Anti-inflammatory effect of rosmarinic acid isolated from Blechnum brasiliense in adult zebrafish brain.

Neuroinflammation has been associated to neurodegenerative disease development, with evidence suggesting that high levels of proinflammatory cytokines promote neuronal dysfunction and death. Therefore, it is necessary to study new compounds that may be used as adjuvant treatments of neurodegenerative diseases by attenuating the inflammatory response in the central nervous system (CNS). The aim of this study was to utilize the lipopolysaccharide (LPS) induction model of neuroinflammation to evaluate the modulation of inflammation by rosmarinic acid (RA) isolated from Blechnum brasiliense in adult zebrafish. First, we investigated the toxicity and antioxidant properties of fractionated B. brasiliense extract (ethyl acetate fraction- EAF) and the isolated RA in zebrafish embryos. Next, we developed a model of neuroinflammation induction by intraperitoneal (i.p.) injection of LPS to observe the RA modulation of proinflammatory cytokines. The median lethal concentration (LC50) calculated was 185.2 ± 1.24 µg/mL for the ethyl acetate fraction (EAF) and 296.0 ± 1.27 µM for RA. The EAF showed free radical inhibition ranging from 23.09% to 63.44% at concentrations of 10-250 µg/mL. The RA presented a concentration-dependent response ranging from 18.24% to 47.63% at 10-250 µM. Furthermore, the RA reduced LPS induction of TNF-α and IL-1ß levels, with the greatest effect observed 6 h after LPS administration. Thus, the data suggested an anti-inflammatory effect of RA isolated from B. brasiliense and reinforced the utility of the new model of neuroinflammation to test the possible neuroprotective effects of novel drugs or compounds.

Stability in clinical use and stress testing of meropenem antibiotic by direct infusion ESI-Q-TOF: Quantitative method and identification of degradation products.

An ESI-MS/MS method through direct infusion was validated for quantitative analysis of meropenem powder for injection. The validation parameters were established in a rapid analysis of 30 s. Drug stability was studied through the submission to stress testing, resulting on four degradation products. Under hydrolytic conditions, in acid, neutral and alkaline media, the major degradation product was formed through the cleavage of the ß-lactam ring. Oxidation of the drug using H2O2 (3%) showed the formation of two degradation products from a decarboxylation reaction and N-oxide formation. Under high humidity conditions, there was detected a dimer product. The stability of meropenem after reconstitution was studied in conditions that simulate its clinical use. In samples reconstituted and diluted in infusion fluids, an extensive degradation was observed. At room temperature meropenem maintained its content > 90% for up to 4 h when prepared in 5% glucose and for up to 12 h when prepared in 0.9% NaCl. Through ESI-MS/MS analyzes it was observed a degradation product formed by ß-lactam ring cleavage, detected in all conditions studied. It was also identified a degradation product formed only in 5% glucose, generated by the hydrolysis of ß-lactam followed by the attachment of a glucose molecule to the nitrogen of the pyrrolidine ring. In general, all the results obtained in the stability studies contribute to the knowledge about this antibiotic and future candidates of this class.

Application of Quality by Design to optimize a stability-indicating LC method for the determination of ticagrelor and its impurities.

Simultaneous analysis of drug compounds and their impurities of degradation and synthesis became constant in the modern pharmaceutical analysis. Likewise, analytical techniques must improve sensitivity and selectivity for the monitoring of pharmaceutical products, allowing a full assessment of impurities in drug products and, therefore, ensure safety and efficacy of pharmacological treatments. The application of Quality by Design (QbD) principles has proved to be feasible on the elaboration of analytical methods, allowing the comprehensive evaluation and measurement of different analytical parameters and their effects on critical properties of the methodology in development. QbD approach was applied to the development of a fast and selective HPLC method for the analysis of the antiplatelet aggregation drug ticagrelor and its degradation products in presence of three impurities of synthesis. Fractional factorial resolution V was the screening experimental design applied to five method parameters. Response surface methodology was carried by central composite star face design on the two critical method parameters selected. Analytical design space, established after the application of Monte-Carlo simulations, verified whether predicted results were in accordance with critical quality attributes. The developed and validated HPLC method with DAD detection at 225 nm was able to resolve eight related compounds in less than three minutes.

