Dosing-time, feeding, and sex-dependent variations of everolimus pharmacokinetics in mice.

BACKGROUND: Everolimus is an oral mammalian target of rapamycin (mTOR) inhibitor used as an immunosuppressant and anticancer. Its pharmacokinetics is highly variable, it has a narrow therapeutic window and shows chronotoxicity with the best time at ZT13 and worst time at ZT1 (ZT; Zeitgeber time, time after light onset) in the preclinical setting. OBJECTIVES: In the present study, we aimed to investigate whether the pharmacokinetics of everolimus vary according to dosing time and whether sex and feeding status interfere with the chronopharmacokinetics. METHOD: A single dosage of 5 mg/kg everolimus was administered orally to C57BL/6J male and female mice, in fed or fasted states at ZT1-rest and ZT13-activity times and blood and tissue samples were collected at 0.5, 1, 2, 4, 12, and 24 h following drug administration. Ileum, liver, plasma, and thymus concentrations of everolimus were determined. RESULTS: Females had a greater ileum AUC0-24h than males when fed (P = 0.043). Everolimus AUC0-24h in the liver was substantially greater at ZT1 than at ZT13 in a fasted state (P = 0.001). Plasma Cmax , AUC0-24h , and AUCtotal were not statistically significant between the groups (P = 0.098). In one of the target organs of everolimus, the thymus, males had considerably higher amounts at ZT1 than females (P = 0.029). CONCLUSION: Our findings imply that the pharmacokinetics of everolimus in mice may differ according to dosing time, sex, and feeding. Greater tissue distribution of everolimus at ZT1 may be associated with the worst tolerated time of everolimus. Our research suggests that oral chronomodulated everolimus therapy may be more effective and safer for cancer patients.

LC-MS/MS Application in Pharmacotoxicological Field: Current State and New Applications.

Nowadays, it is vital to have new, complete, and rapid methods to screen and follow pharmacotoxicological and forensic cases. In this context, an important role is undoubtedly played by liquid chromatography-tandem mass spectrometry (LC-MS/MS) thanks to its advanced features. This instrument configuration can offer comprehensive and complete analysis and is a very potent analytical tool in the hands of analysts for the correct identification and quantification of analytes. The present review paper discusses the applications of LC-MS/MS in pharmacotoxicological cases because it is impossible to ignore the importance of this powerful instrument for the rapid development of pharmacological and forensic advanced research in recent years. On one hand, pharmacology is fundamental for drug monitoring and helping people to find the so-called "personal therapy" or "personalized therapy". On the other hand, toxicological and forensic LC-MS/MS represents the most critical instrument configuration applied to the screening and research of drugs and illicit drugs, giving critical support to law enforcement. Often the two areas are stackable, and for this reason, many methods include analytes attributable to both fields of application. In this manuscript, drugs and illicit drugs were divided in separate sections, with particular attention paid in the first section to therapeutic drug monitoring (TDM) and clinical approaches with a focus on central nervous system (CNS). The second section is focused on the methods developed in recent years for the determination of illicit drugs, often in combination with CNS drugs. All references considered herein cover the last 3 years, except for some specific and peculiar applications for which some more dated but still recent articles have been considered.

Diurnal Changes in Capecitabine Clock-Controlled Metabolism Enzymes Are Responsible for Its Pharmacokinetics in Male Mice.

