Synthesis and evaluation of febrifugine derivatives as anticoccidial agents.

A series of new febrifugine derivatives with a 4(3H)-quinazolinone scaffold were synthesized and evaluated for their anticoccidial activity both in vitro and in vivo. The targets' in vitro activity against Eimeria tenella was studied using quantitative real-time reverse transcription polymerase chain reaction and Madin-Darby bovine kidney cells. Most of these compounds demonstrated anticoccidial efficacy, with inhibition ratios ranging from 3.3% to 85.7%. Specifically, compounds 33 and 34 showed significant inhibitory effects on the proliferation of E. tenella and exhibited lower cytotoxicity compared to febrifugine. The IC50 values of compounds 33 and 34 were 3.48 and 1.79 µM, respectively, while the CC50 values were >100 µM for both compounds. Furthermore, in a study involving 14-day-old chickens infected with 5 × 104 sporulated oocysts, treatment with five selected compounds (22, 24, 28, 33, and 34), which exhibited in vitro inhibition rate of over 50% at 100 µM, at a dose of 40 mg/kg in daily feed for 8 consecutive days showed that compound 34 possessed moderate in vivo activity against coccidiosis, with an anticoccidial index of 164. Structure-activity relationship studies suggested that spirocyclic piperidine may be a preferable substructure to maintain high effectiveness in inhibiting Eimeria spp., when the side chain 1-(3-hydroxypiperidin-2-yl)propan-2-one was replaced.

Response Surface Optimization of Dispersive Solid-Phase Extraction Combined with HPLC for the Rapid Analysis of Multiple Coccidiostats in Feed.

Since antimicrobials were banned as feed additives, coccidiostats with favorable anticoccidial action and growth promotion have been widely used in the breeding industry. The monitoring of coccidiostats in feed is necessary, while the current methods based on mass-spectrometer analysis have limited applicability and matrix effects could interfere with the results. Accordingly, in the present paper, a rapid analytical strategy for the simultaneous determination of six synthetic coccidiostats in feed using high-performance liquid chromatography coupled with diode-array detection was developed. Coccidiostats in chicken feeds were extracted with the trichloroacetic acid-acetonitrile solution. The cleanup was performed by dispersive solid-phase extraction after the optimization of the response surface methodology. The method exhibited good linearity for target coccidiostats within the range of 0.05~20 µg/mL. Recoveries for six compounds in fortified feed samples were from 67.2% to 107.2% with relative standard deviations less than 9.6%. The limit of detection was 0.2~0.3 mg/kg. The successful application of the method in commercial feed verified that it is effective and sensitive for the rapid determination of multiple coccidiostats in chicken feeds.

Evaluation of Mongolian compound library for potential antimalarial and anti-Toxoplasma agents.

179 compounds in a Mongolian compound library were investigated for their inhibitory effect on the in vitro growth of Plasmodium falciparum and Toxoplasma gondii. Among these compounds, brachangobinan A at a half-maximal inhibition concentration (IC50) of 2.62 µM and a selectivity index (SI) of 27.91; 2-(2'-hydroxy-5'-O-methylphenyl)-5-(2″,5″-dihydroxyphenyl)oxazole (IC50 3.58 µM and SI 24.66); chrysosplenetin (IC50 3.78 µM and SI 15.26); 4,11-di-O-galloylbergenin (IC50 3.87 µM and SI 13.38); and 2-(2',5'-dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole (IC50 6.94 µM and SI 11.48) were identified as potential inhibitors of P. falciparum multiplication. Additionally, tricin (IC50 12.94 µM and SI > 23.40) was identified as a potential inhibitor of T. gondii multiplication. Our findings represent a good starting point for developing novel antimalarial and anti-Toxoplasma therapeutics from Mongolian compounds.

PHARMACOKINETICS OF A SINGLE ORAL DOSE OF PONAZURIL IN THE INDIAN PEAFOWL (PAVO CRISTATUS).

