Avaliação da atividade anti-inflamatória do extrato aquoso, frações e compostos identificados nos frutos de Hancornia Speciosa Gomes (Apocynaceae)

Hancornia speciosa Gomes (Apocynaceae), popularly known as ‘mangabeira’, has been used in folk medicine to treat inflammatory disorders, hypertension, dermatitis, diabetes, liver diseases and stomach disorders. Regarding the Hancornia speciosa fruits, the ethnobotany indicates its use especially for treating inflammation and tuberculosis. However, no study has been done so far to prove such biological activities. The objective was evaluation anti-inflammatory activity from the fruits of Hancornia speciosa Gomes (mangabeira). Aqueous extract was prepared by decoction, subsequently submitted the liquid-liquid fractionation. The secondary metabolites were identified by high performance liquid chromatography coupled with detector diode array (HPLC-DAD) and liquid chromatography diode array detector coupled with mass spectrometry (LC-DAD-MS). The anti-inflammatory properties of the aqueous extract, dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and n-butanol (n-BuOH) fractions of the fruits from H. speciosa, as well as rutin and chlorogenic acid were investigated using in vitro and in vivo models. In vivo tests comprised the xylene-induced ear edema that was measured the formation of edema, carrageenan-induced peritonitis was evaluated the total leukocytes at 4h and zymosan-induced air pouch was measured the total leukocytes and differential cell count at 6, 24 and 48 hours, whereas in vitro tests were evaluated levels of cytokines IL-1β, IL-6, IL-12 and TNF-α using ELISA obtained of carrageenan-induced peritonitis model. The results showed the presence of rutin and chlorogenic acid were detected in the aqueous extract from H. speciosa fruits by HPLC-DAD and LC-DAD-ME. Furthermore, the aqueous extracts and fractions, as well as rutin and chlorogenic acid significantly inhibited the xilol-induced ear edema and reduced cell migration in the animal models such as carrageenan-induced peritonitis and zymosan-induced air pouch. In addition, reduced levels of cytokines IL-1β, IL-6, IL-12 and TNF-α were observed. This is the first study that demonstrated the anti-inflammatory effect of aqueous extract from Hancornia speciosa fruits against different inflammatory agents in animal models, suggesting that their bioactive molecules, especially rutin and chlorogenic acid contributing, at least in part, to the anti-inflammatory effect of aqueous extract. These findings support the widespread use of Hancornia speciosa in popular medicine and demonstrate that this aqueous extract has therapeutic potential for the development of a herbal drugs with anti-inflammatory properties.

Otimização evalidação de métodos analíticos para a determinação simultânea de tuberculostáticos (4-FDC) por CLAE/DAD e CLUE/ DAD

Tuberculosis is a serious disease, but curable in practically 100% of new cases, since complied the principles of modern chemotherapy. Isoniazid (ISN), Rifampicin (RIF), Pyrazinamide (PYR) and Chloride Ethambutol (ETA) are considered first line drugs in the treatment of tuberculosis, by combining the highest level of efficiency with acceptable degree of toxicity. Concerning USP 33 - NF28 (2010) the chromatography analysis to 3 of 4 drugs (ISN, PYR and RIF) last in average 15 minutes and 10 minutes more to obtain the 4th drug (ETA) using a column and mobile phase mixture different, becoming its industrial application unfavorable. Thus, many studies have being carried out to minimize this problem. An alternative would use the UFLC, which is based with the same principles of HPLC, however it uses stationary phases with particles smaller than 2 μm. Therefore, this study goals to develop and validate new analytical methods to determine simultaneously the drugs by HPLC/DAD and UFLC/DAD. For this, a analytical screening was carried out, which verified that is necessary a gradient of mobile phase system A (acetate buffer:methanol 94:6 v/v) and B (acetate buffer:acetonitrile 55:45 v/v). Furthermore, to the development and optimization of the method in HPLC and UFLC, with achievement of the values of system suitability into the criteria limits required for both techniques, the validations have began. Standard solutions and tablets test solutions were prepared and injected into HPLC and UFLC, containing 0.008 mg/mL ISN, 0.043 mg/mL PYR, 0.030 mg.mL-1 ETA and 0.016 mg/mL RIF. The validation of analytical methods for HPLC and UFLC was carried out with the determination of specificity/selectivity, analytical curve, linearity, precision, limits of detection and quantification, accuracy and robustness. The methods were adequate for determination of 4 drugs separately without interfered with the others. Precise, due to the fact of the methods demonstrated since with the days variation, besides the repeatability, the values were into the level required by the regular agency. Linear (R> 0,99), once the methods were capable to demonstrate results directly proportional to the concentration of the analyte sample, within of specified range. Accurate, once the methods were capable to present values of variation coefficient and recovery percentage into the required limits (98 to 102%). The methods showed LOD and LOQ very low showing the high sensitivity of the methods for the four drugs. The robustness of the methods were evaluate, facing the temperature and flow changes, where they showed robustness just with the preview conditions established of temperature and flow, abrupt changes may influence with the results of methods

