Synthesis, computational simulations and biological evaluation of new dual 5HT1A/5HT7 receptor ligands based on purine-2,6-dione scaffold.

The new dual 5HT1A/5HT7 receptor ligands were designed based on the purine-2,6-dione scaffold with the fluorine atom. Twenty-one new derivatives were synthesized, and their structure-activity relationship was summarized. Compound 11 (7-(2-(3-fluorophenyl)-2-oxoethyl)-8-((4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione) showed the highest affinity to 5HT1AR and 5HT7R, and was the most potent antagonist of 5-HT1AR (Kb = 0.26 ± 0.1 nM) which activity can be to reference compound NAN-190 (Kb = 0.26 ± 0.1 nM). The experimentally established physicochemical parameters of compound 11 showed that compound, as slightly ionized in the blood, could penetrate the blood-brain barrier. A molecular docking study showed that the fluorine substitution introduces additional stabilization effects on binding to 5HT1A/5HT7Rs. In animal assays of depression and anxiety, compound 11 revealed activity in terms of dosage compared to marketed psychotropics such as fluoxetine, citalopram, and sertraline.

Concentration of Polycyclic Aromatic Hydrocarbons (PAHs) in Human Serum and Adipose Tissues and Stimulatory Effect of Naphthalene in Adipogenesis in 3T3-L1 Cells.

Polycyclic aromatic hydrocarbons (PAHs) are one of the most prevalent classes of environmental pollutants. Some evidence shows that PAHs could be involved in human obesity. However, little is known about the distribution patterns of PAHs in human adipose tissue (AT) and the role of PAHs on adipogenesis/lipogenesis. The aims of this pilot study were to determine concentrations of 16 PAHs defined as high-priority pollutants in the plasma and adipose tissue of French and Polish bariatric patients, as well as their correlation with body mass index (BMI), plasma and AT adipokines expression levels. We finally investigated the role of naphthalene on cell proliferation, viability, and differentiation in 3T3-L1 preadipocytes. The concentration of most PAHs was similar in the three types of AT and it was significantly higher in AT as compared to plasma, suggesting bioaccumulation. Polish patients had higher PAH levels in AT than French ones. Only the concentration of naphthalene in AT was positively correlated with the BMI and serum or adipose chemerin, adiponectin and resistin expression, in French but not in Polish patients, who had significantly higher BMIs. Moreover, naphthalene exposure increased the cell proliferation of 3T3-L1 preadipocytes and lipogenesis, and increased the expression of genes involved in adipogenesis after cell differentiation. Taken together, PAHs and more particularly naphthalene could be an obesogenic molecule and increase the risk of obesity.

Development and Validation of Stability-Indicating HPLC Methods for the Estimation of Lomefloxacin and Balofloxacin Oxidation Process under ACVA, H2O2, or KMnO4 Treatment. Kinetic Evaluation and Identification of Degradation Products by Mass Spectrometry.

The oxidation of lomefloxacin (LOM) and balofloxacin (BAL) under the influence of azo initiator of radical reactions of 4,4'-azobis(4-cyanopentanoic acid) (ACVA) and H2O2 was examined. Oxidation using H2O2 was performed at room temperature while using ACVA at temperatures: 40, 50, 60 °C. Additionally, the oxidation process of BAL under the influence of KMnO4 in an acidic medium was investigated. New stability-indicating HPLC methods were developed in order to evaluate the oxidation process. Chromatographic analysis was carried out using the Kinetex 5u XB-C18 100A column, Phenomenex (Torrance, CA, USA) (250 × 4.6 mm, 5 µm particle size, core shell type). The chromatographic separation was achieved while using isocratic elution and a mobile phase with the composition of 0.05 M phosphate buffer (pH = 3.20 adjusted with o-phosphoric acid) and acetonitrile (87:13 v/v for LOM; 80:20 v/v for BAL). The column was maintained at 30 °C. The methods were validated according to the ICH guidelines, and it was found that they met the acceptance criteria. An oxidation process followed kinetics of the second order reaction. The most probable structures of LOM and BAL degradation products formed were assigned by the UHPLC/MS/MS method.