Ponazuril, a novel coccidiocidal triazinetrione, has shown promise in addressing apicomplexan diseases in mammals and birds. This study describes the pharmacokinetics of ponazuril in healthy adult Indian peafowl (Pavo cristatus) following a single oral dose administered at two different dosages. Peafowl (four males and four females) were administered compounded ponazuril at 20 or 40 mg/kg orally in a double crossover design, with a 2-wk washout period. Blood was collected from each bird at 2, 4, 8, 24, 48, 72, 96, and 120 h after administration for plasma concentration of ponazuril using high-pressure liquid chromatography. Fecals were evaluated for coccidial shedding for 3 consecutive d prior to the ponazuril trial, 1 wk after the first dose of ponazuril, and 1 wk after the second dose of the trial. After the first trial, one peafowl administered 20 mg/kg ponazuril was shedding coccidia, but no coccidia were detected by the end of the second trial. Ponazuril reached peak concentrations (Tmax) at 21.38 h + 5.25 and 22.04 h + 7.39, and peak concentration (Cmax) were 11.82 µg/ml + 3.01 and 18.42 µg/ml + 4.13, for 20 and 40 mg/kg doses, respectively. Ponazuril was detected at 120 h with a concentration of 9.48 µg/ml + 2.59 and 12.25 µg/ml + 2.89 and a half-life of 219.4 + 58.7 h and 186.7 + 58.7 h, for and 40 mg/kg doses, respectively. Ponazuril in peafowl was well absorbed orally, plasma concentrations increased with dose, and elimination was slower than current dosages for birds would suggest. No obvious adverse effects were observed at either dosage.

Optimization of Adsorbent Layer Type and Developing Solvent in Coccidiostats Sample Preparation with Procedure of Solvent Front Position Extraction.

Coccidiostats are drugs used against coccidiosis, a common disease among breeding animals. Their widespread application leads to the appearance of their residues in food, which is potentially harmful for human health and life. The European Union has established limits of concentrations of these drugs in premixtures and food. Nowadays, there are many methods for monitoring coccidiostats' presence in market products, but their frequent weakness is sample preparation. Solvent Front Position Extraction is a planar chromatography-based sample preparation method that allows for effective assay of samples with coccidiostats when coupled with LC-MS/MS. The purpose of this research was to find common conditions for the effective isolation of eight coccidiostats from biological sample components with both lower and higher retention than the substances of interest. The acquired results were used for effective isolation of monensin and salinomycin from the premixture samples and allowed for their quantitative determination. The application of a semi-automatic device for the development of chromatograms positively impacted the results, confirming the effectiveness of the method for determining coccidiostats in biological samples.

Evaluation of optimum conditions for decoquinate nanoliposomes and their anticoccidial efficacy against diclazuril-resistant Eimeria tenella infections in broilers.

Decoquinate (DQ) is used for prophylaxis against coccidian infections within the digestive tract of chickens, but DQ is extremely insoluble in water. Hence, improving the water solubility of DQ is extremely important. First, decoquinate nanoliposomes (DQNLs) were prepared by the thin-film dispersion-ultrasonic method. The preparation conditions of DQNLs were optimized using the orthogonal test. The optimal preparation conditions of DQNLs were: a ratio of egg-yolk lecithin:drug (w/w) of 10:1, ratio of egg-yolk lecithin:cholesterol (w/w) of 5:1, rotary-evaporation temperature of 50 ℃, and ultrasound duration of 15 min. The encapsulation efficiency of DQNLs prepared under these conditions reached 99.24 % and drug loading was 5.67 %. The characterization of optimized DQNLs was also done. Transmission electron microscopy of DQNLs showed that they had the characteristic structure of liposomes. The mean particle size was 115.6 nm. The polydispersity index was 0.175. The zeta potential was -39.1 mV. The stability of DQNLs was high upon storage at 4 ℃. In vivo studies demonstrated that the lower dose (5 mg/L) of DQNLs in drinking water obtained the similar anticoccidial efficacy to that of 40 mg/kg DQ in feed against diclazuril-resistance Eimeria tenella isolate. The in vitro inhibitory effect of DQNLs on the sporulation of Eimeria tenella oocysts was dose-dependent. Therefore, the anticoccidial efficacy of DQ was enhanced significantly after being encapsulated into nanoliposomes.