Anti-inflammatory activity and chemical analysis of extracts from Trifolium riograndense

Abstract Aiming to investigate new therapeutic agents with fewer side effects, the number of studies about natural products has increased. Phenolic compounds comprise a well-studied class of abundant plant-derived compounds, whose anti-inflammatory activity has been described. Isoflavones are phenolic compounds that occur mainly in the Leguminosae family, and can be found in many species, such as Trifolium riograndense Burkart, Leguminosae (clover). In this study an HPLC method was used to determine and quantify four isoflavones (genistein, daidzein, formononetin, and biochanin A) in hydrolyzed leaf, flower, stolon, and root extracts of T. riograndense. In vivo anti-inflammatory activity was investigated using the rat paw edema method and in vitro chemotaxis model with a dry extract from the leaves, which had the highest amount of isoflavones. The major isoflavone found in all parts of the plant was formononetin. The chemotaxis assay revealed that the different concentrations (0.2-50 µg/ml) of the dry extract significantly inhibited neutrophil migration in a concentration-dependent manner (more than 90%). In the rat paw edema test, oral administration of clover extract 100 mg/kg was able to significantly inhibit the edema formation induced by carrageenan. In conclusion, chemical analyses showed that Trifolium riograndense is a plant rich in isoflavones and a new interesting option as isoflavone source. The results of the biological tests taken together show that the extract of T. riograndense has anti-inflammatory effect in rodents.

Stability of doripenem in reconstituted solution - thermal and oxidative decomposition kinetics and degradation products by LC-MS.

A ultra-fast liquid chromatography method applied to quantitation of doripenem in powder for injection was validated. Validation parameters were assayed and a rapid analysis was established by a reversed-phase system comprising a C18 column endcapped (50 × 4.0 mm, 2.0 µm), mobile phase consisting of phosphoric acid 0.01% (pH 3.8) and acetonitrile (98:02, v/v) and a flow rate of 0.4 mL min-1 . Drug stability was studied through submission to forced conditions, allowing the major degradation products to be detected and the kinetics parameters to be established. Thermal and oxidative degradation were determined, and indicated a kinetic decomposition following first and second order, respectively. The main degradation products were identified by LC-MS analysis, and the results were evaluated in order to suggest the chemical structures corresponding to respective masses and fragmentations. Six compounds were identified, with m/z 411, 427, 437, 634, 650 and 664. All of them resulted from cleavage of ß-lactam ring and alcoholic chain and/or dimerization. These experimental results provide valuable information about the stability of doripenem reconstituted solution and procedures for its handling and storage.

Hydrogel containing adapalene- and dapsone-loaded lipid-core nanocapsules for cutaneous application: development, characterization, in vitro irritation and permeation studies.

Lipid-core polymeric nanocapsule suspensions containing adapalene and dapsone (AD-LCNC) were developed and incorporated in a Carbopol 940® hydrogel (AD-LCNC HG). A nanoemulsion (AD-NE), similarly prepared but omitting the polymer, was developed and also incorporated in a Carbopol 940® hydrogel (AD-NE HG) to evaluate the polymer effect. Physicochemical characteristics were evaluated. AD-LCNC suspensions containing 0.07% of dapsone and 0.025% of adapalene presented an average size of 194.9 ± 0.42 nm, zeta potential of -15 ± 1.2 mV and polydispersity index of 0.12 ± 0.02, using electrophoretic light scattering (n = 3). The granulometric profiles showed unimodal size distributions for AD-LCNC suspensions, demonstrating that no microscopic population is present in the formulation. No instability phenomena were observed by multiple light-scattering analysis. Photomicrographs obtained by TEM showed homogeneous- and spherical-shaped particles. The encapsulation efficiency was 99.99% for dapsone and 100% for adapalene. The pH values for AD-LCNC suspensions were 5.1 and 7.6 for AD-LCNC HG. Formulations were classified as nonirritant in the HET-CAM test. Rheological analysis demonstrated a non-Newtonian pseudoplastic profile. The in vitro skin permeation studies showed a higher amount of adapalene in epidermis (130.52 ± 25.72 ng/mg) and dermis (4.66 ± 2.5 ng/mg) for AD-NE HG. The AD-LCNC HG presented higher amount of dapsone in both the skin layers (73.91 ± 21.64 ng/mg in epidermis and 4.08 ± 0.85 ng/mg in dermis). The assay showed significant difference between AD-LCNC HG and AD-NE HG (p < 0.05), and drug was not found in the receptor medium.