Phytochemical and biotechnological studies on Schisandra chinensis cultivar Sadova No. 1-a high utility medicinal plant.

In the presented work, raw materials (fruits and leaves) and in vitro biomass of a highly productive Schisandra chinensis Sadova No. 1 cultivar (SchS) were evaluated for the production of therapeutically useful schisandra lignans (SL). In vitro cultures of SchS were initiated, followed by extensive optimization studies focused on maximizing secondary metabolite production, with the aim of establishing a sustainable source of SL. Different cultivation systems (agar, agitated, bioreactor), experiment times (10, 20, 30, 40, 50 and 60 days) and plant growth regulators (6-benzyladenine-BA and 1-naphthaleneacetic acid-NAA, from 0 to 3 mg/l) in Murashige-Skoog (MS) medium were tested. Moreover, an elicitation procedure was applied to bioreactor-grown microshoots in order to increase SL production. Validated HPLC-DAD protocol enabled to detect fourteen SL in the extracts from in vitro and in vivo materials. The main compounds in the in vitro cultures were as follows: schisandrin (max. 176.3 mg/100 g DW), angeloylgomisin Q (max. 85.1 mg/100 g DW), gomisin A (max. 71.4 mg/100 g DW) and angeloylgomisin H (max. 67.0 mg/100 g DW). The highest total SL content (490.3 mg/100 g DW) was obtained in extracts from the biomass of agar cultures cultivated for 30 days on the MS medium variant containing 3 mg/l BA and 1 mg/l NAA. This amount was 1.32 times lower than in fruit extracts (646.0 mg/100 g DW) and 2.04 times higher than in leaf extracts (240.7 mg/100 g DW). The study demonstrated that SchS is a rich source of SL, thus proving its value for medical, cosmetic and food industry.

Determination of losartan potassium, quinapril hydrochloride and hydrochlorothiazide in pharmaceutical preparations using derivative spectrophotometry and chromatographic-densitometric method.

Two methods, spectrophotometric and chromatographic-densitometric ones, were developed for determination of losartan potassium, quinapril hydrochloride and hydrochlorothiazide in pharmaceutical preparations. Spectrophotometric method involved derivative spectrophotometry and zero order spectrophotometry. The measurements were carried out at lambda = 224.0 nm for quinapril, lambda = 261.0 nm for hydrochlorothiazide and lambda = 270.0 nm for losartan when the derivative spectrophotometry was applied and lambda = 317.0 nm when zero order spectrophotometry was applied for the determination of hydrochlorothiazide. In chromatographic-densitometric studies high performance thin layer chromatography (HPTLC) plates were used as stationary phase and a mixture of solvents n-butanol : acetic acid : water (15 : 5 : 1, v/v/v) as mobile phase. Under the established conditions good resolution of examined constituents was obtained. Retardation factor for quinapril hydrochloride was R(f) - 0.70, for losartan potassium R(f) - 0.85 and for hydrochlorothiazide R(f) - 0.78. The developed methods are characterized by high sensitivity and accuracy. For quantitative analysis, densitometric measurements were carried out at lambda = 218.0 nm for quinapril, lambda = 275.0 nm for hydrochlorothiazide and = 232.0 nm for losartan.

Optimization of method for zinc analysis in several bee products on renewable mercury film silver based electrode.

Zinc is an interesting target for detection as it is one of the elements necessary for the proper functioning of the human body, its excess and deficiency can cause several symptoms. Several techniques including electrochemistry have been developed but require laboratory equipment, preparative steps and mercury or complex working electrodes. We here described the development of a robust, simple and commercially available electrochemical system. Differential pulse (DP) voltammetry was used for this purpose with the cyclic renewable mercury film silver based electrode (Hg(Ag)FE) and 0.05 M KNO3 solution as a supporting electrolyte. The effect of various factors such as: preconcentration potential and time, pulse amplitude and width, step potential and supporting electrolyte composition are optimized. The limits of detection (LOD) and quantification (LOQ) were 1.62 ng/mL and 4.85 ng/mL, respectively. The repeatability of the method at a concentration level of the analyte as low as 3 ng/mL, expressed as RSD is 3.5% (n = 6). Recovery was determined using certified reference material: Virginia Tobacco Leaves (CTA-VTL-2). The recovery of zinc ranged from 96.6 to 106.5%. The proposed method was successfully applied for determination of zinc in bee products (honey, propolis and diet supplements) after digestion procedure.