Pharmacokinetics, Activity, and Residue Elimination of R- and S-Diclazuril in Broiler Chickens.

Diclazuril (DIC) is widely used as a racemic mixture to prevent and treat coccidiosis in farm animals, while the pharmacokinetics, bioactivity, and toxicity of DIC enantiomers are not known at all. This study first established a simple, sensitive, and reliable liquid chromatography tandem mass spectrometry method for separation of R-DIC and S-DIC and their analyses. Then, it was applied to investigate the stereoselective pharmacokinetics and residual elimination of individual enantiomers, and their anticoccidial activity was also evaluated in broiler chickens. The results indicated that the area under the concentration-time curve (AUC) and elimination half-life (t1/2ß) were significantly different (p < 0.05) for two enantiomers in chicken plasma. The AUC and t1/2ß of S-DIC were approximately 2 and 1.4 times those of R-DIC, respectively. The residual elimination of DIC enantiomers in chicken tissues was also stereoselective. The concentrations of S-DIC in chicken muscle and liver were greater than those of R-DIC, and it is the opposite in the kidney. There was no significant difference (p > 0.05) in the anticoccidial activity of racemate and enantiomers when a single enantiomer in feed was added above 0.5 mg kg-1. However, the anticoccidial activity of R-DIC (0.25 mg kg-1) was significantly higher (p < 0.05) than that of S-DIC (0.25 mg kg-1) in the diet. It should be mentioned that in chicken small intestine and cecum, the enantiomerization rate of each enantiomer in the infection group was faster than that in the uninfected group.

Influence of Eimeria spp. Infection on Chicken Jejunal Microbiota and the Efficacy of Two Alternative Products Against the Infection.

Eimeria spp. are important intestinal pathogens of chickens (Gallus gallus domesticus). Anticoccidial feed additives, chemicals, and ionophores have traditionally been used to control Eimeria infections in broiler production. Thus, the trend toward antibiotic-free and organic production requires new approaches to coccidiosis prevention. Two not mutually exclusive methods are the use of plant extracts with antiparasitic activity and manipulation of the intestinal microbiota by pre- and probiotics. In the present study, birds were inoculated with a combination of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. We profiled the jejunal microbiome at multiple time points postinfection to investigate the changes in jejunum microbiota and to identify the time point of the maximum difference between infected and noninfected birds. Additionally, we assessed the anticoccidial effects of two anecdotal treatment methods, green tea and apple cider vinegar, as well as amprolium. Green tea and apple cider vinegar had no effect on oocyst shedding, but green tea reduced the mild unspecific lesions in coccidia-infected birds; there was no influence on unspecific lesions in uninfected controls. Jejunal contents were collected on the day of the infection and 1, 2, 4, 6, 10, and 14 days postinfection (dpi) for investigation of the intestinal microbiota by 16S ribosomal (r)RNA gene sequencing. Comparison of the untreated-uninfected and the untreated-infected groups showed a maximum community dissimilarity of 10 dpi. From 4 days after infection, Clostridiales were significantly enriched at the expense of Lactobacillales in infected compared with uninfected birds. Interestingly, treatment with green tea prevented proliferation of Clostridiales induced by the coccidia and increased the relative abundance of Melainabacteria.