Antifungal activity and mechanism of action of monoterpenes against dermatophytes and yeasts

Dermatomycosis causes highly frequent dermal lesions, and volatile oils have been proven to be promising as antifungal agents. The antifungal activity of geraniol, nerol, citral, neral and geranial (monoterpenes), and terbinafine and anidulafungin (control drugs) against seven opportunistic pathogenic yeasts and four dermatophyte species was evaluated by the Clinical and Laboratory Standards Institute microdilution tests. Monoterpenes were more active against dermatophytes than yeasts (geometric mean of minimal inhibitory concentration (GMIC) of 34.5 and 100.4 µg.ml-1, respectively). Trichophyton rubrum was the fungal species most sensitive to monoterpenes (GMIC of 22.9 µg.ml-1). The trans isomers showed higher antifungal activity than the cis. The mechanism of action was investigated evaluating damage in the fungal cell wall (Sorbitol Protection Assay) and in the cell membrane (Ergosterol Affinity Assay). No changes were observed in the MIC of monoterpenes in the sorbitol protection assay.The MIC of citral and geraniol was increased from 32 to 160 µg.ml-1 when the exogenous ergosterol concentrations was zero and 250 µg.ml-1, respectively. The monoterpenes showed an affinity for ergosterol relating their mechanism of action to cell membrane destabilization.

HPLC-DAD for the determination of three different classes of antifungals: method characterization, statistical approach, and application to a permeation study.

This study describes and characterizes methods for high-performance liquid chromatography diode array detection (HPLC-DAD) analysis of formulations containing molecules with antifungal activity of three different classes: terbinafine and butenafine (allylamines), miconazole and fluconazole (azoles), and geraniol, neral and geranial (monoterpenes). All methods used the same chromatographic column (RP18 ), enabling the analysis to be performed in a single batch. The specificity was extensively discussed through the establishment of purity peak methods. The analytical parameters (linearity, precision and accuracy) were calculated and discussed in detail using specific statistical approaches. All substances showed satisfactory results for chromatographic and analytical parameters. Limits of 1.3% to mean repeatability and 2.0% for intermediate precision are suggested as acceptance criteria in validation of methods by HPLC-DAD, in situations where there is no extensive pretreatment of the samples. The methods proved to be robust and significant factors were discussed regarding their influence on chromatographic parameters (retention time, resolution, tailing factor and column efficiency). Finally, the application of the developed methods was demonstrated by the results of a permeation study of the antifungal agents through bovine hoof membranes.

Highly selective colorimetric method to determine gemifloxacin mesylate in the presence of a synthetic impurity.

A simple visible spectrophotometric method was developed for the determination of gemifloxacin mesylate (GFM) in tablets. The method was based on the formation of a yellow ion-pair complex between the basic nitrogen of the drug and the sulfonphthalein acid dye in phthalate buffer. The method was validated by the study of its specificity, linearity, precision, accuracy, and robustness. The assay was compared with an LC and a microbiological method, and the analysis of variance showed no significant difference between the methods (P>0.05). The results demonstrated that the visible spectrophotometric method is suitable for determination of GFM in tablets, even in the presence of a synthetic impurity.

Quantitative evaluation of besifloxacin ophthalmic suspension by HPLC, application to bioassay method and cytotoxicity studies.