Stability studies of clonazepam, diazepam, haloperidol, and doxepin with diverse polarities in an acidic environment.

Stability of clonazepam, diazepam, haloperidol, and doxepin was determined in acidic solutions. In addition, determination of the kinetic and thermodynamic properties of this stability was carried out. Reaction rate constants (k), half-life times (t(0.1) and t(0.5)), and activation energy (Ea) were estimated for the drugs, which differed in polarity expressed with log P values. It was observed that estimated Ea values increased from 42.13 to 125.03 kJ/mol with an increase of lipophilicity (log P) beginning from the most hydrophilic drug (clonazepam, 2.70 log P) to the most lipophilic drug (doxepin, 4.10 log P). All degradation products were studied using an HPLC/electrospray ionization-MS technique in the positive ionization mode.

Analysis of indole compounds in Armillaria mellea fruiting bodies.

A TLC method with densitometric detection for identification and quantitation of indole compounds in methanolic extracts of fruiting bodies of Armillaria mellea (Vahl.) P. Kumm. (Basidiomycota)--Honey mushroom has been developed. The TLC method with densitometric detection was validated for determination of tryptamine and serotonin contents. The use of this chromatographic-densitometric method for analysis of extracts allowed for a precise, easy and quick determination of the compounds under study. We identified three physiologically active indole compounds: tryptamine. L- tryptophan and serotonin. This analysis demonstrated the highest contents of L-tryptophan (4.467 mg/100 g d.w.). The contents of tryptamine were comparable with the contents of serotonin and amounted to: 2.740 and 2.207 mg/100 g d. w.

Analysis of hypotensive compounds occurring in complex agents.

This is a review of analytical methods, such as spectrophotometry, derivative spectrophotometry and various chromatographic (gas chromatography-GC, high-performance liquid chromatography-HPLC, thin-layer chromatography-TLC, high-performance thin-layer chromatography-HPTLC, liquid chromatography-tandem mass spectrometry-LC-MS, microchip electrophoresis-MCE, capillary electrophoresis-CE) and electroanalytical methods (differential pulse polarography-DPP, cathodic stripping voltammetry-CSV, anodic stripping voltammetry-ASV, differential pulse voltammetry-DPV, cyclic voltammetry-CV, stripping voltammetry-SV, square wave voltammetry-SWV, square wave polarography-SWP) that are used in the analysis of hypotensive complex agents. This review is based on representative publications that were published between 1995 and 2009.

Determination of risperidone in tablets in the presence of its degradation products and placebo-derived constituents.

New chromatographic-densitometric assay was developed for identification and determination of risperidone in pharmaceutical formulations. Thin-layer chromatographic plates (TLC-F254) as a stationary phase and n-butanol-acetic acid-water (12:3:5 v/v/v) as a mobile phase were used for separation. Densitometric measurements were done for all constituents at lambda = 280 nm. A decrease in stability of risperidone was observed in acidic, basic and antioxidant solutions. Degradation of active pharmaceutical ingredient was consistent with first-order kinetics and unrelated to the solution. This assay is specific for risperidone. No interference of tablet origin adjuvants and degradation products were observed. Moreover, high sensitivity, limit of detection (0.22 microg/spot), limit of quantitation (0.67 microg/spot), recovery (98.2-100.82%), broad linear range (0.09 microg/spot to 1.40 microg/spot) and accuracy (1.87% RSD) are characteristic traits of the chromatographic-densitometric assay.

Mycoremediation of azole antifungal agents using in vitro cultures of Lentinula edodes.