Influencia de la infección de Eimeria spp. en el microbiota yeyunal del pollo y en la eficacia de dos productos alternativos contra la infección. Los protozoarios del género Eimeria spp. son importantes patógenos intestinales de los pollos (Gallus gallus domesticus). Los aditivos anticoccidiales en el alimento, los productos químicos y los ionóforos se han utilizado tradicionalmente para controlar las infecciones por Eimeria en la producción de pollos de engorde. Por lo tanto, la tendencia hacia la producción orgánica y libre de antibióticos requiere nuevos enfoques para la prevención de la coccidiosis. Dos métodos no mutuamente excluyentes son el uso de extractos de plantas con actividad antiparasitaria y la manipulación de la microbiota intestinal por prebióticos y probióticos. En el presente estudio, las aves fueron inoculadas con una combinación de Eimeria acervulina, Eimeria maxima y Eimeria tenella. Se determinó el perfil del microbioma yeyunal en múltiples puntos de tiempo después de la infección, para investigar los cambios en la microbiota del yeyuno e identificar el momento donde se observa la diferencia máxima entre las aves infectadas y no infectadas. Además, se evaluaron los efectos anticoccidiales de dos métodos de tratamiento basados por datos anecdóticos, el té verde y el vinagre de sidra de manzana, así como el amprolium. El té verde y el vinagre de sidra de manzana no tuvieron ningún efecto sobre el desprendimiento de oocistos, pero el té verde redujo las lesiones leves inespecíficas en aves infectadas con coccidia; no hubo influencia en lesiones inespecíficas en controles no infectados. Los contenidos yeyunales se recogieron el día de la infección y a los días 1, 2, 4, 6, 10 y 14 después de la infección (dpi) para la investigación de la microbiota intestinal mediante secuenciación del gene del ARN ribosómico 16S. La comparación de los grupos no tratados-no infectados y no tratados-infectados mostró una diferencia máxima en la comunidad a los diez días después de la inoculación. A partir de los cuatro días después de la infección, los agentes Clostridiales se enriquecieron significativamente a expensas de los Lactobacillales en aves infectadas en comparación con las aves no infectadas. Curiosamente, el tratamiento con té verde evitó la proliferación de Clostridiales inducida por los coccidios y aumentó la abundancia relativa de melainabacterias.

Evaluation of ursolic acid derivatives with potential anti-Toxoplasma gondii activity.

Toxoplasma gondii is an important pathogen that causes serious public health problems. Currently, therapeutic drugs for toxoplasmosis cause serious side effects, and more effective and novel substances with relatively low toxicity are urgently needed. Ursolic acid (UA) has many properties that can be beneficial to healthcare. In this study, we synthesized eight series of UA derivatives bearing a tetrazole moiety and evaluated their anti-T. gondii activity in vitro using spiramycin as a positive control. Most of the synthesized derivatives exhibited better anti-T. gondii activity in vitro than UA, among which compound 12a exhibited the most potent anti-T. gondii activity. Furthermore, the results of biochemical parameter determination indicated that 12a effectively restored the normal body weight of mice infected with T. gondii, reduced hepatotoxicity, and exerted significant anti-oxidative effects compared with the findings for spiramycin. Additionally, our molecular docking study indicated that the synthesized compounds could act as potential inhibitors of T. gondii calcium-dependent protein kinase 1 (TgCDPK1), with 12a possessing strong affinity for TgCDPK1 via binding to the key amino acids GLU129 and TYR131.

Anticoccidial effects of Khaya senegalensis aqueous stem bark extract on broiler chickens experimentally infected with Eimeria species.

