Hypericum perforatum extract and hyperforin inhibit the growth of neurotropic parasite Toxoplasma gondii and infection-induced inflammatory responses of glial cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum perforatum L. has been widely used as a natural antidepressant. However, it is unknown whether it is effective in treating infection-induced neuropsychiatric disorders. AIM OF THE STUDY: In order to evaluate the effectiveness of H. perforatum against infection with neurotropic parasite Toxoplasma gondii, which has been linked to neuropsychiatric disorders, this study investigated the anti-Toxoplasma activity using in vitro models. MATERIALS AND METHODS: Dried alcoholic extracts were prepared from three Hypericum species: H. perforatum, H. erectum, and H. ascyron. H. perforatum extract was further separated by solvent-partitioning. Hyperforin and hypericin levels in the extracts and fractions were analyzed by high resolution LC-MS. Anti-Toxoplasma activities were tested in vitro, using cell lines (Vero and Raw264), murine primary mixed glia, and primary neuron-glia. Toxoplasma proliferation and stage conversion were analyzed by qPCR. Infection-induced damages to the host cells were analyzed by Sulforhodamine B cytotoxicity assay (Vero) and immunofluorescent microscopy (neurons). Infection-induced inflammatory responses in glial cells were analysed by qPCR and immunofluorescent microscopy. RESULTS: Hyperforin was identified only in H. perforatum among the three tested species, whereas hypericin was present in H. perforatum and H. erectum. H. perforatum extract and hyperforin-enriched fraction, as well as hyperforin, exhibited significant anti-Toxoplasma property as well as inhibitory activity against infection-induced inflammatory responses in glial cells. In addition, H. perforatum-derived hyperforin-enriched fraction restored neuro-supportive environment in mixed neuron-glia culture. CONCLUSIONS: H. perforatum and its major constituent hyperforin are promising anti-Toxoplasma agents that could potentially protect neurons and glial cells against infection-induced damages. Further study is warranted to establish in vivo efficacy.

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.

Anticoccidial effect of Fructus Meliae toosendan extract against Eimeria tenella.

CONTEXT: Fructus Meliae toosendan extracts (FMTE) have a good therapeutic effect on coccidiosis, but there is no relevant research on its prophylactic effect on coccidiosis. OBJECTIVE: This study comprehensively evaluates the anticoccidial effect of FMTE. MATERIALS AND METHODS: In vitro, the unsporulated oocysts were treated with serial dilutions of FMTE and incubated for 7 d, and the sporulated oocysts were counted for calculating the median lethal concentration (LC50) of FMTE. In vivo, 180 10-day-old broiler chickens free of coccidiosis were weighted and randomly distributed into six groups: normal group, untreated group, 4 protective groups (positive group and three FMTE groups). From day 10 to day 21, chickens in the three FMTE groups were pre-treated with FMTE at the dosage of 2.5, 5 and 10 g/kg/d, respectively, and chickens in the positive group were pre-treated with qiuliling (10 g/kg/d). On day 14, chickens in all groups except the normal group were orally infected with 1.5 × 104 sporulated oocysts. The clinical symptoms were observed from day 10 to day 21, the anticoccidial index (ACI), tissue lesions, and intestinal microflora were determined on day 21. RESULTS: FMTE showed anti-sporulation effect against E. tenella and the LC50 value was 245.83 µg/mL in vitro. In vivo, FMTE at the dosage of 10 g/kg/d was effective against E. tenella infection, and its ACI value was 162.56, which was higher than the value of positive drug qiuliling (128.81). Discussion and conclusions: FMTE have potent anticoccidial effects, and it presents an alternative anticoccidial agent for avian coccidiosis control.

A new sample treatment strategy based on simultaneous supramolecular solvent and dispersive solid-phase extraction for the determination of ionophore coccidiostats in all legislated foodstuffs.

A single-step sample treatment, for the simultaneous extraction and clean-up for the determination of ionophore coccidiostats in EU legislated foodstuffs, is here proposed. The treatment is based on the combination of: (i) a supramolecular solvent with restricted access properties (SUPRAS-RAM), spontaneously formed by the addition of hexanol, water and THF to the sample; and (ii) dispersive solid phase extraction (dSPE). The SUPRAS-RAM extract was directly compatible with LC-MS/MS and no further re-extraction, evaporation or cleanup procedures were necessary. SUPRAS-RAM efficiently extracted the ionophores (recoveries in milk, eggs, fat, liver, kidney, and chicken and beef muscle were in the range 71-112%) and removed proteins and carbohydrates, whereas dSPE removed fats and other lipophilic compounds. The method was validated following the European Commission Decision 2002/657/EC. Detection limits (0.004-0.07 µg kg-1) were far below the maximum residue limits (1-150 µg kg-1). Method analytical and operational characteristics were suitable for routine determination of ionophores.