The circadian timing system controls absorption, distribution, metabolism, and elimination processes of drug pharmacokinetics over a 24-h period. Exposure of target tissues to the active form of the drug and cytotoxicity display variations depending on the chronopharmacokinetics. For anticancer drugs with narrow therapeutic ranges and dose-limiting side effects, it is particularly important to know the temporal changes in pharmacokinetics. A previous study indicated that pharmacokinetic profile of capecitabine was different depending on dosing time in rat. However, it is not known how such difference is attributed with respect to diurnal rhythm. Therefore, in this study, we evaluated capecitabine-metabolizing enzymes in a diurnal rhythm-dependent manner. To this end, C57BL/6J male mice were orally treated with 500 mg/kg capecitabine at ZT1, ZT7, ZT13, or ZT19. We then determined pharmacokinetics of capecitabine and its metabolites, 5'-deoxy-5-fluorocytidine (5'DFCR), 5'-deoxy-5-fluorouridine (5'DFUR), 5-fluorouracil (5-FU), in plasma and liver. Results revealed that plasma Cmax and AUC0-6h (area under the plasma concentration-time curve from 0 to 6 h) values of capecitabine, 5'DFUR, and 5-FU were higher during the rest phase (ZT1 and ZT7) than the activity phase (ZT13 and ZT19) (p < 0.05). Similarly, Cmax and AUC0-6h values of 5'DFUR and 5-FU in liver were higher during the rest phase than activity phase (p < 0.05), while there was no significant difference in liver concentrations of capecitabine and 5'DFCR. We determined the level of the enzymes responsible for the conversion of capecitabine and its metabolites at each ZT. Results indicated the levels of carboxylesterase 1 and 2, cytidine deaminase, uridine phosphorylase 2, and dihydropyrimidine dehydrogenase (p < 0.05) are being rhythmically regulated and, in turn, attributed different pharmacokinetics profiles of capecitabine and its metabolism. This study highlights the importance of capecitabine administration time to increase the efficacy with minimum adverse effects.

Fabric phase sorptive extraction combined with high performance liquid chromatography for the determination of favipiravir in human plasma and breast milk.

A fast procedure obtained by the combination of fabric phase extraction (FPSE) with high performance liquid chromatography (HPLC) has been developed and validated for the quantification of favipiravir (FVP) in human plasma and breast milk. A sol-gel polycaprolactone-block-polydimethylsiloxane-block-polycaprolactone (sol-gel PCAP-PDMS-PCAP) coated on 100% cellose cotton fabric was selected as the most efficient membrane for FPSE in human plasma and breast milk samples. HPLC-UV analysis were performed using a RP C18 column under isocratic conditions. Under these optimezed settings, the overall chromatographic analysis time was limited to only 5 min without encountering any observable matrix interferences. Following the method validation procedure, the herein assay shows a linear calibration curve over the range of 0.2-50 µg/mL and 0.5-25 µg/mL for plasma and breast milk, respectively. The method sensitivities in terms of limit of detection (LOD) and limit of quantification (LOQ), validated in both the matrices, have been found to be 0.06 and 0.2 µg/mL for plasma and 0.15 and 0.5 µg/mL for milk, respectively. Intraday and interday precision and trueness, accordingly to the International Guidelines, were validated and were below 3.61% for both the matrices. The herein method was further tested on real samples in order to highlight the applicability and the advantage for therapeutic drug monitoring (TDM) applications. To the best of our knowledge, this is the first validated FPSE-HPLC-UV method in human plasma and breast milk for TDM purposes applied on real samples. The validated method provides fast, simple, cost reduced, and sensitive assay for the direct quantification of favipiravir in real biological matrices, also appliyng a well-known rugged and cheap instrument configuration.

Fungal biotransformation of carvone and camphor by Aspergillus flavus and investigation of cytotoxic activities of naturally obtained essential oils.

In this study, the biotransformation of carvone and camphor by Aspergillus flavus and the products were investigated. The biotransformation reaction of carvone by A. flavus resulted in the production of neodihydrocarveol, dihydrocarvone, 2-cyclohexene-1-one,2-methyl-5-(1-methylethenyl), limonene-1,2-diol, trans-p-mentha-1(7),8-dien-2-ol, p-menth-8(10)-ene-2,9-diol, and the biotransformation reaction of camphor resulted in the production of 2 -campholenic acid, 2-cyclohexene-1-one,2-hydroxy-4,4,6,6-tetramethyl, α-campholene aldehyde. The naturally produced essential oils by biotransformation of carvone and camphor were observed to be cytotoxic to breast cancer cells while no significant inhibition was seen in the healthy cell line. Additionally, biotransformation products had the highest inhibition (74%) against aflatoxin B1. The bioactivities of biotransformation products are promising, and they can be further investigated for their therapeutic potential as active agents.

Remineralization potential of P11-4 and fluoride on secondary carious primary enamel: A quantitative evaluation using microcomputed tomography.

