Umweltchemische Analysen von Pharma-Produkten: Methodenentwicklung zur Analytik von Benzimidazol-Entwurmungsmitteln und deren Stoffwechselprodukten in Schweinegülle und damit gedüngten Böden

Benzimidazole anthelmintics enter agricultural land by using manure of treated animals as soil amendment. Efficient analytical methods are required to identify and reliably quantify the residues of these compounds. In this study an analytical method based on liquid chromatographic coupled to tandem mass spectrometry was developed for the simultaneous determination of fenbendazole, flubendazole and 8 of their corresponding metabolites in surface water, liquid pig manure, soil and manured soil. The quantification was conducted using electrospray ionization (ESI) in positive mode. Target compounds were extracted from surface water using solid phase extraction (SPE) at pH 2.3 with recovery rates 84%. For target compound extraction from manure samples, direct solvent extraction using ethyl acetate at pH 9.5 or lyophilization followed by methanol extraction at original pH as well as ultrasound-assisted solvent extraction (USE) of lyophilized manure samples using a methanol/ethyl acetate mixture (1:4, v/v) at pH 9.5, were successfully used. Size exclusion chromatography (SEC) followed by SPE were used to clean-up the raw extracts. For manure samples, the overall recovery rates ranged from 78 to 118 % with relative standard deviations of ≤ 19 %. Soil and manured soil samples were treated via direct solvent extraction or USE technique using different solvents and solvent mixtures. At alkaline pH using methanol/ethyl acetate mixture (1:4, v/v), all compounds were efficiently extracted from soil and manured soil with overall recovery rates ranged from 70 to 119 % and relative standard deviations were ≤ 21 %. The method detection and quantitation limits ranged from 0.5 to 1 and from 1.6 to 3.1 µg/kg fresh manure. They were between 0.7 to 2.5 µg/kg and 2.4 to 7.9 µg/kg for soil and manured soil samples, respectively. Incubated spiked manure samples for 30 days at 4 and 20 ºC, were analyzed in order to check the aging effect on the extractability of the target compounds. Matrix effects were investigated using different calibration approaches. Standard addition method was efficient to compensate these effects. Therefore, it was used to quantify the concentrations of detected compounds in the real samples. Flubendazole and 3 of its metabolites were detected in 7 real manure samples. Confirmation of positive finding was successfully achieved meeting the current requirements of analytical quality assurance in accordance with European Commission Decision 2002/657/EC.Durch Behandlung von Nutztieren mit Benzimidazol-Entwurmungsmitteln gelangen diese Substanzen über das Aufbringen von Gülle in Ackerböden. Es sind leistungsfähige Analysenmethoden nötig um die Rückstände der Mittel zuverlässig zu bestimmen. In dieser Arbeit wurde eine analytische Methode entwickelt, die auf der Flüssigkeitschromatographie kombiniert mit Tandemmassenspektrometrie (LC/MS/MS) basiert und die Simultanbestimmung von Fenbendazol, Flubendazol und 8 Metaboliten in Oberflächenwasser, Schweinegülle und gedüngten Böden gestattet. Die Quantifizierung wurde unter Verwendung der LC/MS/MS mittels Elektrospray-Ionisierung (ESI+) durchgeführt. Die Zielkomponenten wurden aus Oberflächenwasser mittels Festphasenextraktion (SPE) bei pH 2.3 mit einer Wiederfindungsrate von > 84 % extrahiert. Bei Gülleproben wurde die direkte Flüssig-Extraktion mit Ethylacetat bei pH 9.5 oder die Gefriertrocknung gefolgt von einer Extraktion mit MeOH bei unverändertem pH-Wert sowie die Ultraschallextraktion (USE) gefriergetrockneter Gülleproben mit einer Mischung MeOH/Ethylacetat (1: 4, v/v) bei pH 9.5 eingesetzt. Die Rohextrakte wurden mit der Größenausschlußchromatographie (SEC) gefolgt von der Festphasenextraktion (SPE) gereinigt. Bei den Gülleproben lag die Wiederfindungsrate im Bereich von 78 bis 118 % mit relativen Standardabweichungen von ≤ 19 %. Böden bzw. güllegedüngte Böden wurden über die direkte Flüssigextraktion oder mittels Ultraschalltechnik unter Einsatz verschiedener Lösungsmittel bzw. Lösungsmittelgemische extrahiert. Im alkalischen pH-Bereich wurden mit einem MeOH/Ethylacetat-Gemisch (1:4, v/v) alle Komponenten bei einer Wiederfindungsrate von 70 bis 119 % und einer relativen Standardabweichung von ≤ 21 % aus den Böden extrahiert. Die Bestimmungsgrenzen reichten von 0.5 bis 1 bzw. 1.6 bis 3.1 µg/kg für frische Gülle. Bei Böden bzw. bei güllegedüngten Böden wurden 0.7 bis 2.5 µg/kg und 2.4 bis 7.9 µg/kg erreicht. Dotierte und anschließend bei 4 °C und 20 °C für 30 Tage inkubierte Gülleproben wurden analysiert um Alterungseffekte auf die Extrahierbarkeit der Zielkomponenten zu überprüfen. Matrixeffekte, welche durch die Coeluation von Matrixbestandteilen der Proben entstanden, wurden durch Verwendung verschiedener Kalibrierungsansätze untersucht. Mittels einer Standardadditionsmethode konnten die Effekte effizient kompensiert werden. Schließlich wurden Flubendazol und 3 Metabolite in 7 realen Gülleproben bestimmt und die Ergebnisse gemäss den Richtlinien der analytischen Qualitätssicherung (European Commission Decision 2002/657/EC) bestätigt