Graded concentrations (200, 400 and 800 mg/kg) of the aqueous stem bark extract of Khaya senegalensis was evaluated for its therapeutic efficacy against experimentally induced coccidiosis in broiler chicken. The phytochemical analysis shows the presence of tannins, saponins, cardiac glycosides and steroids. There was significant reduction in oocyst count across the groups in a graded dose manner with 800 mg/kg being the most efficacious dose. There was also weight gain across the treatment groups with immuno-modulatory and erythropoetic activities observed. Also, a significant (p < 0.05) graded dose-dependent reduction in the oocyst count in the treatment groups. A significant (p < 0.05) increase in mean weight gain was also recorded across the experimental groups except the negative control. The haematology also showed a dose-dependent increase in red blood cells, haemoglobin and packed cell volume of the treatment groups. The extract had no significant difference (p > 0.05) on the white blood cells, but a slight decrease in the white blood cells and heterophil counts was observed at 400 mg/kg. Furthermore, the aspartate amino transaminase level showed a significant difference (p < 0.05). Fluctuating levels of other serum biochemical parameters such as total protein, albumin and potassium were observed. No significant difference (p > 0.05) in the sodium concentration was observed. In addition, oxidative stress biomarkers such as catalase significantly increased (p < 0.05) in all the experimental groups in addition to the concomitant increase in reduced gluthathione (GSH) and superoxide dismutase (SOD) levels. Conclusively, the aqueous extract of K. senegalensis was effective in the management of coccidiosis thus supporting its folkloric use.

Stability of antibacterial and coccidiostat drugs on chicken meat burgers upon cooking and in vitro digestion.

The impact of culinary practices - oven or microwave cooking combined with herbs and/or beer - on antibacterial and coccidiostat drugs stability and bioaccessibility in chicken meat was evaluated. Fourteen compounds from 6 classes (ß-lactams, tetracyclines, sulfonamides, fluoroquinolones, macrolides, and coccidiostats) were monitored after cooking and in vitro digestion (INFOGEST protocol) at two fortification levels. Depending on their reduction, the presence of transformation products derived from cooking or digestion was investigated. In general, compounds were stable during cooking except amoxicillin, chlortetracycline and tylosin (reductions > 50%). Molecular rearrangement and dechlorination reactions are the most probable transformations derived from cooking. Adding herbs/beer does not benefit their reductions. During in vitro digestion, maximum bioaccessibilities of 60% were observed for all quantified compounds. As drugs and bile salts interact, increasing the absorption of lipophilic drugs, their bioaccessibility predictions must not be based only on the determination of their free form using LC-MS/MS.

Rat two-generation reproductive toxicity and teratogenicity studies of a novel coccidiostat - Ethanamizuril.

In the current study, to support the safety assessment of ethanamizuril as a new potent anticoccidial agent of triazine compounds, a reproductive toxicity and teratogenic potential assay of ethanamizuril was investigated. Groups of 30 males and 30 females were administered 0, 0.02, 0.1 or 0.2 mg/ml ethanamizuril by gastric incubation through a 10-week prebreed period as well as during mating, gestation, parturition and lactation in any generation. Compared to the control group, no test compound-related changes in copulation index, fertility index, gestation length, litter size, pup weight, pup sex ratio, pup viability, epididymal sperm counts or motility or other functional reproductive measures were noted in any generation, except few significant changes in high dose group in the number of sperm motility at III level in F0 males and the body weights of GD14 and GD21 in F1 rats. There were no compound-related necropsy findings or effects on organ weight. Histopathologic examinations revealed no evidence of compound-related changes in any organs including the reproductive organs of both sexes. In conclusion, long-term administrated 0.2, 1.0 and 2.0 mg/kg of ethanamizuril by means of oral gavage did not affect the reproduction of Sprague-Dawley rats and the development ability of their offspring under the experimental conditions.

Multispectral and molecular docking investigations on the interaction of primethamine/trimethoprim with BSA/HSA.

Primethamine (PMA) and trimethoprim (TMP) were investigated as traditional coccidiostats on the binding of bovine serum albumin (BSA) and human serum albumin (HSA) by multispectral and molecular docking techniques. The Stern-Volmer plots and time-resolved fluorescence measurement declared that PMA/TMP quenching the intrinsic fluorescence of BSA/HSA was static quenching process. The binding constants (Ka) and binding sites (n) were calculated at different temperatures. Meanwhile, thermodynamic parameters showed electrostatic forces played a leading role in the interaction of PMA/TMP with BSA/HSA. Some metal ions such as K+, Mg2+, Cu2+, Ca2+, Zn2+ and Fe3+ had no effects on the binding system. The UV-vis absorption spectra confirmed that the interaction between PMA/TMP and BSA/HSA did happen. The analyses of synchronous fluorescence, FT-IR and circular dichroism spectra illustrated that PMA/TMP changed the secondary structures of BSA/HSA. According to Förster non-radiative energy transfer theory, the binding distance between PMA/TMP and BSA/HSA was calculated. The binding location of PMA/TMP to BSA/HSA was identified as sub-domain IIA, which was further confirmed by molecular docking.Communicated by Ramaswamy H. Sarma.