The aim of this study was to assess the ability of self-assembling peptide (P11-4) diffusion, assembly, and remineralization to effect artificial secondary caries-like lesions in human primary teeth in vitro. Enamel-dentin blocks obtained from extracted human primary molars were embedded into epoxy resin blocks. Cavities (approximately 1 × 1 × 2 mm) were prepared on the surface using a high-speed diamond bur under constant water cooling and filled with composite restorative material (Filtek Z250; 3 M ESPE). The samples were immersed in demineralizing solution (20 ml) for 96 h to produce secondary caries lesions and divided into two groups according to the testing materials: fluoride varnish (Duraphat; Colgate, UK) and P11-4 (Curodont Repair; Credentis, Switzerland). Except for the control areas, all samples were remineralized for 3-5 min using the remineralizing agents, and then all the sections were placed in a pH-cycling system for 5 days at 35°C. The pH cycling procedure was followed by micro-CT analysis for the qualitative evaluation of surface changes. The Mann-Whitney U test was used to compare two independent groups. In the comparison of more than two dependent groups, Bonferroni smoothed pairwise analyses were used to determine the source of the Kruskal-Wallis H test difference. The results of the study revealed that the remineralization depths of the peptide group were higher than those of the fluoride group (p < .01). There was a statistically significant difference in remineralization effects between the fluoride and peptide groups. P11-4 can be considered as an effective remineralizing agent for secondary caries lesions.

Simultaneous determination of febuxostat and montelukast in human plasma using fabric phase sorptive extraction and high performance liquid chromatography-fluorimetric detection.

In the present work, a new sensitive and selective high-performance liquid chromatography-fluorimetric detection (HPLC-FLD) method was developed and validated to quantify febuxostat (FBX) and montelukast (MON) in human plasma. The developed procedure was successfully applied to a study aimed at evaluating the pharmacokinetic profiles of febuxostat and montelukast in human plasma. A sol-gel poly (caprolactone)-block-poly(dimethylsiloxane)-block-poly(caprolactone) (sol-gel PCAP-PDMS-PCAP) extraction sorbent coated fabric phase sorptive extraction membrane was used in the extraction process. The entire chromatographic analysis was performed with isocratic elution of the composition of the mobile phase (acetonitrile:water, 60:40, v:v, 0.032% glacial acetic acid) on the C18 column. The flow rate is varied during the analysis, particularly from 0.5 mL min-1 at the start and linearly increased to 1.5 mL min-1 in 7 min. The detection and quantification of the analytes was carried out by means of a fluorimetric detector at 320 nm and 350 nm as absorption wavelengths and at 380 and 400 nm as emission wavelengths for FBX and MON, respectively. The calibration curves demonstrated linearity in the range 0.3-10 ng mL-1 and 5-100 ng mL-1 for FBX and MON, respectively, while the LOD and LOQ values were 0.1 and 0.3 ng mL-1 for FBX and 1.5 and 5 ng mL-1 for MON. Intraday and interday RSD% values were found lower than 5.79%. As reported, the method was applied to real plasma samples obtained from a volunteer who was co-administered both the drugs. Pharmacokinetic data reveal that the concentration of both the drugs reaches the plateau approximately at the same time, but exhibits an elimination phase at different rates. This study demonstrated the usefulness of the new method and its applicability in therapeutic drug monitoring (TDM).

Development of sol-gel phenyl/methyl/poly (dimethylsiloxane) sorbent coating for fabric phase sorptive extraction and its application in monitoring human exposure to selected polycyclic aromatic hydrocarbons using high performance liquid chromatography-fluorescence detection.