Besifloxacin (BSF) is a synthetic chiral fluoroquinolone developed for the topical treatment of ophthalmic infections. The present study reports the development and validation of a microbiological assay, applying the cylinder-plate method, for determination of BSF in ophthalmic suspension. To assess this methodology, the development and validation of the method was performed for the quantification of BSF by high performance liquid chromatography (HPLC). The HPLC method showed specificity, linearity in the range of 20-80 µg mL(-1) (r=0.9998), precision, accuracy and robustness. The microbiological method is based on the inhibitory effect of BSF upon the strain of Staphylococcus epidermidis ATCC 12228 used as a test microorganism. The bioassay validation method yielded excellent results and included linearity, precision, accuracy, robustness and selectivity. The assay results were treated statistically by analysis of variance (ANOVA) and were found to be linear (r=0.9974) in the range of 0.5-2.0 µg mL(-1), precise (inter-assay: RSD=0.84), accurate (101.4%), specific and robust. The bioassay and the previously validated high performance liquid chromatographic (HPLC) method were compared using Student's t test, which indicated that there was no statistically significant difference between these two methods. These results confirm that the proposed microbiological method can be used as routine analysis for the quantitative determination of BSF in an ophthalmic suspension. A preliminary stability study during the HPLC validation was performed and demonstrated that BSF is unstable under UV conditions. The photodegradation kinetics of BSF in water showed a first-order reaction for the drug product (ophthalmic suspension) and a second-order reaction for the reference standard (RS) under UVA light. UVA degraded samples of BSF were also studied in order to determine the preliminary in vitro cytotoxicity against mononuclear cells. The results indicated that BSF does not alter the cell membrane and has been considered non-toxic to human mononuclear cells in the experimental conditions tested.

Biological safety studies of gemifloxacin mesylate and related substances.

PURPOSE: The aim of this study was to evaluate the cytotoxic, phototoxic, genotoxic and photogenotoxic potential of gemifloxacin mesylate (GFM), its main synthetic impurity (SI) and one isolated and structurally elucidated degradation product (DP). METHODS: The neutral red uptake (NRU) and reduction of 2,5-diphenyl-3,-(4,5-dimethyl-2-thiazolyl)tetrazolium bromide (MTT) assays were performed as in vitro endpoints to evaluate cytotoxicity and phototoxicity in a 3T3 cell line, and predict toxicity and/or phototoxicity after systemic administration of the drug. The in vitro alkaline single-cell electrophoresis (comet) assay was used to evaluate the genotoxic and photogenotoxic potential of the substances using the same cell line. RESULTS: The results showed that the SI and the DP are more cytotoxic and phototoxic than the drug GFM using the 3T3 cell line. In the comet assay, the drug GFM was found to be more genotoxic and photogenotoxic than its related substances. CONCLUSIONS: Our findings highlight the relevance of the biological safety studies to increase the knowledge regarding the toxic potential of the related substances, which can be associated with the drug side effects and toxicity.

In vivo and in vitro anti-inflammatory activity of red clover Trifolium pratense dry extract

Red clover Trifolium pratense L., Fabaceae, contains four isoflavones, mainly formononetin and biochanin A, and in smaller concentrations, daidzein and genistein. These compounds have gained a lot interest due to its human health benefits, such as estrogenic and progestogenic activities, antioxidant, anti-cancer and others. The objective of this study was to determine in vivo and in vitro anti-inflammatory activity of red clover dry extract. The in vitro anti-inflammatory activity was assayed by the technique using the Boyden chamber method, evaluating the leukocyte migration inhibition (chemotaxis). The in vivo anti-inflammatory activity was tested by a carrageenan-induced rat paw edema test. The results of anti-inflammatory in vitro test showed that there was a significant inhibition of leukocyte migration at the concentrations of 100, 50, 25, 10 and 5 µg/mL of red clover dry extract, these doses resulted in 94.73, 95.39, 94.73, 84.68 and 78.75 percent of inhibition for each dose, respectively. The anti-inflammatory in vivo test resulted in a significant activity in both tested doses (100 and 50 mg/kg of red clover dry extract) and at each tested time. The average percentage of edema inhibition was 63.37 percent. The findings of this study suggested that red clover extract might be suitable for the treatment of inflammatory diseases.

Gemifloxacin mesylate (GFM) stability evaluation applying a validated bioassay method and in vitro cytotoxic study.