Analytical and CFD Methods Investigating Shroud Blade Tip Leakage.

This study deals with the leakage flow over a shrouded turbine stage, its interaction with the main passage flow, and the associated losses. The study addressed these topics by providing an analytical correlation loss model and detailed CFD simulations. An analytical model of leakage flow loss over a shrouded turbine stage has been developed. The analytical model uses directly measurable flow quantities to predict the effect of some of the over-shroud design parameters on stage performance. The model displays good predictive ability for the mass leakage fraction and the mixing losses. The model resolves the negative incidence angle induced by mixing the leakage flow with the main stream and predicts the increment in the total mixing loss coefficient at increasing leakage jet injection angles. The main contributions of this model to the leakage jet models documented in the open literature are the effect of the leakage jet injection angle on the mixing loss and the accounting of the effect of the number of fins on the leakage mass fraction in an explicit way. The present model exhibits a good qualitative and quantitative agreement with comparative benchmark data. An in-house three-dimensional turbomachinery CFD code was developed and validated against six test cases, showing its ability to capture the salient flow features in each test case. This work makes an innovative use of Detached Eddy Simulation as an advanced Reynolds Averaged Navier-Stokes (RANS) model. A detailed leakage flow structure over the rotor shroud and its interaction with the main passage flow were modeled for seven test cases to investigate the effect of the number of fins, the clearance gap ratio, and the leakage jet injection angle on the flow. The results showed that reducing the injection 90° to 30° leads to a reduction in entropy mixing loss coefficient by up to 24.7% and gives a 0.2% increase in the rotor static to static efficiency and highlighted that reducing the leakage jet injection angle is a promising concept to control most of the adverse effects of the leakage flow