Following the convenient, yet very powerful pathway to create designer extraction sorbent using sol-gel chemistry, a novel sol-gel phenyl/methyl/poly(dimethylsiloxane) sorbent coating was created on polyester fabric substrate for fabric phase sorptive extraction (FPSE) and was subsequently applied to monitor human exposure to selected polycyclic aromatic hydrocarbons (PAHs) including pyrene, chrysene, and benzo[a]pyrene in plasma samples obtained from tobacco smoker volunteers using high performance liquid chromatography-fluorescence detector (HPLC-FLD). A rapid FPSE-HPLC-FLD method was developed that adequately resolved the PAHs chromatographically, after their successful extraction from human plasma using fabric phase absorption extraction (FPSE) and subsequently analysed in the liquid chromatographic system by means of an analytical column (InterSustain C-18 column 150 × 4.6 mm, 5 µm) using acetonitrile (ACN) and water as mobile phases in gradient elution mode. With the optimized conditions, the retention times were found to be 6.168, 7.214, and 10.404 min for pyrene, chrysene, and benzo[a]pyrene, respectively. The total chromatographic runtime was limited to 12.5 min. The method, validated through the calculation of all the analytical parameters according to the International Guidelines, was applied to the analysis of real samples collected from informed volunteers. The proposed approach which included the use of sol-gel phenyl/methyl/poly(dimethylsiloxane) immobilized on hydrophobic polyester substrate and C18 stationary phase used in HPLC, has shown a high potential as a rapid tool for future clinical, forensic and toxicological applications, also in the light of the LOD and LOQ values comparable to those normally obtainable with more sophisticated, and expensive instruments that often require highly trained personnel. The results reported here further consolidate the application of FPSE in the analysis of biological samples for both diagnostic and analytical-clinical purposes.

Triterpenoids and steroids isolated from Anatolian Capparis ovata and their activity on the expression of inflammatory cytokines.

CONTEXT: Capparis L. (Capparaceae) is grown worldwide. Caper has been used in traditional medicine to treat various diseases including rheumatism, kidney, liver, stomach, as well as headache and toothache. OBJECTIVE: To isolate and elucidate of the secondary metabolites of the C. ovata extracts which are responsible for their anti-inflammatory activities. MATERIALS AND METHODS: Buds, fruits, flowers, leaves and stems of C. ovata Desf. was dried, cut to pieces, then ground separately. From their dichloromethane/hexane (1:1) extracts, eight compounds were isolated and their structures were elucidated by NMR, mass spectroscopic techniques. The effects of compounds on the expression of inflammatory cytokines in SH-SY5Y cell lines were examined by qRT-PCR ranging from 4 to 96 µM. Cell viability was expressed as a percentage of the control, untreated cells. RESULTS: This is a first report on isolation of triterpenoids and steroids from C. ovata with anti-inflammatory activity. One new triterpenoid ester olean-12-en-3ß,28-diol, 3ß-pentacosanoate (1) and two new natural steroids 5α,6α-epoxycholestan-3ß-ol (5) and 5ß,6ß-epoxycholestan-3ß-ol (6) were elucidated besides known compounds; oleanolic acid (2), ursolic acid (3), ß-sitosterol (4), stigmast-5,22-dien-3ß-myristate (7) and bismethyl-octylphthalate (8). mRNA expression levels as EC10 of all the tested seven genes were decreased, particularly CXCL9 (19.36-fold), CXCL10 (8.14-fold), and TNF (18.69) by the treatment of 26 µM of compound 1 on SH-SY5Y cells. DISCUSSION AND CONCLUSIONS: Triterpenoids and steroids isolated from C. ovata were found to be moderate-strong anti-inflammatory compounds. Particularly, compounds 1 and 3 were found to be promising therapeutic agents in the treatment of inflammatory and autoimmune diseases.

Rheological characterization of starch gels: A biomass based sorbent for removal of polycyclic aromatic hydrocarbons (PAHs).

Environmental awareness increased the demand for the biomass based materials with superior properties instead of petroleum products. This study aims to prepare starch networks as sorbents for the removal of polycyclic aromatic hydrocarbons (PAHs). Two types of crosslinker, epichlorohydrine (ECH) and glutaraldehyde (GA), were choosed for the preparation of Gel-E and Gel-G networks, respectively. Rheological, swelling and morphological properties of the resulted materials were investigated as a function of various reaction parameters as starch, crosslinker and base concentration and also reaction temperature. The rheological measurements showed that while network formation of Gel-E hydrogels was strongly affected by the NaOH and starch concentration, the strength of the Gel-G hydrogels mainly depends on the crosslinker amount. Starch networks showed high PAH sorption capacities up to 1.42 g per gram sorbent with three model PAH molecules. Although PAH sorption capacities of the Gel-E networks are higher than those of Gel-G gels due to the pore sizes differences of the gel samples, both of them are promising materials as biosorbent for the PAH sorption applications due to the relatively high sorption capacities, low cost and simple preparation methods.