Azole antifungal agents are widely used as active ingredients in antifungal pharmaceuticals and agricultural fungicides. An increase in the use of azole antifungals has resulted in an increase in the concentration of these compounds in wastewater and surface water, with potential implications for agriculture. In the present study, bifonazole (BIF) and clotrimazole (CTZ) were selected for investigation because of their widespread use in topical formulations and persistence in the environment. The mycoremediation capacity of BIF and CTZ by mycelia of Lentinula edodes in in vitro culture was evaluated. The main aim of this study was to identify the presumable biodegradation products of the investigated active pharmaceutical substances using the LC/MS/MS method. For this purpose, the media were enriched with the following active pharmaceutical ingredients selected for this study: BIF powder, CTZ powder, and BIF cream, each of them at the same concentration of 0.1 mg/mL. Subsequently, thin-layer chromatography coupled with densitometry was used to evaluate the content of BIF and CTZ in mycelium from in vitro cultures of L. edodes. The degradation process was found to affect primarily the imidazole moiety of both investigated compounds. In addition, the amounts of undegraded investigated compounds were found to be 4.98, 9.26, and 4.56 mg/g dry weight for BIF powder, CTZ powder, and BIF cream, respectively. Therefore, the findings of this study revealed that L. edodes could be considered for remediation of pollution caused by azole antifungal agents.

Application of derivative spectrophotometry for determination of enalapril, hydrochlorothiazide and walsartan in complex pharmaceutical preparations.

A derivative spectrophotometry method was developed to determine enalapril, hydrochlorothiazide, candesartan and walsartan in complex antihypertensive drugs. The pharmaceutical preparations containing hydrochlorothiazide and one of the angiotensin convertase inhibitors were investigated. It was found that the developed method enables the constituents of the investigated drugs to be determined directly despite evident interference of the zero order absorption spectra. For determination of enalapril and hydrochlorothiazide as well as candesartan and hydrochlorothiazide the first derivative was used, while for walsartan and hydrochlorothiazide the second derivative was employed. The method was of high sensitivity; the LOD accuracy for enalapril was 2.81 microg x mL(-1), 0.56 microg x mL(-1) for candesartan, 4.02 microg x mL(-1) for walsartan and ranged from 0.31 microg x mL(-1) to 1.78 microgxmL(-1) for hydrochlorothiazide, depending on preparation under investigation. The recovery of individual constituents was within the limit of 100% +/- 5%, RSD varied from 1.11% to 2.94%, and the linearity range was from 4.1 microg x mL(-1) to 20.5 microg x mL(-1) for enalapril, from 6.45 microg x mL(-1) to 32.25 microg x mL(-1) for walsartan, from 2.36 microg x mL(-1) to 11.80 microg x mL(-1) for candesartan, and from 0.96 microg x mL(-1) to 26.00 microg x mL(-1) for hydrochlorothiazide.

Seasonal variations of PCDD/Fs congeners in air, soil and eggs from a Polish small-scale farm.

The transfer of dioxin from the environment to the food is a problem in a consumers' health protection. The study aimed to determine the concentration of dioxins in free-range chicken eggs, air and soil samples, collected during 12 months on an individual small farm, located in Malopolska region, Poland. In the majority of analyzed eggs, the concentrations of dioxin exceeded several times the legal limit of 2.5 pg WHO-TEQ g-1fat. Seasonal changes in the PCDD/Fs congeners in egg, air and soil samples were studied. During the winter season, when the combustion processes of the solid fuel in domestic furnaces are intensive, the PM10 concentration in the Malopolska region exceeds the legal limit (50µg/m3) even eight times. In this period, eggs, air and soil samples showed a higher share of PCDFs with a specific contribution of 2,3,7,8-TCDF. During the summer months, in the egg, air and soil samples, the share of PCDDs is higher with dominant OCDD and 1,2,3,4,6,7,8-HpCDDs, showing the effect of other combustion processes such as grass utilization or burning plastic wastes in controlled fires. In August, the month of the highest average air temperature and lowest rainfall amount, the highest toxicity of PCDD/Fs in eggs (9.52pgWHO-TEQ g-1fat) was found. Due to the similarity of the shares of PCDD/Fs congeners in total WHO-TEQ value we can take into account the influence of toxicity of PCDD/Fs in the air and soil on the toxicity in the eggs.