Detecção de metamidofós em solos por métodos ecotoxicológico e cromatografia líquida acoplada à espectrometria de massas sequencial (LC-MS/MS)

Soil contamination by pesticides is an environmental problem that needs to be monitored and avoided. However, the lack of fast, accurate and low cost analytical methods for discovering residual pesticide in complex matrices, such as soil, is a problem still unresolved. This problem needs to be solved before we are able to assess the quality of environmental samples. The intensive use of pesticides has increased since the 60s, because the dependence of their use, causing biological imbalances and promoting resistance and recurrence of high populations of pests and pathogens (upwelling). This has contributed to the appearance of new pests that were previously under natural control. To develop analytical methods that are able to quantify residues pesticide in complex environment. It is still a challenge for many laboratories. The integration of two analytical methods one ecotoxicological and another chemical demonstrates the potential for environmental analysis of methamidophos. The aim of this study was to evaluate an ecotoxicological method as "screening" analytical methamidophos in the soil and perform analytical confirmation in the samples of the concentration of the analyte by chemical method LC-MS/MS In this work we tested two soils: a clayey and sandy, both in contact with the kinetic methamidophos model followed pseudo-second order. The clay soil showed higher absorption of methamidophos and followed the Freundlich model, while the sandy, the Langmuir model. The chemical method was validated LC-MS/MS satisfactory, showing all parameters of linearity, range, precision, accuracy, and sensitivity adequate. In chronic ecotoxicological tests with C. dubia, the NOEC was 4.93 and 3.24 for ng L-1 of methamidophos to elutriate assays of sandy and clay soils, respectively. The method for ecotoxicological levels was more sensitive than LC-MS/MS detection of methamidophos, loamy and sandy soils. However, decreasing the concentration of the standard for analytical methamidophos and adjusting for the validation conditions chemical acquires a limit of quantification (LOQ) in ng L-1, consistent with the provisions of ecotoxicological test. The methods described should be used as an analytical tool for methamidophos in soil, and the ecotoxicological analysis can be used as a "screening" and LC-MS/MS as confirmatory analysis of the analyte molecule, confirming the objectives of this work

Desenvolvimento de métodos para quantificação de benzilpenicilina em medicamentos veterinários e seus resíduos em leite bovino e caprino por UFLC-DAD

The Benzylpenicillin (PENG) have been as the active ingredient in veterinary medicinal products, to increase productivity, due to its therapeutic properties. However, one of unfortunate quality and used indiscriminately, resulting in residues in foods exposed to human consumption, especially in milk that is essential to the diet of children and the ageing. Thus, it is indispensable to develop new methods able to detect this waste food, at levels that are toxic to human health, in order to contribute to the food security of consumers and collaborate with regulatory agencies in an efficient inspection. In this work, were developed methods for the quality control of veterinary drugs based on Benzylpenicillin (PENG) that are used in livestock production. Additionally, were validated methodologies for identifying and quantifying the antibiotic residues in milk bovine and caprine. For this, the analytical control was performed two steps. At first, the groups of samples of medicinal products I, II, III, IV and V, individually, were characterized by medium infrared spectroscopy (4000 – 600 cm-1). Besides, 37 samples, distributed in these groups, were analyzed by spectroscopy in the ultraviolet and near infrared region (UV VIS NIR) and Ultra Fast Liquid Chromatograph coupled to linear arrangement photodiodes (UFLC-DAD). The results of the characterization indicated similarities, between PENG and reference standard samples, primarily in regions of 1818 to 1724 cm-1 of ν C=O that shows primary amides features of PENG. The method by UFLC-DAD presented R on 0.9991. LOD of 7.384 × 10-4 μg mL-1. LOQ of 2.049 × 10-3 μg mL-1. The analysis shows that 62.16% the samples presented purity ≥ 81.21%. The method by spectroscopy in the UV VIS NIR presented medium error ≤ 8 – 12% between the reference and experimental criteria, indicating is a secure choice for rapid determination of PENG. In the second stage, was acquiring a method for the extraction and isolation of PENG by the addition of buffer McIlvaine, used for precipitation of proteins total, at pH 4.0. The results showed excellent recovery values PENG, being close to 92.05% of samples of bovine milk (method 1). While samples of milk goats (method 2) the recovery of PENG were 95.83%. The methods for UFLC-DAD have been validated in accordance with the maximum residue limit (LMR) of 4 μg Kg-1 standardized by CAC/GL16. Validation of the method 1 indicated R by 0.9975. LOD of 7.246 × 10-4 μg mL-1. LOQ de 2.196 × 10-3 μg mL-1. The application of the method 1 showed that 12% the samples presented concentration of residues of PENG > LMR. The method 2 indicated R by 0.9995. LOD 8.251 × 10-4 μg mL-1. LOQ de 2.5270 × 10-3 μg mL-1. The application of the method showed that 15% of the samples were above the tolerable. The comparative analysis between the methods pointed better validation for LCP samples, because the reduction of the matrix effect, on this account the tcalculs < ttable, caused by the increase of recovery of the PENG. In this mode, all the operations developed to deliver simplicity, speed, selectivity, reduced analysis time and reagent use and toxic solvents, particularly if compared to the established methodologies.

