were assessed using the paper disk diffusion and broth dilution methods. Both the octyl and dodecyl 2-deoxy beta-D-glycosides inhibited significantly Enterococcus faecalis, a microbe also highly susceptible to dodecyl 2,6-dideoxy-alpha-L-arabino-hexopyranoside. This compound was particularly active against Bacillus cereus and Bacillus subtilis, presenting for both Bacillus species a minimal inhibitory concentration of the same order of magnitude and a minimal lethal concentration even smaller than that obtained for chloramphenicol, a bioactivity which remained unaltered after 1 year solution storage at 4 degrees C. In addition, activity over Listeria monocytogenes was also observed. Direct cytotoxicity and genotoxicity of the glycosides were determined by proliferative index (mitotic index) evaluation in peripheral human lymphocytes of healthy donors. All compounds induced acute toxicity effects, and the response was dose dependent for the alpha-anomer of both the alkyl 2-deoxy-arabino-hexopyranosides and for the corresponding dodecyl beta-anomer, what suggests that non-toxic but still bioactive concentrations may be found for these compounds.

Facile synthesis of oxo-/thioxopyrimidines and tetrazoles C–C linked to sugars as novel non-toxic antioxidant acetylcholinesterase inhibitors

Microwave-assisted synthesis of oxo-/thioxopyrimidines and tetrazoles linked to furanoses with D-xylo and D-ribo configuration, and to a D-galacto pyranose is reported and compared to conventional methods. Reaction of dialdofuranoses and dialdopyranoses with a β-keto ester and urea or thiourea under microwave irradiation at 300 W gave in 10 min the target molecules containing the 2-oxo- or 2-thioxo-pyrimidine ring in high yield. The tetrazole-derived compounds were obtained in two steps by reaction of the formyl group with hydroxylamine hydrochloride, copper sulfate, triethylamine and dicyclohexylcarbodiimide to give an intermediate nitrile, which was then treated with sodium azide. The use of microwave irradiation in the latter step also resulted in a considerably shorter reaction time (10 min) compared to hours under conventional heating to obtain a complete starting materials conversion. Acetylcholinesterase inhibition ranged from 20% to 80% for compounds concentration of 100 μg/mL, demonstrating the potential of this family of compounds for the control of Alzheimers disease symptoms. Most of the compounds showed antioxidant activity in the β-carotene/linoleic acid assay, some of them exhibiting IC(50) values in the same order of magnitude as those of gallic acid. The bioactive compounds did not show cytotoxic effects to human lymphocytes using the MTT method adapted for non-adherent cells, nor genotoxicity determined by the short-term in vitro chromosomal aberration assay.

Synthesis and Antimicrobial Evaluation of Oxazole-2(3H)-thione and 2-Alkylsulfanyl-1,3-oxazole Derivatives

The preparation of oxazole-2(3H)-thiones (OXTs) by condensation of thiocyanic acid on α-hydroxycarbonyl substrates has been revisited. Extension to more complex scaffolds afforded chiral OXTs, whereas carbohydrate-fused 2-alkylsulfanyl-1,3-oxazolines led to original hemiaminal structures. A survey of the reactivity of OXTs with various electrophiles showed Sor N-chemoselectivity based on HSAB parameters. Antimicrobial evaluation of selected synthesized compounds was carried out, from which the hemiaminal 15 emerged as a promising antifungal agent. INTRODUCTION Treatment of infectious diseases still represents a topical challenge. The emergent infections as well as the increasing number of multi-drug resistant microbial pathogens specially Gram-positive bacteria have been a serious concern both in hospital and community settings. Linezolid (1) (ZyvoxTM) (Figure 1) is a member of the oxazolidinone drugs active against most Gram-positive pathogens, namely Staphylococcus aureus (MRSA) and Staphylococcus epidermis (MRSE). Although it has been approved HETEROCYCLES, Vol. 88, No. 2, 2014 1013