Development of a micelle‐enhanced high‐throughput fluorometric method for determination of niclosamide using a microplate reader

The present paper describes the development and validation of a simple and sensitive micelle‐enhanced high‐throughput fluorometric method for the determination of niclosamide (NIC) in 96‐microwell plates. The proposed method is based on the reduction of the nitro group of niclosamide to an amino group using Zn/HCl to give a highly fluorescent derivative that was developed simultaneously and measured at λ_em 444 nm after excitation at λ_ex 275 nm. Tween‐80 and carboxymethylcellulose (CMC) have been used as fluorescence enhancers and greatly enhanced the fluorescence by factors of 100–150%. The different experimental conditions affecting the fluorescence reaction were carefully investigated and optimized. The proposed method showed good linearity (r²≥ 0.9997) over the concentration ranges of 1–5 and 0.5–5 μg/ml with lower detection limits of 0.01 and 0.008 μg/ml and lower quantification limits of 0.04 and 0.03 μg/ml on using Tween‐80 and or CMC, respectively. The developed high‐throughput method was successfully applied for the determination of niclosamide in both tablets and spiked plasma. The capability of the method for measuring microvolume samples made it convenient for handling a very large number of samples simultaneously. In addition, it is considered an environmentally friendly method with lower consumption of chemicals and solvents.     

Utilization of N,S-doped carbon dots as a fluorescent nanosensor for determination of cromolyn based on inner filter effect: application to aqueous humour

A simple and eco-friendly hydrothermal technique is used to prepare water soluble N- and S-co-doped carbon quantum dots probes (N,S-CQDs) from thiosemicarbazide and citric acid. Several characterization techniques were performed to ensure the successful synthesis of highly luminescent N,S-CQDs. The prepared probe exhibited analytical potential as an optical nanosensor for the spectrofluorimetric determination of cromolyn sodium (CRO) in its pharmaceutical dosage forms and aqueous humour. The emission intensity of the synthesized N,S-CQDs was measured at 411 nm after excitation at 345 nm. Addition of increasing concentrations of CRO to N,S-CQDs led to quenching of its fluorescence intensity. CRO was investigated within a wide concentration range 10.0–150.0 μM with a limit of detection of 2.0 μM and a limit of quantification of 6.0 μM. The quenching of fluorescent N,S-CQDs occurred through the inner filter effect (IFE). The developed spectrofluorimetric method was successfully optimized and validated according to the International Council of Harmonisation guidelines (ICH). The method greenness is proved through using both Eco-Scale and AGREE approaches.     

Breast-Feeding Protects Infantile Diarrhea Caused by Intestinal Protozoan Infections

This study investigated the effect of breast-feeding in protection against protozoan infection in infants with persistent diarrhea. Infants were classified into 2 groups; 161 breast-fed infants and the same number of non-breast-fed infants. Microscopic examinations of stool were done for detection of parasites and measuring the intensity of infection. Moreover, serum levels of IgE and TNF-α were measured by ELISA. Cryptosporidium spp., Entamoeba histolytica/Entamoeba dispar, Giardia lamblia, and Blastocystis sp. were demonstrated in infants with persistent diarrhea. The percentage of protozoan infections was significantly lower in breast-fed infants than that in the non-breast-fed infants. The levels of IgE and TNF-α were significantly lower in the breast-fed group than in the non-breast-fed group. There were significant positive associations between the serum levels of IgE and TNF-α and the intensity of parasite infection in the breast-fed group. It is suggested that breast-feeding has an attenuating effect on the rate and intensity of parasite infection.     

Micelle‐enhanced spectrofluorimetric method for determination of sitagliptin and identification of potential alkaline degradation products using LC‐MS

A novel, quick, simple and highly sensitive spectrofluorimetric method was developed and validated for the determination of sitagliptin (SG) in its pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behavior of sitagliptin in an SDS micellar system. In an aqueous solution of phosphate buffer pH 4.0, the fluorescence intensity of SG in the presence of SDS was greatly enhanced, by 200%, i.e. twofold enhancement. The fluorescence intensity of SG was measured at 300 nm after excitation at 270 nm. The method showed good linearity in the range 0.03–10.0 µg/mL with a good correlation coefficient (r = 0.9998). The limits of detection and quantitation values were 5.31 and 16.1 ng/mL, respectively. The proposed method was successfully applied to the analysis of SG in its single and co‐formulated commercial tablets; the results were in good agreement with those obtained using a reference method. Application of the proposed method was extended to stability studies of SG after exposure to different forced degradation conditions according to the ICH guidelines, such as acidic, alkaline, thermal, photo‐ and oxidative stress. The chemical structure of certain potential degradation products (DPs) were investigated using LC‐MS. Copyright © 2013 John Wiley & Sons, Ltd.     