Desenvolvimento de métodos para quantificação de benzilpenicilina em medicamentos veterinários e seus resíduos em leite bovino e caprino por UFLC-DAD

The Benzylpenicillin (PENG) have been as the active ingredient in veterinary medicinal products, to increase productivity, due to its therapeutic properties. However, one of unfortunate quality and used indiscriminately, resulting in residues in foods exposed to human consumption, especially in milk that is essential to the diet of children and the ageing. Thus, it is indispensable to develop new methods able to detect this waste food, at levels that are toxic to human health, in order to contribute to the food security of consumers and collaborate with regulatory agencies in an efficient inspection. In this work, were developed methods for the quality control of veterinary drugs based on Benzylpenicillin (PENG) that are used in livestock production. Additionally, were validated methodologies for identifying and quantifying the antibiotic residues in milk bovine and caprine. For this, the analytical control was performed two steps. At first, the groups of samples of medicinal products I, II, III, IV and V, individually, were characterized by medium infrared spectroscopy (4000 – 600 cm-1). Besides, 37 samples, distributed in these groups, were analyzed by spectroscopy in the ultraviolet and near infrared region (UV VIS NIR) and Ultra Fast Liquid Chromatograph coupled to linear arrangement photodiodes (UFLC-DAD). The results of the characterization indicated similarities, between PENG and reference standard samples, primarily in regions of 1818 to 1724 cm-1 of ν C=O that shows primary amides features of PENG. The method by UFLC-DAD presented R on 0.9991. LOD of 7.384 × 10-4 μg mL-1. LOQ of 2.049 × 10-3 μg mL-1. The analysis shows that 62.16% the samples presented purity ≥ 81.21%. The method by spectroscopy in the UV VIS NIR presented medium error ≤ 8 – 12% between the reference and experimental criteria, indicating is a secure choice for rapid determination of PENG. In the second stage, was acquiring a method for the extraction and isolation of PENG by the addition of buffer McIlvaine, used for precipitation of proteins total, at pH 4.0. The results showed excellent recovery values PENG, being close to 92.05% of samples of bovine milk (method 1). While samples of milk goats (method 2) the recovery of PENG were 95.83%. The methods for UFLC-DAD have been validated in accordance with the maximum residue limit (LMR) of 4 μg Kg-1 standardized by CAC/GL16. Validation of the method 1 indicated R by 0.9975. LOD of 7.246 × 10-4 μg mL-1. LOQ de 2.196 × 10-3 μg mL-1. The application of the method 1 showed that 12% the samples presented concentration of residues of PENG > LMR. The method 2 indicated R by 0.9995. LOD 8.251 × 10-4 μg mL-1. LOQ de 2.5270 × 10-3 μg mL-1. The application of the method showed that 15% of the samples were above the tolerable. The comparative analysis between the methods pointed better validation for LCP samples, because the reduction of the matrix effect, on this account the tcalculs < ttable, caused by the increase of recovery of the PENG. In this mode, all the operations developed to deliver simplicity, speed, selectivity, reduced analysis time and reagent use and toxic solvents, particularly if compared to the established methodologies.