Synthesis, antioxidant activity and SAR study of novel spiro-isatin-based Schiff bases.

A new series of 21 Schiff bases of spiro-isatin was synthesized, and their DPPH, CUPRAC and ABTS cation radical scavenging abilities were investigated for antioxidant activity. The results showed that all the synthesized compounds exhibited antioxidant activity for each assay. 5̍-(2,3-Dihydroxybenzylideneamino)spiro[[1,3] dioxolane-2,3̍-indoline]-2̍-on (5c) (IC50 = 4.49 µM, for DPPH; IC50 = 0.39 µM, for ABTS.+; and A0.50 = 0.42 µM, for CUPRAC) showed significantly better ABTS, CUPRAC and DPPH radical scavenging ability than quercetin (IC50 = 8.69 µM, for DPPH; IC50 = 15.49 µM, for ABTS.+; and A0.50 = 18.47 µM, for CUPRAC), which is used as a standard. SAR study showed that the synthesized compounds had higher ABTS.+ activity than DPPH and CUPRAC activities. Moreover, the compounds (5c and 5d), containing two hydroxyl groups, exhibited the highest antioxidant activities for all assays. Quantum chemical calculations were also carried out to support SAR results.

Anticholinesterase, Antioxidant, Antiaflatoxigenic Activities of Ten Edible Wild Plants from Ordu Area, Turkey.

Turkey has highly rich floras of medicinal and aromatic plants because of having various climate conditions in different regions. One of these regions is Middle Black Sea Region, especially Ordu Province. Extracts of 10 edible plants (Arum maculatum L., Hypericum orientale L., Ornithogalum sigmoideum Freyn et Sint., Silene vulgaris Garcke var. macrocarpa, Plantago lanceolata L., Achillea millefolium L. subsp. pannonica, Rumex crispus L., Rumex acetosella L., Capsella bursa-pastoris L., Coronopus squamatus Asch.), grown in Ordu, Turkey, were prepared with different solvents (hexane, ethanol and water, separately) and their anticholinestrase and antiaflatoxigenic activities were evaluated. Additionally, the cupric reducing antioxidant capacities (CUPRAC) and ABTS cation radical scavenging abilities of the extracts were assayed. The ethanol extract of R. acetosella exhibited the highest antioxidant activity (A0.5 value of 25.31 µg/mL, for CUPRAC activity; IC50 value of 23.73 µg/mL, for ABTS activity). The hexane extract of C. bursa-pastoris showed the strongest inhibition against AChE enzyme with IC50 value of 7.24 µg/mL, and the hexane extract of A. millefolium subsp. pannonica had the highest BChE activity with IC50 value of 6.40 µg/mL. The ethanol extract of P. lanceolata exhibited the strongest inhibition against aflatoxin with 88% inhibition.

Comparison of antioxidant, anticholinesterase, and antidiabetic activities of three curcuminoids isolated from Curcuma longa L.

Antioxidant, anticholinesterase and antidiabetic activities of three curcuminoids isolated from the Curcuma longa were simultaneously tested and compared in this study. The highest antioxidant power was detected for curcumin with the applied methods. The drug potentials of curcuminoids for Alzheimer's disease were controlled. Bisdemethoxycurcumin (BDMC) showed substantial inhibitory activity. The activity of demethoxycurcumin (DMC) followed BDMC, whereas curcumin showed very little acetylcholinesterase inhibition activity. Antidiabetic activity of curcuminoids was evaluated by their α-glucosidase inhibitory activities. All curcuminoids show activities with decreasing order as BDMC > curcumin > DMC. The significant activities of BDMC compared to its isomers and examination of chemical structures of isomers might be a starting point in designing new drugs for Alzheimer's and Diabetes Mellitus.

Microwave assisted synthesis of novel hybrid tacrine-sulfonamide derivatives and investigation of their antioxidant and anticholinesterase activities.