Determination of ethanamizuril, a novel triazine coccidiostat, in rat plasma by ultra-performance liquid chromatography system-tandem mass spectrometry and its application in a toxicological study.

Ethanamizuril is a new triazine compound that has the potential to be a novel anticoccidial drug. Toxicological studies in experimental rats were performed to understand the safety profile of ethanamizuril for drug product development. In this study, a novel, selective and accurate ultra-performance liquid chromatography tandem mass spectrometry method has been developed for the determination of ethanamizuril concentrations in rat plasma. With 4-nitro-o-cresol as an internal standard, sample pretreatment involved a one-step extraction with acetonitrile of 100 µL plasma. The detection was carried out by electrospray ionization mass spectrometry in negative ion mode with selected ion recording. The standard curves were linear (r2 ≥ 0.999) over the concentration range of 0.1-100 µg/mL. The relative standard deviations of intra- and inter-day precisions were less than 8.4 and 8.87%, respectively. The mean extraction recovery of ethanamizuril from rat plasma was 97.68-102.57%. The method was fully validated and successfully applied to monitor plasma concentrations of ethanamizuril in a short-term toxicity study and two-generation reproduction toxicity study. The result of the study confirmed that the elimination of ethanamizuril in rats is slow.

Detection of p-Nitroaniline Released from Degradation of 4,4'-Dinitrocarbanilide in Chicken Breast during Thermal Processing.

The diphenylurea 4,4'-dinitrocarbanilide (DNC) is the residue of concern left in edible tissues of broilers fed diets containing the anticoccidial nicarbazin. When chicken meat is submitted to thermal processing, p-nitroaniline (p-NA) is expected from DNC degradation. This work aimed at evaluating whether thermal processing of DNC-containing chicken meat induces p-NA appearance. First, a hydrolysis assay was performed in aqueous solutions at 100 °C in different pH, confirming that DNC cleavage yields p-NA. Then a novel LC-MS/MS method was used to detect traces of this aromatic amine in DNC-containing chicken breast fillets subjected to cooking methods. Our evidence showed p-NA occurrence in such chicken meat samples, which corroborated results from hydrolysis assay. The p-NA appearance in fillets was rather discrete during boiling treatment, but its concentration became pronounced over time for grilling, frying, and roasting, achieving respectively 326.3, 640.0, and 456.9 µg/kg. As far as we are concerned, no other research identified degradation products from DNC residue in heat-processed chicken fillets. Therefore, this study leads to additional approaches to assess impacts on food safety.

Efficacy of a commercial herbal formula in chicken experimental coccidiosis.