The validation of a microbiological assay applying the cylinder-plate method to determine the quinolone gemifloxacin mesylate (GFM) content is described. Using a strain of Staphylococcus epidermidis ATCC 12228 as the test organism, the GFM content in tablets at concentrations ranging from 0.5 to 4.5 µg mL(-1) could be determined. A standard curve was obtained by plotting three values derived from the diameters of the growth inhibition zone. A prospective validation showed that the method developed is linear (r=0.9966), precise (repeatability and intermediate precision), accurate (100.63%), specific and robust. GFM solutions (from the drug product) exposed to direct UVA radiation (352 nm), alkaline hydrolysis, acid hydrolysis, thermal stress, hydrogen peroxide causing oxidation, and a synthetic impurity were used to evaluate the specificity of the bioassay. The bioassay and the previously validated high performance liquid chromatographic (HPLC) method were compared using Student's t test, which indicated that there was no statistically significant difference between these two validated methods. These studies demonstrate the validity of the proposed bioassay, which allows reliable quantification of GFM in tablets and can be used as a useful alternative methodology for GFM analysis in stability studies and routine quality control. The GFM reference standard (RS), photodegraded GFM RS, and synthetic impurity samples were also studied in order to determine the preliminary in vitro cytotoxicity to peripheral blood mononuclear cells. The results indicated that the GFM RS and photodegraded GFM RS were potentially more cytotoxic than the synthetic impurity under the conditions of analysis applied.

Anti-inflammatory activity of essential oil from leaves of Myrciaria tenella and Calycorectes sellowianus.

The GC-MS analysis revealed that the leaf essential oils of Myrciaria tenella (DC.) Berg and Calycorectes sellowianus O. Berg (Myrtaceae) were composed of 34 and 37 compounds, respectively. The main constituents of M. tenella oil were beta-caryophyllene (25.1%), and spathulenol (9.7%), while for C. sellowianus were guaiol (13.1%) and beta-caryophyllene (8.6%). The anti-inflammatory effect of both essential oils was investigated in vitro and in vivo. Both oils reduced significantly (p < 0.005) the treated neutrophils chemotaxis with 93% and 91% inhibition for M. tenella and C. sellowianus, respectively. However, in the systemic treatment with the essential oils (50 mg/kg p.o.) only the M. tenella oil was able to significantly reduce the carrageenan-induced paw edema with a similar effect to that observed for indomethacin (10 mg/kg), the positive control.

Stress degradation studies and kinetic determinations of duloxetine enteric-coated pellets by HPLC.

A stability-indicating HPLC assay method was developed for the quantitative determination of duloxetine (DLX) in a pharmaceutical dosage form in the presence of its degradation products, and kinetic determinations were evaluated in acid conditions and UV-C radiation exposure. Chromatographic separation was achieved by use of an ACE C18 column (250 x 4.0 mm id, 5 microm particle size). The mobile phase was prepared by mixing aqueous 50 mM potassium phosphate buffer (pH 6.0 containing 0.3% triethylamine) and acetonitrile (60 + 40, v/v). DLX was rapidly degraded in an acid medium and in the presence of hydrogen peroxide and UV-C radiation; it was more stable in alkaline medium. The described method was linear over a range of 4.0-14.0 microg/mL for determination of DLX (r = 0.9998). The precision was demonstrated by the RSD of intraday (0.79-1.07%) and interday (0.85%) studies. The mean recovery was found to be 100.56%. The acid degradation of DLX in 0.1 M HCI solution showed an apparent zero-order kinetics (k = 0.177 microg/mL/min), and the photodegradation demonstrated an apparent first-order kinetics (k = 0.082 microg/mL/min). The developed method was found to be simple, specific, robust, linear, precise, and accurate for the determination of DLX in enteric-coated pellets.

LC method for telithromycin in tablets: a stability-indicating assay.

A liquid chromatographic (LC) method for the quantitative determination of telithromycin, the first member of the ketolides, which is a new class of macrolides, was developed. Analytical parameters were studied according to International Conference on Harmonization (ICH) guidelines. An Ace RP-18 octadecyl silane column (250 mm x 4.6 mm, 5 microm) maintained at 50 degrees C was used as the stationary phase, and methanol and 0.067 M potassium monobasic phosphate buffer pH 4.0 (55:45, v/v) were used as the mobile phase with UV detection at 265 nm. In forced degradation studies, the effects of acid, base, oxidation, UV light and temperature were investigated showing no interference in the drug peak. The method was linear (r=0.9999) at concentration ranging from 10.0 to 40.0 microg/mL, precise (intra-day relative standard deviation [RSD] and inter-day RSD values<2.0%), accurate (mean recovery=100.76%), specific and robust. Detection and quantitation limits were 0.0027 and 0.0082 microg/mL, respectively. The results showed the proposed method is suitable for its intended use. The validated method may be used to quantify telithromycin tablets and to determine the stability of the drug. The method is able to separate telithromycin from its degradation products and tablet excipients for its sensitivity and reproducibility. These results are in accordance with a previous microbiological assay study, which used the same tested conditions showing that the methods can be interchangeable.