A novel series of tacrine derivatives containing sulfonamide group were synthesized and their inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were evaluated. The result showed that all the synthesized tacrine-sulfonamides (VIIIa-o) exhibited inhibitory activity on both cholinesterases. VIIIg showed the highest inhibitory activity on AChE IC50=0.009µM. This value is 220-fold greater than that of galantamine (IC50=2.054µM) and 6-fold greater than tacrine (IC50=0.055µM). VIIIf displayed the strongest inhibition of BuChE (IC50=2.250µM), which is close to donepezil (IC50=2.680µM) and 8-fold greater than that of galantamine (IC50=18.130µM) Furthermore, all of the synthesized tacrine derivatives showed higher inhibition of BuChE than that of galantamine. In addition, the cupric reducing antioxidant capacities (CUPRAC) and ABTS cation radical scavenging abilities of the synthesized compounds were investigated for the antioxidant activity. Among them, VIIIb (IC50=94.390±2.310µM) showed significantly better ABTS cation radical scavenging ability than all of the new synthesized compounds.

Design, synthesis and docking study of novel coumarin ligands as potential selective acetylcholinesterase inhibitors.

New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC50 value of 43 nM) was the best AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c showed negligible cell death. Molecular docking studies were also carried out to clarify the inhibition mode of the more active compounds. Best pose of compound 6c is positioned into the active site with the coumarin ring wedged between the residues of the CAS and catalytic triad of AChE. In addition, the coumarin ring is anchored into the gorge of the enzyme by H-bond with Tyr130.

Synthesis, anticholinesterase activity and molecular modeling study of novel carbamate-substituted thymol/carvacrol derivatives.

New thymol and carvacrol derivatives with the carbamate moiety were synthesized and their inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were evaluated. 5-isopropyl-2-methylphenyl(3-fluorophenyl)carbamate (29) was found to be the most potent AChE inhibitor with IC50 values of 2.22µM, and 5-isopropyl-2-methylphenyl (4-fluorophenyl)carbamate (30) exhibited the strongest inhibition against BuChE with IC50 value of 0.02µM. Additionally, the result of H4IIE hepatoma cell toxicity assay for compounds 18, 20, 29, 30 and 35 showed negligible cell death at 0.07-10µM. Moreover in order to better understand the inhibitory profiles of these molecules, molecular modeling studies were applied. Binding poses of studied compounds at the binding pockets of AChE and BuChE targets were determined. Predicted binding energies of these compounds as well as structural and dynamical profiles of molecules at the target sites were estimated using induced fit docking (IFD) algorithms and post-processing molecular dynamics (MD) simulations methods (i.e., Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approaches).

Development and validation of a HPLC method for the determination of benzo(a)pyrene in human breast milk.

A simple analytical procedure was developed for the quantitation of benzo(a)pyrene in human breast milk using solid phase extraction (SPE) combined with high performance liquid chromatography. Before the chromatographic process, SPE, including C18 functional groups in silicagel cartridges, was conducted for sample preparation. A C18 column (100×4.6 mm id, 3 µm particle size) was used with acetonitrile:water (80:20) as the mobile phase at a flow rate 1mL/min at 30°C. Fluorimetric detection was performed for excitation and emission at 290 and 406 nm, respectively. It was observed that the calibration curve was linear over the range of 0.5-80 ng/mL. The limit of detection and limit of quantitation were found to be 0.5 and 1.07 ng/mL, respectively. Intraday and interday relative standard deviation values were less than 5.15%. Moreover, the newly developed method provides a fast, simple, cost effective, and sensitive assay to detect an important carcinogen substance, benzo(a)pyrene, in human breast milk.

Capparis ovata treatment suppresses inflammatory cytokine expression and ameliorates experimental allergic encephalomyelitis model of multiple sclerosis in C57BL/6 mice.