A new sensitive method for the simultaneous chromatographic separation and tandem mass spectrometry detection of anticoccidials, including highly polar compounds, in environmental waters.

A sensitive and selective method was developed and validated for the determination of 26 anticoccidial compounds (six ionophores and twenty chemical coccidiostats) in surface and groundwater samples at parts-per-quadrillion (pg L-1) to parts-per-trillion (ng L-1) levels by ultra-high performance liquid chromatography with tandem mass spectrometry detection (UHPLC-MS/MS). A range of different analytical columns and mobile phase compositions were evaluated to enhance selectivity and retention of a number of highly polar and basic anticoccidials along with other non-polar coccidiostats. A combined separation, including these problematic polar compounds, was achieved on a phenyl-hexyl column, by binary gradient elution with water/acetonitrile using ammonium formate and formic acid as additives. The anticoccidial residues were extracted from raw, unfiltered, water samples (250 mL) using polymeric divinylbenzene solid phase extraction (SPE) cartridges, with subsequent elution (methanol:acetonitrile:ethyl acetate, 40:40:20, v/v) and concentration prior to determination. The method recovery (at a concentration representative of realistic expected environmental water concentrations based on literature review) ranged from 81% to 105%. The method was successfully validated for 26 anticoccidials, at four concentration levels, in accordance to Commission Decision 2002/657/EC and SANTE/11813/2017 guidelines. Trueness and precision, under within-laboratory reproducibility conditions, ranged from 88% to 111% and 0.9% to 10.3% respectively.

Multiple response optimization of a QuEChERS extraction and HPLC analysis of diclazuril, nicarbazin and lasalocid in chicken liver.

A method for the simultaneous determination of three commonly used coccidiostats in chicken liver was developed, comprising a multi-residue QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction step, and a liquid chromatography-ultra violet-fluorescence (HPLC-UV/FL) analysis. The QuEChERS extraction was optimized using an experimental design approach that includes a screening step to obtain the critical variables, an optimization step using multiple response surface analysis and the calculation of a desirability parameter. The optimized method was validated with fortified samples, reaching an average recovery of 91% and an overall precision of 5.5% (mean of three analytes at three levels). Limits of detection calculated on fortified samples were 20 µg kg-1 for lasalocid, 15 µg kg-1 for nicarbazin and 120 µg kg-1 for diclazuril. These values resulted at least one order of magnitude lower than the maximum allowed residue limit (MRL) of the studied coccidiostats for chicken liver.

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.

Anticoccidial effects of Artemisia annua ethanolic extract: prevention, simultaneous challenge-medication, and treatment.

The effect of Artemisia annua ethanolic extract (AE) as a potential source of herbal anticoccidial activity was investigated on experimental coccidiosis in chicken. One hundred ninety-two one-day-old chicks were divided in to 8 groups (n = 24) including AE prevention group, AE-treated group, simultaneously challenged AE-medicated group, challenged-untreated group (positive control), unchallenged-untreated group (negative control), salinomycine prevention group, salinomycine-treated group, and simultaneously challenged salinomycine-medicated group, in a completely randomized design. Oral challenge carried out by a suspension containing a mixture of 200,000 oocysts Eimeria acervulina, 30,000 oocysts Eimeria necatrix, and 20,000 oocysts Eimeria tenella on day 21 of age. Weight gain in AE prevention group significantly increased compared to positive control group (p < 0.05). Unlike salinomycine prevention group, the food conversion ratio (FCR) of AE prevention group was not significantly higher than negative control. Oocyst per gram (OPG) in simultaneously challenged AE-medicated group had no significant difference, while for 38% of the days, in simultaneously challenged salinomycine-medicated group significantly decreased (p < 0.05). The food intake of AE-treated group had no significant difference with salinomycine-treated group (p > 0.05). In half of the days of OPGs sampling, AE-treated group was reduced significantly compared to positive control group (p < 0.05). Collectively, the in vivo study of anticoccidial effects of AE in the prevention section was more effective than the treatment section, while the treatment section was more effective than the simultaneous section. We concluded that AE has a potential value to use as an herbal medicine for preventive measure in chicken coccidiosis.