Simultaneous Determination of Acetylsalicylic Acid, Hydrochlorothiazide, Enalapril, and Atorvastatin in a Polypill-Based Quaternary Mixture by TLC.

A new chromatographic-densitometric method has been developed for the qualitative and quantitative determination of the active ingredients in a simulated mixture corresponding to the PolyIran polypill, composed of acetylsalicylic acid, hydrochlorothiazide (HCT), enalapril (ENA), and atorvastatin (ATR), whose efficacy in the treatment and prevention of cardiovascular disease has been documented in clinical trials. Chromatographic separation was performed using TLC silica gel 60 plates with fluorescent indicator F254 as the stationary phase and a mixture of n-hexane-ethyl acetate-methanol-water-acetic acid (8.4 + 8 + 3 + 0.4 + 0.2, v/v/v/v/v) as the mobile phase. Densitometric measurements were carried out at λ = 210 nm when determining ENA and at λ = 265 nm in the case of the other drugs. Peaks of examined substances were well separated in the recorded chromatograms, enabling the evaluation of the results in terms of both qualitative and quantitative analysis. The method was specific for the analyzed components and was characterized by high sensitivity. The LOD was between 0.043 and 0.331 µg/spot, and LOQ was between 0.100 and 0.942 µg/spot. Recovery was in the range of 97.02-101.34%. The linearity range was broad and ranged from 0.600 to 6.000 µg/spot for acetylsalicylic acid, from 0.058 to 1.102 µg/spot for HCT, from 0.505 to 6.560 µg/spot for ENA, and from 0.100 to 1.000 µg/spot for ATR. The method was characterized by good precision, with RSD values that ranged from 0.10 to 2.26%.

Nitric oxide spin-trapping and NADPH-diaphorase activity in mature rat brain after injury.

Traumatic brain injury (TBI) induces inflammatory reactions, and one of the essential mediators of this reaction is nitric oxide (NO). The action of this compound is still under study because no clear consensus has been reached about its exact action in the central nervous system. Further, it is unknown if, in the damaged brain, its neuroprotective activity outweighs its putative neurodegenerative properties. Using ferrous-diethyldithiocarbamate chelate, a lipophilic spin trap for NO detection by electron paramagnetic resonance (EPR) spectroscopy, we followed NO production in injured brain of mature Wistar rats. To relate changes in the amount of NO in the lesioned brain to the activity of NO synthase (NOS), this study also used NADPH-diaphorase staining. Our data show a rapid drop of NO concentration in the damaged brain below control values. This phenomenon persisted over several hours postinjury and varied with brain region. This decrease in NO concentration was accompanied by a simultaneous increase in the number of NADPH-diaphorase-positive cells, perhaps indicative of increased NOS activity. It is therefore assumed that, in the lesioned brain, a very rapid removal of NO occurs via its transformation to other reactive species such as peroxynitrite, which further adversely influence the damaged tissue.

Physiologically Active Compounds in Four Species of Phellinus.

The content of two groups of compounds with biological activity (non-hallucinogenic indole compounds and free phenolic acids) were analyzed in extracts of fruiting bodies of four species of Phellinus: P. igniarius, P. pini, P. pomaceus and P. robustus. The presence of indole compounds in methanolic extracts was analyzed by high-performance liquid chromatography and thin-layer chromatography coupled with densitometric detection. Three metabolites (serotonin, tryptamine, and L-tryptophan) were identified. The contents of individual indole compounds ranged from 1.70 (tryptamine in P. robustus) to 8.32 mg x 100 g⁻¹ dry weight (L-tryptophan in P. robustus). Four free phenolic acids were detected in methanolic extracts by the HPLC method. The total content ranged from 9.9 mg x 100 g⁻¹ DW (P. igniarius) to 32.5 mg x 100 g⁻¹ DW (P. robustus).

Spectrophotometric method for simultaneous determination of valsartan and substances from the group of statins in binary mixtures.