Analytical and CFD methods investigating shroud blade tip leakage

This study deals with the leakage flow over a shrouded turbine stage, its interaction with the main passage flow, and the associated losses. The study addressed these topics by providing an analytical correlation loss model and detailed CFD simulations. An analytical model of leakage flow loss over a shrouded turbine stage has been developed. The analytical model uses directly measurable flow quantities to predict the effect of some of the over-shroud design parameters on stage performance. The model displays good predictive ability for the mass leakage fraction and the mixing losses. The model resolves the negative incidence angle induced by mixing the leakage flow with the main stream and predicts the increment in the total mixing loss coefficient at increasing leakage jet injection angles. The main contributions of this model to the leakage jet models documented in the open literature are the effect of the leakage jet injection angle on the mixing loss and the accounting of the effect of the number of fins on the leakage mass fraction in an explicit way. The present model exhibits a good qualitative and quantitative agreement with comparative benchmark data. An in-house three-dimensional turbomachinery CFD code was developed and validated against six test cases, showing its ability to capture the salient flow features in each test case. This work makes an innovative use of Detached Eddy Simulation as an advanced Reynolds Averaged Navier-Stokes (RANS) model. A detailed leakage flow structure over the rotor shroud and its interaction with the main passage flow were modeled for seven test cases to investigate the effect of the number of fins, the clearance gap ratio, and the leakage jet injection angle on the flow. The results showed that reducing the injection 90° to 30° leads to a reduction in entropy mixing loss coefficient by up to 24.7% and gives a 0.2% increase in the rotor static to static efficiency and highlighted that reducing the leakage jet injection angle is a promising concept to control most of the adverse effects of the leakage flow.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Nano-Structured Lipid Carrier-Based Oral Glutathione Formulation Mediates Renoprotection against Cyclophosphamide-Induced Nephrotoxicity, and Improves Oral Bioavailability of Glutathione Confirmed through RP-HPLC Micellar Liquid Chromatography

The study aimed to develop a new glutathione (GSH) oral formulation to enhance the delivery of GSH and counter the nephrotoxicity of the anticancer drug, cyclophosphamide (CP). A nanostructured lipid carrier glutathione formulation (GSH-NLCs) composed of glutathione (500 mg), stearic and oleic acid (300 mg, each), and Tween® 80 (2%, w/v) was prepared through the emulsification-solvent-evaporation technique, which exhibited a 452.4 ± 33.19 nm spheroidal-sized particulate material with narrow particle size distributions, −38.5 ± 1.4 mV zeta potential, and an entrapment efficiency of 79.8 ± 1.9%. The GSH formulation was orally delivered, and biologically tested to ameliorate the CP-induced renal toxicity in a rat model. Detailed renal morphology, before and after the GSH-NLCs administration, including the histopathological examinations, confirmed the ameliorating effects of the prepared glutathione formulation together with its safe oral delivery. CP-induced oxidative stress, superoxide dismutase depletion, elevation of malondialdehyde levels, depletion of Bcl-2 concentration levels, and upregulated NF-KB levels were observed and were controlled within the recommended and near normal/control levels. Additionally, the inflammatory mediator marker, IL-1β, serum levels were marginally normalized by delivery of the GHS-NLCs formulation. Oral administration of the pure glutathione did not exhibit any ameliorating effects on the renal tissues, which suggested that the pure glutathione is reactive and is chemically transformed during the oral delivery, which affected its pharmacological action at the renal site. The protective effects of the GSH-NLCs formulation through its antioxidant and anti-inflammatory effects suggested its prominent role in containing CP-induced renal toxicity and renal tissue damage, together with the possibility of administrating higher doses of the anticancer drug, cyclophosphamide, to achieve higher and effective anticancer action in combination with the GSH-NLCs formulation

Application of the Box–Behnken design for the production of soluble curcumin: Skimmed milk powder inclusion complex for improving the treatment of colorectal cancer

The main objective of this study was to develop a soluble product of the practically insoluble curcumin (CMN) to treat colorectal cancer more effectively than with pure CMN. To improve the solubility of CMN, various hydrophilic carriers of skimmed milk powder (SMP), polyvinylpyrrolidone (PVP), and mannitol (MNT) were utilized to prepare solid dispersion (SD) binary complexes. The prepared complexes were characterized in terms of their aqueous solubility and in vitro drug release and analyzed by Fourier transform infrared spectrophotometry, powder X-ray diffractometry, scanning electron microscopy, dynamic light scattering, and the novel dyeing test. Based on this characterization, the best SD complex was optimized using the Box–Behnken design (RSM-BBD). These results showed that the solubility of CMN was greatly improved in combination with SMP. The SD of CMN with SMP produced significantly improved solubility (0.646 ± 0.024 mg/ml) and dissolution (54.94 ± 3.21% at 5 min). Further, solid-state characterization revealed that the complex exhibited intermolecular inclusion of the drug and carrier. Also, the complex did not undergo any chemical modification owing to its amorphous form, and the novel dye test showed better coloring impact, indicating the solubility of CMN. The in vitro cytotoxicity of the complex showed that 50% inhibition (IC50) of SW480 and Caco-2 cells was achieved at a considerably lower concentration than that of pure CMN. Flow cytometry analysis confirmed that the cell cycle arrest was at G2/M phase (43.26% and 65.14%), and DNA fragmentation analysis investigation confirmed that the complex induced more DNA damage during apoptosis