Multiresidue method for the simultaneous determination of veterinary medicinal products, feed additives and illegal dyes in eggs using liquid chromatography-tandem mass spectrometry.

A multiclass method was developed for the simultaneous determination of 120 analytes in fresh eggs. The method covers the analytes from the groups of tetracyclines (6), fluoroquinolones (11), sulphonamides (17), nitroimidazoles (9), amphenicols (2), cephalosporins (7), penicillins (8), macrolides (8), benzimidazoles (20), coccidiostats (14), insecticides (3), dyes (12) and others (3). Samples were extracted using 0.1% formic acid in acetonitrile:water (8:2) with the addition of EDTA and cleaned using solid phase extraction with Hybrid SPE cartridges. The chromatographic separation was achieved on C8 column using mobile phase consisting of (A) methanol:acetonitrile (8:2) - (B) 0.1% formic acid in a gradient mode. Validation results according to the Commission Decision 2002/657/EC are as follows: linearity (r⩾0.99), recovery (75-108%), repeatability (CV 1.60-15.9%), reproducibility (CV 2.60-15%), decision limit (CCα 2.25-1156 µg/kg) and detection capability (CCß 2.04-1316 µg/kg). The presented method was used for analysis of 150 real eggs samples taken from monitoring control program.

Insights into anti-parasitism induced by a C-type lectin from Bothrops pauloensis venom on Toxoplasma gondii.

Here we evaluate the effects of BpLec, a C-type lectin isolated from Bothrops pauloensis snake venom, on Toxoplasma gondii parasitism. BpLec (0.195-12.5 µg/mL) did not interfere with HeLa (host cell) viability by MTT assay, whereas higher doses decreased viability and changed HeLa morphology. In addition, the host cell treatment before infection did not influence adhesion and proliferation indexes. BpLec did not alter T. gondii tachyzoite viability, as carried out by trypan blue exclusion, but decreased both adhesion and parasite replication, when tachyzoites were treated before infection. Galactose (0.4 M) inhibited the BpLec effect on adhesion assays, suggesting that BpLec probably recognize some glycoconjugate from T. gondii membrane. Additionally, we performed cytokine measurements from supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BpLec. MIF and IL-6 productions by HeLa cells were increased by BpLec treatment. Also, TGF-ß1 secretion was diminished post-infection, although this effect was not dependent on BpLec treatment. Taken together, our results show that BpLec is capable of reducing T. gondii parasitism after tachyzoite treatment and may represent an interesting tool in the search for parasite antigens involved in these processes.

The effects of combining Artemisia annua and Curcuma longa ethanolic extracts in broilers challenged with infective oocysts of Eimeria acervulina and E. maxima.

Due to an increasing demand for natural products to control coccidiosis in broilers, we investigated the effects of supplementing a combination of ethanolic extracts of Artemisia annua and Curcuma longa in drinking water. Three different dosages of this herbal mixture were compared with a negative control (uninfected), a positive control (infected and untreated), chemical coccidiostats (nicarbazin+narazin and, later, salinomycin), vaccination, and a product based on oregano. Differences in performance (weight gain, feed intake, and feed conversion rate), mortality, gross intestinal lesions and oocyst excretion were investigated. Broilers given chemical coccidiostats performed better than all other groups. Broilers given the two highest dosages of the herbal mixture had intermediate lesion scores caused by Eimeria acervulina, which was higher than in broilers given coccidiostats, but less than in broilers given vaccination, oregano and in negative controls. There was a trend for lower mortality (P = 0·08) in the later stage of the growing period (23-43 days) in broilers given the highest dosage of herbal mixture compared with broilers given chemical coccidiostats. In conclusion, the delivery strategy of the herbal extracts is easy to implement at farm level, but further studies on dose levels and modes of action are needed.

In vivo anticoccidial activity of berberine [18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium]--an isoquinoline alkaloid present in the root bark of Berberis lycium.