Applicability of derivative spectrophotometry for the determination of valsartan in the presence of a substance from the group of statins was checked. The obtained results indicate that the proposed method may be effective by using appropriate derivatives: for valsartan and fluvastatin - D1, D2 and D3, for valsartan and pravastatin - D1 and D3, for valsartan and atorvastatin - D2 and D3. The method was characterized by high sensitivity and accuracy. Linearity was maintained in the following ranges: 9.28-32.48 mg mL-1 for valsartan, 8.16-28.56 mg mL-1 f or fluvastatin, 14.40-39.90 mg mL-1 for atorvastatin and 9.60-48.00 mg mL-1 for pravastatin. Determination coefficients were in the range of 0.989-0.999 depending on the analyte and the order of derivative. The precision of the method was high with RSD from 0.1 to 2.5 % and recovery of individual components was within the range of 100 ± 5 %. The developed method was successfully applied to the determination of valsartan combined with fluvastatin, atorvastatin and pravastatin in laboratory prepared mixtures and in pharmaceutical preparations.

In vitro culture of Boletus badius as a source of indole compounds and zinc released in artificial digestive juices.

The objective of this study was to obtain the in vitro cultures of Boletus badius under controlled conditions and investigate the release of indole compounds and zinc from the mycelium of B. badius to artificial digestive juices under conditions similar to those in the human gastrointestinal tract. Biomass was obtained from cultures grown using both only the Oddoux medium as well as the same medium with added zinc hydroaspartate and zinc sulfate. The release of 5-hydroxy-L-tryptophan, L-tryptophan, and serotonin from the B. badius biomass extracts to the artificial digestive juices was determined. Differential pulse anodic stripping voltammetry was used to demonstrate that zinc is released from each of the extracted materials. The total amount of zinc in the materials under study was estimated to be between 7.12 and 44.15 mg/100 g dry weight. It was demonstrated that in vitro cultures of B. badius grown using appropriately selected media may supplement zinc and indole compounds.

Densitometric high performance thin-layer chromatography identification and quantitative analysis of psychotropic drugs.

A thin-layer chromatography (TLC)-densitometry method has been developed to identify and quantify haloperidol, amitriptyline, sulpiride, promazine, fluphenazine, doxepin, diazepam, trifluoperazine, clonazepam, and chlorpromazine in selected psychotropic drugs. Separation was performed on precoated silica gel 60 F254 TLC plates. Chromatograms were developed in various mobile phases, and 8 of 30 tested phases were selected based on spot location and developing time. The identification and quantification were carried out based on ultraviolet densitometric measurements at chosen wavelengths. In addition to retention coefficients, the absorption spectra recorded directly from chromatograms were also used in qualitative analysis. Under established experimental conditions, high sensitivity of the method was achieved. The limit of detection ranged from 0.009 to 0.260 microg, depending on the wavelength selected for measuring. A satisfactory recovery, ranging from 92.99 to 104.70%, was achieved for individual constituents.

Identification and quantitation of polymyxin B, framycetin, and dexamethasone in an ointment by using thin-layer chromatography with densitometry.

A new thin-layer chromatographic-densitometric method has been developed for rapid identification and quantitative determination of polymyxin B, framycetin, and dexamethasone in a dental ointment. Silica gel 60 and F254 silica gel 60 plates were used for separating antibiotics and dexamethasone acetate, respectively. When determining framycetin and polymyxin B, chromatograms were developed by using 2 mobile phases, namely methanol and methanol-n-butanol-ammonia (25%)-chloroform (14 + 4 + 9 + 12, v/v/v/v/). The densitometric measurements were made at 550 nm after detection with 0.3% ninhydrin solution. Dexamethasone was determined by using the mobile phase cyclohexane-ethyl acetate (2 + 3, v/v) and ultraviolet densitometric recording at 245 nm. The results obtained for individual constituents with the chromatographic-densitometric method demonstrate similar accuracy, relative standard deviation values from 1.49 to 2.47%, and relative error values from 0.02 to 0.81% and are comparable to those obtained with the reference methods.