In silico toxicity evaluation for transformation products of antimicrobials, from aqueous photolysis degradation.

This study investigates degradation processes of three antimicrobials in water (norfloxacin, ciprofloxacin, and sulfamethoxazole) by photolysis, focusing on the prediction of toxicity endpoints via in silico quantitative structure-activity relationship (QSAR) of their transformation products (TPs). Photolysis experiments were conducted in distilled water with individual solutions at 10 mg L-1 for each compound. Identification of TPs was performed by means of LC-TOF-MS, employing a method based on retention time, exact mass fragmentation pattern, and peak intensity. Ten main compounds were identified for sulfamethoxazole, fifteen for ciprofloxacin, and fifteen for norfloxacin. Out of 40 identified TPs, 6 have not been reported in the literature. Based on new data found in this work, and TPs already reported in the literature, we have proposed degradation pathways for all three antimicrobials, providing reasoning for the identified TPs. QSAR risk assessment was carried out for 74 structures of possible isomers. QSAR predictions showed that all 19 possible structures of sulfamethoxazole TPs are non-mutagenic, whereas 16 are toxicant, 18 carcinogenic, and 14 non-readily biodegradable. For ciprofloxacin, 28 out of the 30 possible structures for the TPs are mutagenic and non-readily biodegradable, and all structures are toxicant and carcinogenic. All 25 possible norfloxacin TPs were predicted mutagenic, toxicant, carcinogenic, and non-readily biodegradable. Results obtained from in silico QSAR models evince the need of performing risk assessment for TPs as well as for the parent antimicrobial. An expert analysis of QSAR predictions using different models and degradation pathways is imperative, for a large variety of structures was found for the TPs.

Identification of transformation products of rosuvastatin in water during ZnO photocatalytic degradation through the use of associated LC-QTOF-MS to computational chemistry.

Rosuvastatin (RST), a synthetic statin, is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, with a number of pleiotropic properties, such as anti-inflammation, antioxidation and cardiac remodelling attenuation. According to IMS Health, rosuvastatin was the third best-selling drug in the United States in 2012. RST was recently found in European effluent samples at a detection frequency of 36%. In this study, we evaluate the identification process of major transformation products (TPs) of RST generated during the heterogeneous photocatalysis process with ZnO. The degradation of the parent molecule and the identification of the main TPs were studied in demineralised water. The TPs were monitored and identified by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS). Ten TPs were tentatively identified and some of them originated from the hydroxylation suffered by the aromatic ring during the initial stages of the process. Structural elucidation of some of the most abundant or persistent TPs was evaluated by computational analysis, which demonstrated that this approach can be used as a tool to help the elucidation of structures of unknown molecules. The analysis of the parameters obtained from ab initio calculations for different isomers showed the most stable structures and, consequently, the most likely to be found.

Photocatalytic degradation of rosuvastatin: analytical studies and toxicity evaluations.

Photocatalytic degradation of rosuvastatin, which is a drug that has been used to reduce blood cholesterol levels, was studied in this work employing ZnO as catalyst. The experiments were carried out in a temperature-controlled batch reactor that was irradiated with UV light. Preliminary the effects of the photocatalyst loading, the initial pH and the initial rosuvastatin concentration were evaluated. The experimental results showed that rosuvastatin degradation is primarily a photocatalytic process, with pseudo-first order kinetics. The byproducts that were generated during the oxidative process were identified using nano-ultra performance liquid chromatography tandem mass spectrometry (nano-UPLC-MS/MS) and acute toxicity tests using Daphnia magna were done to evaluate the toxicity of the untreated rosuvastatin solution and the reactor effluent.

Hydrolysates of sheep cheese whey as a source of bioactive peptides with antioxidant and angiotensin-converting enzyme inhibitory activities.

Enzymatic proteolysis may be employed to release bioactive peptides, which have been investigated for potential benefits from both technological and human health perspectives. In this study, sheep cheese whey (SCW) was hydrolyzed with a protease preparation from Bacillus sp. P7, and the hydrolysates were evaluated for antioxidant and angiotensin I-converting enzyme (ACE)-inhibitory activities. Soluble protein and free amino acids increased during hydrolysis of SCW for up to 4h. Antioxidant activity of hydrolysates, evaluated by the 2,2'azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid radical scavenging method, increased 3.2-fold from 0 h (15.9%) to 6h of hydrolysis (51.3%). Maximum Fe(2+) chelation was reached in 3h hydrolysates, and the reducing power peaked at 1h of hydrolysis, representing 6.2 and 2.1-fold increase, respectively, when compared to that of non-hydrolyzed SCW. ACE inhibition by SCW (12%) was improved through hydrolysis, reaching maximal values (55% inhibition) in 4h, although 42% inhibition was already observed after 1h hydrolysis. The peptide LAFNPTQLEGQCHV, derived from ß-lactoglobulin, was identified from 4-h hydrolysates. Such a biotechnological approach might be an interesting strategy for SCW processing, potentially contributing to the management and valorization of this abundant dairy byproduct.