Coccidiosis, caused by various Eimeria species, is a major parasitic disease in chicken. However the increasing resistance of these parasites to currently used anticoccidial drugs has stimulated the search for new methods of control. As part of this effort we investigated the root bark of Berberis lycium (barberry) as a potential source of compounds with anticoccidial activity. In the present study anticoccidial activity of different solvent extracts of the root bark of B. lycium and berberine was evaluated in vivo using broiler chicken. Results of the study demonstrated equipotent efficacy of pure berberine in comparison to that of standard drug amprolium on the basis of reduction in coccidian oocyst output, body weight gain of chicken and feed conversion ratio. Among the extracts crude methanolic extract showed highest anticoccidial activity tested at 300 mg/kg body weight which could be due to the presence of alcohol-soluble active ingredients in root bark of B. lycium. Toxicological studies revealed that B. lycium extracts as well as berberine were not lethal up to dosage of 2000 mg/kg body weight. LD(50) was not determined as mortalities were not recorded in any of the five groups of chicken. From the present study it can be concluded that root bark of B. lycium has the immense potential to contribute to the control of coccidian parasites of chicken. Our results corroborate the use of berberine for treatment of severe diarrhoea, amoebiasis and intestinal infections and could justify its use in folk medicine for treatment of haemorrhagic dysentery.

Quantification of four ionophores in soil, sediment and manure using pressurised liquid extraction.

A multi-residue pressurised liquid extraction (PLE) methodology has been established for the determination of the four ionophores: lasalocid, monensin, salinomycin and narasin in solid environmental matrices. The PLE methodology is combined with solid phase extraction as clean-up using liquid chromatography coupled to tandem mass spectrometry applying electrospray ionisation for detection. The samples were freeze-dried prior to extraction. The absolute recoveries for soil and sediment ranged from 71 to 123% (relative standard deviation (RSDs) below 16%) and in the range 94-133% (RSDs 9-35%) for poultry manure. The final method allowed for the detection of four ionophores down to a few hundred ngkg(-1) in natural solid matrices with limit of quantifications (LOQs) being 0.96, 0.87, 0.98, and 0.64µgkg(-1) in soil for lasalocid, monensin, salinomycin, and narasin, respectively. Corresponding LOQs in sediment were 1.28, 1.34, 1.39, and 0.78µgkg(-1) for the respective ionophores, while in manure the LOQs were 0.98, 1.01, 1.45, and 1.01µgkg(-1).

Development and validation of a multi-residue liquid chromatography-tandem mass spectrometry confirmatory method for eleven coccidiostats in eggs.

A confirmatory method for the determination of residues of eleven coccidiostats including ionophore antibiotics: lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin and chemical coccidiostats: decoquinate, diclazuril, halofuginone, nicarbazin and robenidine in poultry eggs was developed and validated. The sample was extracted with acetonitrile, defatted with hexane and cleaned-up on a silica SPE cartridge. The analytes were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method performance characteristics required by Commission Decision 2002/657/EC were estimated adopting a more flexible and simple validation design. In this alternative approach the experimental study focuses on a larger dynamic range with progressively increasing validation levels. Each of them presents equal concentrations of all the analytes. On the contrary the classical Decision plan investigates a restricted concentration range necessarily different for each analyte being determined by the individual permitted limit (0.5-1.5 times the permitted limit). As a consequence each validation level involves the simultaneous fortification with complex mixtures containing different concentrations of each analyte. Adopting this alternative strategy the validation of several substances with significantly different permitted limits is considerably simplified and a deeper knowledge of the method is achieved. The results proved that the method enables the confirmation of regulated coccidiostats in eggs at the levels required in the official control of residues (CCα in the range of 2.2-174 µg kg(-1), depending on the coccidiostat). The repeatability (CV(r) in the range of 1.1-19%) and within-laboratory reproducibility (CV(Rw) in the range of 1.8-30%) are also acceptable. The procedure was successfully verified in the proficiency test and implemented in the national residue control plan.

Determination of residual clopidol in chicken muscle by capillary gas chromatography-negative chemical ionization-mass spectrometry.

A selective, sensitive gas chromatography-mass spectrometry (GC-MS) method with negative chemical ionization (NCI) was developed for the detection and quantification of clopidol in chicken muscle. Chicken muscle samples were extracted with acetonitrile and concentrated to dryness; the residue was redissolved in ethyl acetate and applied to an Alumina B cartridge for cleanup. The residue was derivatized with Sylon BFT and analyzed by GC-NCI-MS. The selected-ion monitoring mode was performed at m/z values of 156, 158, 191, and 193. The differences in the ratios for the standards and spikes in chicken muscle were within the acceptability criteria. All recoveries of the drug from chicken muscle spiked at 0.5, 5.0 and 50.0 microg/kg were 74.5-95.6% intra-day, and 71.6-94.8% inter-day, respectively, with relative standard deviations being lower than 15%. The limits of detection and quantitation were 0.1 and 0.5 microg/kg, respectively. The NCI mode had better selectivity and sensitivity than the electron impact (EI) mode for clopidol.