BACKGROUND: Coccidiosis represents a serious threat to the poultry industry, affecting production and causing high morbidity, mortality and significant costs resulting from treatment and prophylaxis. In-feed anticoccidials have been used for decades for managing avian coccidiosis and were very effective until drug resistance emerged. The use of natural remedies has become a promising alternative in combating coccidiosis in chickens. Therefore, the purpose of the present study was to assess the efficiency of a commercial herbal formula (H), as oral liquid preparations, in experimental chicken coccidiosis. METHODS: Two independent controlled battery experiments (BE1 and BE2) were designed and the product was tested in 3 different formulas (H1, H2 and H3): H1 contained a propylene glycol extract of Allium sativum and Thymus serpyllum; H2 contained Origanum vulgare, Satureja hortensis and Chelidonium majus; and H3 contained Allium sativum, Urtica dioica, Inula helenium, Glycyrrhiza glabra, Rosmarinus officinalis, Chelidonium majus, Thymus serpyllum, Tanacetum vulgare and Coriandrum sativum. Chickens were divided into five groups for each BE as follows: (i) uninfected untreated control (UU1, UU2); (ii) infected untreated control (IU1, IU2); (iii) infected treated with amprolium (ITA1, ITA2); and (iv, v) two experimental groups infected treated with H1 (ITH1) and H2 (ITH2) formulas in the BE1 and with H3 (ITH3-5 and ITH3-10) formula in the BE2. The chickens from infected groups were challenged with 5000 (BE1) and 50,000 (BE2) sporulated oocysts of Eimeria spp. (E. acervulina, E. tenella and E. maxima), respectively. The anticoccidial efficacy was assessed by recording the following: oocysts output (OPG), lesion score (LS), weight gain (WG), feed conversion ratio (FCR) and anticoccidial index (ACI). Additionally, polyphenolics and flavonoids (caffeic-chlorogenic acid, apigenin, kaempferol, luteolin, quercitin, quercitrin) from herb extracts found in H3 formula were determined by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. RESULTS: H1 and H2 reduced the WG, and increased the FCR and OPG compared with controls. H1 reduced the duodenal lesions, whilst H2 reduced the caecal lesions, compared with control. H3 decreased the OPG of Eimeria spp., reduced the total lesion score and improved the zootechnical performance (weight gain and feed conversion ratio). According to ACI value, H1 and H2 had no efficacy on Eimeria spp. infection, but H3 had good to marked anticoccidial effect, the ACI being slightly greater in the group ITH3-5. According to the results of LC-MS/MS, the concentration of polyphenols in H3 formula was the highest, the sum of chlorogenic acid and caffeic acid being 914.9 µg/ml. CONCLUSIONS: H3 formula is a promising natural anticoccidial and field trials are recommended in order to validate the obtained data.

A screen-printed electrode modified with tungsten disulfide nanosheets for nanomolar detection of the arsenic drug roxarsone.

A sensitive electrochemical (voltammetric; DPV) sensor has been developed for the determination of coccidiostat drug (roxarsone) based on the use of an SPCE (screen-printed carbon electrode) modified with tungsten disulfide nanosheets (WS2 NSs). The electrochemical detection of roxarsone on the WS2-modified SPCE was examined by electrochemical strategies. XPS, XRD, Raman, SEM, TEM, EDS and EIS were used to characterize the nanosheets. The effects of scan rate, pH values (phosphate buffer) and buffer concentration were optimized. A selective roxarsone sensor was developed that works best at -0.64 V (vs. Ag/AgCl) and performs much better than the bare SPCE. Features include (a) a wider linear range (0.05 to 490 µM), (b) a nanomolar detection limit (0.03 µM) and (c) high sensitivity (29 µA·µM-1·cm-2). The modified SPCEs have been successfully applied to the determination of roxarsone in spiked meat samples where they gave high accuracy and good recoveries. Graphical abstract Synthesis of WS2 nanosheets and electrochemical detection of roxarsone.

Multi-residue methodology for the determination of 16 coccidiostats in animal tissues and eggs by hydrophilic interaction liquid chromatography - Tandem mass spectrometry.

A simple, sensitive and efficient confirmatory method was developed and validated for the determination of 16 coccidiostats in animal tissues and eggs using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The sample preparation consisted of a solid-liquid extraction with ACN and dispersive SPE cleanup with MgSO4 and C18. Analysis was realized in an Acquity BEH HILIC silica column, in SRM mode. Both positive and negative ionization was performed, using polarity switching. Isocratic elution was used with a mobile phase of ACN: aqueous ammonium formate 1 mM with 0.1% formic acid (80:20, v/v). Method validation was performed in eggs, poultry, bovine, ovine, porcine and rabbit tissue and exceptionally low LODs were achieved, varying from 0.004 µg kg-1 (decoquinate in porcine tissue) to 0.560 µg kg-1 (halofuginone in eggs). The developed methodology was applied in 82 muscle and egg samples through the Greek National Residue Control Plan for coccidiostats.