Antifungal activity of the lemongrass oil and citral against Candida spp.

Superficial mycoses of the skin are among the most common dermatological infections, and causative organisms include dermatophytic, yeasts, and non-dermatophytic filamentous fungi. The treatment is limited, for many reasons, and new drugs are necessary. Numerous essential oils have been tested for both in vitro and in vivo antifungal activity and some pose much potential as antifungal agents. By using disk diffusion assay, we evaluated the antifungal activity of lemongrass oil and citral against yeasts of Candida species (Candida albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis). This study showed that lemongrass oil and citral have a potent in vitro activity against Candida spp.

New high-performance liquid chromatographic method for determination of clopidogrel in coated tablets.

A new high-performance liquid chromatographic method was developed and validated for clopidogrel determination in pharmaceutical formulations. The system consisted of an ACE 5 octadecylsilane (C18; 150 x 4.6 mm id), 5.0 microm particle size column; methanol-0.1% triethylamine (75 + 25, v/v), pH 5.3, mobile phase at a flow rate of 1.2 mL/min; and a diode array detector set at 220 nm. Specificity, linearity, precision, accuracy, and robustness were the parameters evaluated. The retention time for clopidogrel was 6.8 min. To estimate specificity, an aqueous sample solution was subjected to degradation by ultraviolet light and by acid, alkaline, and oxidation media. The peaks of degradation products did not interfere with the compound signal, and there was no interference when a placebo solution was analyzed. Linearity over a concentration range of 10.0 to 90.0 microg/mL was shown (correlation coefficient = 0.9998). Low values of relative standard deviation indicated the adequate intraday and interday precision. The average recovery was found as 99.16%. In the robustness test, small modifications to the mobile phase composition did not affect the determination of clopidogrel. The proposed method proved to be simple, fast, and cost efficient for the intended use.

Development and validation of column high-performance liquid chromatographic and ultraviolet spectrophotometric methods for citalopram in tablets.

Column high-performance liquid chromatographic (LC) and UV spectrophotometric methods for the quantitative determination of citalopram, a potent and selective serotonin reuptake inhibitor, in tablets were developed. The parameters linearity, precision, accuracy, specificity, robustness, limit of detection, and limit of quantitation were studied according to International Conference on Harmonization guidelines. Chromatography was carried out by the reversed-phase technique on an ACE C18 column with a mobile phase composed of 0.30% triethylamine solution-acetonitrile (55 + 45, v/v) adjusted to pH 6.6 with 10% ortho-phosphoric acid at a flow rate of 1.0 mL/min and 25 degrees C. The UV spectrophotometric method was performed at 239 nm. The linearity of the LC method was in the range of 10.00-70.00 microg/mL, and 2.50-17.50 microg/mL for the UV spectrophotometric method. The interday and intraday assay precision was < 1.5% (relative standard deviation) for the LC and UV spectrophotometric methods. The recoveries were in the range 100.70-101.35% for the LC method and 98.48-98.65% for the UV spectrophotometric method. Statistical analysis by Student's t-test showed no significant difference between the results obtained by the 2 methods. The proposed methods are highly sensitive, precise, and accurate and can be used for the reliable quantitation of citalopram in tablets.

Antifungal activity of the lemongrass oil and citral against Candida spp

Superficial mycoses of the skin are among the most common dermatological infections, and causative organisms include dermatophytic, yeasts, and non-dermatophytic filamentous fungi. The treatment is limited, for many reasons, and new drugs are necessary. Numerous essential oils have been tested for both in vitro and in vivo antifungal activity and some pose much potential as antifungal agents. By using disk diffusion assay, we evaluated the antifungal activity of lemongrass oil and citral against yeasts of Candida species (Candida albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis). This study showed that lemongrass oil and citral have a potent in vitro activity against Candida spp.