Antitumor Activity of Nitazoxanide against Colon Cancers: Molecular Docking and Experimental Studies Based on Wnt/β-Catenin Signaling Inhibition

In colon cancer, wingless (Wnt)/β-catenin signaling is frequently upregulated; however, the creation of a molecular therapeutic agent targeting this pathway is still under investigation. This research aimed to study how nitazoxanide can affect Wnt/β-catenin signaling in colon cancer cells (HCT-116) and a mouse colon cancer model. Our study included 2 experiments; the first was to test the cytotoxic activity of nitazoxanide in an in vitro study on a colon cancer cell line (HCT-116) versus normal colon cells (FHC) and to highlight the proapoptotic effect by MTT assay, flow cytometry and real-time polymerase chain reaction (RT-PCR). The second experiment tested the in vivo cytotoxic effect of nitazoxanide against 1,2-dimethylhydrazine (DMH) prompted cancer in mice. Mice were grouped as saline, DMH control and DMH + nitazoxanide [100 or 200 mg per kg]. Colon levels of Wnt and β-catenin proteins were assessed by Western blotting while proliferation was measured via immunostaining for proliferating cell nuclear antigen (PCNA). Treating HCT-116 cells with nitazoxanide (inhibitory concentration 50 (IC50) = 11.07 µM) revealed that it has a more cytotoxic effect when compared to 5-flurouracil (IC50 = 11.36 µM). Moreover, it showed relatively high IC50 value (non-cytotoxic) against the normal colon cells. Nitazoxanide induced apoptosis by 15.86-fold compared to control and arrested the cell cycle. Furthermore, nitazoxanide upregulated proapoptotic proteins (P53 and BAX) and caspases but downregulated BCL-2. Nitazoxanide downregulated Wnt/β-catenin/glycogen synthase kinase-3β (GSK-3β) signaling and PCNA staining in the current mouse model. Hence, our findings highlighted the cytotoxic effect of nitazoxanide and pointed out the effect on Wnt/β-catenin/GSK-3β signaling

Differential Effect of Three Macrolide Antibiotics on Cardiac Pathology and Electrophysiology in a Myocardial Infarction Rat Model: Influence on Sodium Nav1.5 Channel Expression

Macrolides were reported to have cardiotoxic effects presented mainly by electrocardiogram (ECG) changes with increased risk in cardiac patients. We aimed to determine the impact of three macrolides, azithromycin, clarithromycin and erythromycin, on cardiac electrophysiology, cardiac enzyme activities, histopathological changes, and sodium voltage-gated alpha subunit 5 (Nav1.5) channel expression. We used eight experimental groups of male albino rats: vehicle, azithromycin (100 mg/kg), clarithromycin (100 mg/kg), erythromycin (100 mg/kg), MI + vehicle, MI + azithromycin (100 mg/kg), MI + clarithromycin (100 mg/kg) and MI + erythromycin (100 mg/kg); each group received chronic oral doses of the vehicle/drugs for seven weeks. ECG abnormalities and elevated serum cardiac enzymes were observed particularly in rats with AMI compared to healthy rats. Microscopic examination revealed elevated pathology scores for rats treated with clarithromycin in both experiments following treatment with erythromycin in healthy rats. Although rats with MI did not show further elevations in fibrosis score on treatment with macrolides, they produced significant fibrosis in healthy rats. Downregulation of cardiac Nav1.5 transcript was observed following macrolides treatment in both groups (healthy rats and rats with MI). In conclusion, the current findings suggested the potential cardiotoxic effects of chronic doses of macrolide antibiotics in rats with MI as manifested by abnormal ECG changes and pathological findings in addition to downregulation of Nav1.5 channels. Furthermore, in the current dose ranges, azithromycin produced the least toxicity compared to clarithromycin and erythromycin