Anti-parasitic effect on Toxoplasma gondii induced by a spider peptide lycosin-I.

Toxoplasmosis is a widely distributed parasitic protozoan disease, caused by Toxoplasma gondii (T. gondii). High prevalence of toxoplasmosis and limitations of conventional treatments lead to a search for new therapeutic drugs. Lycosin-I is a linear peptide, derived from the venom of the spider Lycosa singoriensis. The aim of the present study was to determine the anti-parasitic effect of lycosin-Ι against T. gondii. In vitro, the anti-T. gondii activities of lycosin-Ι were evaluated by MTT assay, trypan blue exclusion assay, cell counting assay and plaque assay. Cytokines of IL-6 and IL-8 were measured by quantitative PCR. In addition, the structures of tachyzoites treated with lycosin-Ι were also observed by scanning and transmission electron microscopy. In vivo, mice were challenged with parasites treated by lycosin-I. The results revealed that lycosin-Ι had shown a significant ability to inhibit T. gondii invasion and proliferation. Cytokines of IL-6 and IL-8 were reduced by lycosin-Ι at transcription level in human foreskin fibroblast (HFF) cells infected with T. gondii tachyzoites, but they were increased compared to non-infected cells. For tachyzoites, lycosin-Ι induced their cell membrane alterations with formation of invaginations, some of them appeared to be vacuolated in their cytoplasm. Moreover, lycosin-Ι had prolonged the survival time of mice by controlling T. gondii proliferation. In conclusion, our present study provides the first evidence for anti-T. gondii by using the spider peptide lycosin-Ι. These findings suggest that lycosin-Ι is a potential alternative agent for the treatment of toxoplasmosis.

Effect of micro-environment modification and polymer type on the in-vitro dissolution behavior and in-vivo performance of amorphous solid dispersions.

This study investigates the effects of micro-environment modification and polymer type on the in-vitro dissolution behavior and in-vivo performance of micro-environment pH modifying solid dispersions (pHM-SD) for the poorly water-soluble model drug Toltrazuril (TOL). Various pHM-SDs were prepared using Ca(OH)2 as a pH-modifier in hydrophilic polymers, including polyethylene glycol 6000 (PEG6000), polyvinylpyrrolidone k30 (PVPk30) and hydroxypropyl methylcellulose (HPMC). Based on the results of physicochemical characterizations and in-vitro dissolution testing, the representative ternary (Ca(OH)2:TOL:PEG6000/HPMC/PVPk30=1:8:24, w/w/w) and binary (TOL:PVPk30=1:3, w/w) solid dispersions were selected and optimized to perform in-vivo pharmacokinetic study. The micro-environment pH modification improved the in-vitro water-solubility and in-vivo bioavailability of parent drug TOL. Furthermore, the addition of alkalizers not only enhanced the release and absorption of prototype drug, but also promoted the generation of active metabolites, including toltrazuril sulfoxide (TOLSO) and toltrazuril sulfone (TOLSO2). The in-vitro dissolution profiles and in-vivo absorption, distribution and metabolism behaviors of the pHM-SDs varied with polymer type. Moreover, in-vivo bioavailability of three active pharmaceutical ingredients increased with an increase in in-vitro dissolution rates of the drug from the pHM-SDs prepared with various polymers. Therefore, a non-sink in-vitro dissolution method can be used to predict the in-vivo performance of pHM-SDs formulated with various polymers with trend consistency. In-vitro and in-vivo screening procedures revealed that the pHM-SD composed of Ca(OH)2, TOL and PVPk30 at a weight ratio of 1:8:24, of which the safety was adequately proved via histopathological examination, may be a promising candidate for providing better clinical outcomes.