Since ancient times, Capparis species have been widely used in traditional medicine to treat various diseases. Our recent investigations have suggested Capparis ovata's potential anti-neuroinflammatory application for the treatment of multiple sclerosis (MS). The present study was designed to precisely determine the underlying mechanism of its anti-neuroinflammatory effect in a mouse model of MS. C. ovata water extract (COWE) was prepared using the plant's fruit, buds, and flower parts (Turkish Patent Institute, PT 2012/04,093). We immunized female C57BL/6J mice with MOG35-55/CFA. COWE was administered at a daily dose of 500mg/kg by oral gavage either from the day of immunization (T1) or at disease onset (T2) for 21days. Gene expression analysis was performed using a Mouse Multiple Sclerosis RT² Profiler PCR Array, and further determinations and validations of the identified genes were performed using qPCR. Whole-genome transcriptome profiling was analyzed using Agilent SurePrint G3 Mouse GE 8X60K microarrays. Immunohistochemical staining was applied to brain sections of the control and treated mice to examine the degree of degeneration. COWE was further fractionated and analyzed phytochemically using the Zivak Tandem Gold Triple Quadrupole LC/MS-MS system. COWE remarkably suppressed the development of EAE in T1, and the disease activity was completely inhibited. In the T2 group, the maximal score was significantly reduced compared with that of the parallel EAE group. The COWE suppression of EAE was associated with a significantly decreased expression of genes that are important in inflammatory signaling, such as TNFα, IL6, NF-κB, CCL5, CXCL9, and CXCK10. On the other hand, the expression of genes involved in myelination/remyelination was significantly increased. Immunohistochemical analysis further supported these effects, showing that the number of infiltrating immune cells was decreased in the brains of COWE-treated animals. In addition, differential expression profiling of the transcriptome revealed that COWE treatment caused the down regulation of a group of genes involved in the immune response, inflammatory response, antigen processing and presentation, B-cell-mediated immunity and innate immune response. Collectively, these results suggest anti-neuroinflammatory mechanisms by which COWE treatment delayed and suppressed the development of EAE and ameliorated the disease in mice with persistent clinical signs.

Method Validation for the Quantitative Analysis of Aflatoxins (B1, B2, G1, and G2) and Ochratoxin A in Processed Cereal-Based Foods by HPLC with Fluorescence Detection.

Modified AOAC 991.31 and AOAC 2000.03 methods for the simultaneous determination of total aflatoxins (AFs), aflatoxin B1, and ochratoxin A (OTA) in processed cereal-based foods by RP-HPLC coupled with fluorescence detection were validated. A KOBRA® Cell derivatization system was used to analyze total AFs. One of the modifications was the extraction procedure of mycotoxins. Both AFs and OTA were extracted with methanol-water (75+25, v/v) and purified with an immunoaffinity column before HPLC analysis. The modified methods were validated by measuring the specificity, selectivity, linearity, sensitivity, accuracy, repeatability, reproducibility, recovery, LOD, and LOQ parameters. The validated methods were successfully applied for the simultaneous determination of mycotoxins in 81 processed cereal-based foods purchased in Turkey. These rapid, sensitive, simple, and validated methods are suitable for the simultaneous determination of AFs and OTA in the processed cereal-based foods.

Potential of aryl-urea-benzofuranylthiazoles hybrids as multitasking agents in Alzheimer's disease.

New benzofuranylthiazole derivatives containing the aryl-urea moiety were synthesized and evaluated in vitro as dual acetylcholinesterase (AChE)-butyrylcholinesterase (BuChE) inhibitors. In addition, the cupric reducing antioxidant capacities (CUPRAC) and ABTS cation radical scavenging abilities of the synthesized compounds were assayed. The result showed that all the synthesized compounds exhibited inhibitory activity on both AChE and BuChE with 1-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(2-fluorophenyl)urea (e25, IC50 value of 3.85 µM) and 1-(4-iodophenyl)-3-(4-(5-nitrobenzofuran-2-yl)thiazol-2-yl)urea (e38, IC50 value of 2.03 µM) as the strongest inhibitors against AChE and BuChE, respectively. Compound e38 was 8.5-fold more potent than galanthamine. The selectivity index of e25 and e38 was 2.40 and 0.37 against AChE and BuChE, respectively. Compound e2, e4 and e11 (IC50 = 0.2, 0.5 and 1.13 µM, respectively) showed a better ABTS cation radical scavenging ability than the standard quercetin (IC50 = 1.18 µM). Best poses of compounds e38 on BuChE and e25 on AChE indicate that the thiazole ring and the amidic moiety are important sites of interaction with both ChEs. In addition, the benzofuran ring and phenyl ring are anchored to the side chains of both enzymes by π-π(pi-pi) interactions.