Isolation, structure, and coccidiostat activity of coccidiostatin A.

Coccidiosis is one of the more common and costly diseases in poultry that is caused by various Eimeria species. In our quest to discover coccidiostats from natural products, we discovered a microbial fermentation extract that exhibited in vivo anticoccidial activity. Fractionation of this extract led to the discovery of two potent antiprotozoals, emecorrugatin A (1) and coccidiostatin A (2). The former compound exhibited only in vitro activity, whereas the latter new compound exhibited in vivo activity against Eimeria species in chickens at 150 ppm dosed in chicken feed. The isolation, structure elucidation, relative configuration, and activity of coccidiostatin A (2) are described.

Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase.

Trisubstituted pyrrole inhibitors of the essential coccidian parasite cGMP dependent protein kinase (PKG) block parasite invasion and show in vivo efficacy against Eimeria in chickens and Toxoplasma in mice. An imidazopyridine inhibitor of PKG activity with greater potency in both parasite invasion assays and in vivo activity has recently been identified. Susceptibility experiments with a Toxoplasma knock-out strain expressing a complementing compound-refractory PKG allele ('T761Q-KO'), suggest a role for additional secondary protein kinase targets. Using extracts from this engineered T. gondii strain and a radiolabeled imidazopyridine ligand, a single peak of binding activity associated with calmodulin-like domain protein kinase (CDPK1) has been identified. Like PKG, CDPK1 has been implicated in host cell invasion and exhibits sub-nanomolar sensitivity to the compound. Amino acid sequence comparisons of coccidian CDPKs and a mutational analysis reveal that the binding of the ligand to PKG and CDPK1 (but not other CDPK isoforms) is mediated by similar contacts in a catalytic site hydrophobic binding pocket, and can be blocked by analogous amino acid substitutions. Transgenic strains over-expressing a biochemically active but compound-refractory CDPK1 mutant ('G128Q') fail to show reduced susceptibility to the compound in vivo, suggesting that selective inhibition of this enzyme is not responsible for the enhanced anti-parasitic potency of the imidazopyridine analog. An alternative secondary target candidate, the alpha-isoform of casein kinase 1 (CK1alpha), shows sensitivity to the compound in the low nanomolar range. These results provide an example of the utility of the Toxoplasma model system for investigating the mechanism of action of novel anticoccidial agents.

Microwave extraction of incurred salinomycin from chicken tissues.

A rapid, accurate, environmentally friendly and cost-effective microwave extraction technique was developed for the extraction of spiked and incurred salinomycin from chicken tissues (kidney, liver, muscle, ovarian yolk and fat). Extraction of salinomycin from various tissues was achieved by irradiating the sample in absolute ethanol and 2-propanol (15 + 2) for 9 sec. in a common household microwave oven. The extract was analysed without further cleanup by HPLC on a C18 column (5 microns) and detected at 592 nm via post-column reaction with 4-dimethylaminobenzaldehyde (DMABA) in a heated reactor coil at 86 degrees C. Recoveries of salinomycin from spiked tissues at 30 ng/g level ranged between 87 and 100%. The limit of quantitation was found to be 10 ng/g. The developed method was applied for the analysis of incurred tissues and ovarian yolk of laying chickens given sodium salinomycin in feed at different levels for 14 consecutive days followed by withdrawal periods. Residues were detected in all tissues and ovarian yolk at 0 withdrawal time but declined during the withdrawal period. Highest residue were found in fat and ovarian yolk.

Fudecalone, a new anticoccidial agent produced by Penicillium sp. FO-2030.

Penicillium sp. FO-2030, a soil isolate, was found to produce a new anticoccidial compound. The active compound, designated fudecalone, was isolated from the fermentation broth of the producing strain by solvent extraction, silica gel column chromatography and preparative HPLC. The structure of fudecalone was elucidated to be 3,3a,6,6a,7,8,9,10-octahydro-1-hydroxy-4,7,7-trimethyl-1H-naphtho[1,8a- c]furan-6-one mainly by spectroscopic studies including various NMR measurements. The anticoccidial activity using cell systems indicated that schizont formation of monensin-resistant Eimeria tenella was completely inhibited by fudecalone at concentrations more than 16 microM.