Enhanced Synthesis of Antioxidant Enzymes, Defense Proteins and Leghemoglobin in Rhizobium-Free Cowpea Roots after Challenging with Meloydogine incognita.

The root knot nematodes (RKN), Meloydogine spp., particularly Meloidogyne incognita and Meloidogyne javanica species, parasitize several plant species and are responsible for large annual yield losses all over the world. Only a few available chemical nematicides are still authorized for RKN control owing to environmental and health reasons. Thus, plant resistance is currently considered the method of choice for controlling RKN, and research performed on the molecular interactions between plants and nematodes to identify genes of interest is of paramount importance. The present work aimed to identify the differential accumulation of root proteins of a resistant cowpea genotype (CE-31) inoculated with M. incognita (Race 3) in comparison with mock-inoculated control, using 2D electrophoresis assay, mass spectrometry identification and gene expression analyses by RT-PCR. The results showed that at least 22 proteins were differentially represented in response to RKN challenge of cowpea roots mainly within 4-6 days after inoculation. Amongst the up-represented proteins were SOD, APX, PR-1, ß-1,3-glucanase, chitinases, cysteine protease, secondary metabolism enzymes, key enzymes involved in ethylene biosynthesis, proteins involved in MAPK pathway signaling and, surprisingly, leghemoglobin in non-rhizobium-bacterized cowpea. These findings show that an important rearrangement in the resistant cowpea root proteome occurred following challenge with M. incognita.

Pigment production by filamentous fungi on agro-industrial byproducts: an eco-friendly alternative.

The search for new sources of natural pigments has increased, mainly because of the toxic effects caused by synthetic dyes used in food, pharmaceutical, textile, and cosmetic industries. Fungi provide a readily available alternative source of natural pigments. In this context, the fungi Penicillium chrysogenum IFL1 and IFL2, Fusarium graminearum IFL3, Monascus purpureus NRRL 1992, and Penicillium vasconiae IFL4 were selected as pigments producers. The fungal identification was performed using ITS and part of the ß-tubulin gene sequencing. Almost all fungi were able to grow and produce water-soluble pigments on agro-industrial residues, with the exception of P. vasconiae that produced pigments only on potato dextrose broth. The production of yellow pigments was predominant and the two strains of P. chrysogenum were the largest producers. In addition, the production of pigments and mycotoxins were evaluated in potato dextrose agar using TOF-MS and TOF-MS/MS. Metabolites as roquefortine C, chrysogine were found in both extracts of P. chrysogenum, as well fusarenone X, diacetoxyscirpenol, and neosolaniol in F. graminearum extract. In the M. purpureus extract, the pigments monascorubrin, rubropunctatin, and the mycotoxin citrinin were found. The crude filtrates have potential to be used in the textile industry; nevertheless, additional pigment purification is required for food and pharmaceutical applications.

The presence of sboA and spaS genes and antimicrobial peptides subtilosin A and subtilin among Bacillus strains of the Amazon basin.

This report demonstrates the usefulness of PCR for the genes spaS and sboA as a means of identifying Bacillus strains with a potential to produce subtilin and subtilosin A. One collection strain and five Bacillus spp. isolated from aquatic environments in the Amazon basin were screened by PCR using primers for sboA and spaS designed specifically for this study. The sequences of the PCR products showed elevated homology with previously described spaS and sboA genes. Antimicrobial peptides were isolated from culture supernatants and analyzed by mass spectrometry. For all samples, the mass spectra revealed clusters with peaks at m/z 3300-3500 Da, corresponding to subtilosin A, subtilin and isoforms of these peptides. These results suggest that the antimicrobial activity of these strains may be associated with the production of subtilosin A and/or subtilin. The PCR used here was efficient in identifying novel Bacillus strains with the essential genes for producing subtilosin A and subtilin.

Determination of new immunoadjuvant saponin named QB-90, and analysis of its organ-specific distribution in Quillaja brasiliensis by HPLC.