Pre‐treatment with the synthetic antioxidant T‐butyl bisphenol protects cerebral tissues from experimental ischemia reperfusion injury

Treatments to inhibit or repair neuronal cell damage sustained during focal ischemia/reperfusion injury in stroke are largely unavailable. We demonstrate that dietary supplementation with the antioxidant di‐tert‐butyl‐bisphenol (BP) before injury decreases infarction and vascular complications in experimental stroke in an animal model. We confirm that BP, a synthetic polyphenol with superior radical‐scavenging activity than vitamin E, crosses the blood–brain barrier and accumulates in rat brain. Supplementation with BP did not affect blood pressure or endogenous vitamin E levels in plasma or cerebral tissue. Pre‐treatment with BP significantly lowered lipid, protein and thiol oxidation and decreased infarct size in animals subjected to middle cerebral artery occlusion (2 h) and reperfusion (24 h) injury. This neuroprotective action was accompanied by down‐regulation of hypoxia inducible factor‐1α and glucose transporter‐1 mRNA levels, maintenance of neuronal tissue ATP concentration and inhibition of pro‐apoptotic factors that together enhanced cerebral tissue viability after injury. That pre‐treatment with BP ameliorates oxidative damage and preserves cerebral tissue during focal ischemic insult indicates that oxidative stress plays at least some causal role in promoting tissue damage in experimental stroke. The data strongly suggest that inhibition of oxidative stress through BP scavenging free radicals in vivo contributes significantly to neuroprotection.

     

In vitro antifungal activity of lactic acid bacteria low molecular peptides against spoilage fungi of bakery products

Bio-preservation, a promising preservation method that involves the use of “friendly” microorganisms such as lactic acid bacteria, has recently become a topic of considerable interest. In the present study, 16 lactic acid bacteria isolates were evaluated for antifungal activity against six fungi commonly associated with bread spoilage. The antifungal compounds were heat stable at 121 °C, and only four isolates, DU15, IT10, TE10, and IS10, showed partial loss of activity when supernatants were treated with proteolytic enzymes. The four isolates showed high inhibition activity at pH 3 and were identified using 16S rDNA sequencing as belonging to Leuconostoc mesenteroides DU15, Lactobacillus plantarum TE10, Lactobacillus plantarum IT10, and Lactobacillus plantarum IS10. The minimum germination inhibitions were 30 mg, 50 mg, 40 mg, and 50 mg for TE10, IT10, DU15, and IS10 respectively. The optimum conditions for the strains to produce antifungal compounds were 37 °C for 48 h for IT10, IS10, and TE10, and 30 °C for 24 h for DU15. Antifungal activity was increased threefold when supernatants were filtered using 10 KDa membranes. These findings demonstrate the potential of using lactic acid bacteria antifungal peptides as natural preservatives in bakery products to control the growth of spoilage fungi.     

Modulatory effect of linoleic and oleic acid on cell proliferation and lipid metabolism gene expressions in primary bovine satellite cells

This study was performed to elucidate the effects of linoleic acid (LA), oleic acid (OA) and their combination (LA?+?OA) on cell proliferation, apoptosis, necrosis, and the lipid metabolism related gene expression in bovine satellite cells (BSCs), isolated from bovine muscles. Cell viability was significantly increased with the OA and LA treatment. Furthermore, LA?+?OA enhanced cell proliferation in a dose-dependent manner (10 to 100?µM), whereas it lowered at 250?µM. In addition, a cell-cycle analysis showed that 100?µM of LA and OA markedly decreased the G0/G1 phase proportion (62.58% and 61.33%, respectively), compared to controls (68.02%), whereas the S-phase cells’ proportion was increased. The ratio of G2/M phase cells was not significantly different among the groups. Moreover, analyses with AO/EtBr staining showed that no apoptosis occurred. Necrosis were determined by flow cytometry using Annexin V-FITC/PI staining which revealed no early apoptosis in the cells pretreated with LA or OA, but occurred in the LA?+?OA group. We also analyzed the mRNA expression of lipid metabolizing genes such as peroxisome proliferator receptor alfa (PPARα), peroxisome proliferator receptor gamma (PPARγ), acyl-CoA oxidase (ACOX), lipoprotein lipase (LPL), carnitine palmitoyl transferase (CPT-1), and fatty-acid binding protein4 (FABP4), which were upregulated in LA or OA treated cells compared to the control group. In essence, LA and OA alone promote the cell proliferation without any apoptosis and necrosis, which might upregulate the lipid metabolism related gene expressions, and increase fatty-acid oxidation in the BSCs’ lipid metabolism.