An LC method was developed and validated in order to quantify the saponin purified fraction named QB-90 obtained from the aqueous extracts of Quillaja brasiliensis leaves. Previously, QB-90 was shown to be active as adjuvant in an experimental vaccine for herpesvirus bovine type 1 in mice. The analysis was performed using an RP-8 column with acetonitrile:water isocratic elution at 214 nm. The standard curve for QB-90 was linear over the range of 0.8-10 µg mL(-1) (r = 0.9996). The aqueous extract showed linear response in the range of 50-1000 µg mL(-1) (r = 0.9996). The proposed method showed adequate repeatability and intermediary precision. The limits of detection (LOD) and quantification (LOQ) were 0.074 and 0.248 µg mL(-1), respectively. This method was applied successfully to analyse QB-90 organ-specific distribution in field stands of Q. brasiliensis and in laboratory-grown seedlings. Leaves from young plants accumulated higher QB-90 amounts than leaves from adult trees.

Antithrombotic effect of chikusetsusaponin IVa isolated from Ilex paraguariensis (Maté).

The triterpene chikusetsusaponin IVa was isolated from the fruit of Ilex paraguariensis. Using biochemical and pharmacological methods, we demonstrated that chikusetsusaponin IVa (1) prolongs the recalcification time, prothrombin time, activated partial thromboplastin time, and thrombin time of normal human plasma in a dose-dependent manner, (2) inhibits the amidolytic activity of thrombin and factor Xa upon synthetic substrates S2238 and S2222, (3) inhibits thrombin-induced fibrinogen clotting (50% inhibition concentration, 199.4 ± 9.1 µM), and (4) inhibits thrombin- and collagen-induced platelet aggregation. The results also indicate that chikusetsusaponin IVa preferentially inhibits thrombin in a competitive manner (K(i)=219.6 µM). Furthermore, when administered intravenously to rats, chikusetsusaponin IVa inhibited thrombus formation in a stasis model of venous thrombosis, although it did not induce a significant bleeding effect. Chikusetsusaponin IVa also prolonged the ex vivo activated partial thromboplastin time. Altogether, these data suggest that chikusetsusaponin IVa exerts antithrombotic effects, including minor hemorrhagic events. This appears to be important for the development of new therapeutic agents.

Immobilization of keratinolytic metalloprotease from Chryseobacterium sp. strain kr6 on glutaraldehyde-activated chitosan.

Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support (qm) and dissociation constant (Kd) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at 65 degrees C. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.

Antimicrobial factor from Bacillus amyloliquefaciens inhibits Paenibacillus larvae, the causative agent of American foulbrood.

Bacillus amyloliquefaciens LBM 5006 produces an antimicrobial factor active against Paenibacillus larvae, a major honeybee pathogen. The antagonistic effect and the mode of action of the antimicrobial factor were investigated. The antibacterial activity was produced starting at mid-logarithmic growth phase, reaching its maximum during the stationary phase. Exposure of cell suspensions of P. larvae to this antimicrobial resulted in loss of cell viability and reduction in optical density associated with cell lysis. Scanning electron microscopy showed damaged cell envelope and loss of protoplasmic material. The antimicrobial factor was stable for up to 80°C, but it was sensitive to proteinase K and trypsin. Mass spectrometry analysis indicates that the antimicrobial activity is associated with iturin-like peptides. The antimicrobial factor from B. amyloliquefaciens LBM 5006 showed a bactericidal effect against P. larvae cells and spores. This is the first report on iturin activity against P. larvae. This antimicrobial presents potential for use in the control of American foulbrood disease.

Antioxidant, antihypertensive and antimicrobial properties of ovine milk caseinate hydrolyzed with a microbial protease.

BACKGROUND: Bioactive peptides might be released from precursor proteins through enzymatic hydrolysis. These molecules could be potentially employed in health and food products. In this investigation, ovine milk caseinate hydrolysates obtained with a novel microbial protease derived from Bacillus sp. P7 were evaluated for antioxidant, antimicrobial, and angiotensin I-converting enzyme (ACE)-inhibitory activities. RESULTS: Antioxidant activity measured by the 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid method increased with hydrolysis time up to 2 h, remaining stable for up to 4 h. Hydrolysates showed low 2,2-diphenyl-1-picrylhydrazyl radical-scavenging abilities, with higher activity (31%) reached after 1 h of hydrolysis. Fe(2+) -chelating ability was maximum for 0.5 h hydrolysates (83.3%), decreasing thereafter; and the higher reducing power was observed after 1 h of hydrolysis. ACE-inhibitory activity was observed to increase up to 2 h of hydrolysis (94% of inhibition), declining afterwards. 3 h hydrolysates were shown to inhibit the growth of Bacillus cereus, Corynebacterium fimi, Aspergillus fumigatus, and Penicillium expansum. CONCLUSION: Ovine caseinate hydrolyzed with Bacillus sp. P7 protease presented antioxidant, antihypertensive, and antimicrobial activities. Hydrolysis time was observed to affect the evaluated bioactivities. Such hydrolysates might have potential applications in the food industry.