High performance thin layer chromatographic determination of nifedipine in human serum after liquid-liquid extraction

A method using HPTLC for quantitation of nifedipine in serum was developed and validated. It includes a liquid-liquid extraction, and carbamazepine as internal standard. Chloroform: ethyl acetate: cyclohexane (19:2:2, v/v/v) was the mobile phase. The method showed good relationship (r = 0.996) (2.00 to 25.00 ng/band, corresponding to 0.02 and 0.25 ng/µL in serum). The % RSD of intra-assay and inter-assay, were between 0.57 and 3.56 and 1.16 to 3.60, respectively. LOD and LOQ were 0.72 and 0.86 ng/band, respectively. The recovery values were between 93 and 102%. Rf for nifedipine and carbamazepine were 0.31 and 0.10, respectively.

Separation and characterization of asphaltenic subfractions

The structure of the various asphaltenic subfractions found in crude oil was evaluated. For this purpose, C5 asphaltenes were extracted from an asphaltic residue using n-pentane as the flocculant solvent. The different subfractions were isolated from the C5 asphaltenes by the difference in solubility in different solvents. These were characterized by infrared spectroscopy, nuclear magnetic resonance, X-ray fluorescence, elementary analysis and mass spectrometry. The results confirmed that the subfractions extracted with higher alkanes had greater aromaticity and molar mass. However, small solubility variations between the subfractions were attributed mainly to the variation in the concentrations of cyclical hydrocarbon compounds and metals.

COMPARISON BETWEEN ASPHALTENES (SUB)FRACTIONS EXTRACTED FROM TWO DIFFERENT ASPHALTIC RESIDUES: CHEMICAL CHARACTERIZATION AND PHASE BEHAVIOR

Asphaltenes are blamed for various problems in the petroleum industry, especially formation of solid deposits and stabilization of water-in-oil emulsions. Many studies have been conducted to characterize chemical structures of asphaltenes and assess their phase behavior in crude oil or in model-systems of asphaltenes extracted from oil or asphaltic residues from refineries. However, due to the diversity and complexity of these structures, there is still much to be investigated. In this study, asphaltene (sub)fractions were extracted from an asphaltic residue (AR02), characterized by NMR, elemental analysis, X-ray fluorescence and MS-TOF, and compared to asphaltene subfractions obtained from another asphaltic residue (AR01) described in a previous article. The (sub)fractions obtained from the two residues were used to prepare model-systems containing 1 wt% of asphaltenes in toluene and their phase behavior was evaluated by measuring asphaltene precipitation onset using optical microscopy. The results obtained indicated minor differences between the asphaltene fractions obtained from the asphaltic residues of distinct origins, with respect to aromaticity, elemental composition (CHN), presence and content of heteroelements and average molar mass. Regarding stability, minor differences in molecule polarity appear to promote major differences in the phase behavior of each of the asphaltene fractions isolated.

Extraction and spectrophotometric determination of Hg2+ as complex anion HgI3-

A method has been developed for the extraction and spectrophotometric determination of Hg2+ in a concentration range of 0.2-1.0 mg L-1; following the Lambert-Beer's law using high molecular weight quaternary ammonium salts dissolved in chloroform. The metal complex anion was determined in the extract in the region UV (260 nm).

Thermal comfort in reduced models of broilers' houses, under different types of roofing materials

The research was developed to evaluate the use of different types of roofing materials regularly used in poultry houses. Measurements of thermal comfort were made through the use of techniques such as the Black Globe and Humidity Index (BGHI), the Thermal Heat Load (THL) and Enthalpy (H). Conducted in the State University of Goiás, during the months of April and May, 2011, the experiment was composed of five different treatments: AC - Asbestos cement tiles, BA -Bamboo tiles, BAP - Bamboo tiles painted in white, FB -Vegetable fiber tiles and bitumen, FBP -Vegetable fiber tiles and bitumen painted in white. The experiment consisted in 15 repetitions, which were considered the different days of measurements taken. Throughout the studied period, the time of the day considered the least comfortable was the one observed at 2:00pm, and the coverage of vegetable fiber and bitumen showed the highest value of BGHI (84.1) when compared to other types of coverage, characterizing a situation of lower thermal comfort, and no difference was found for THL and H on treatments in the studied region.

Allelopathic effect of black mustard tissues and root exudates on some crops and weeds

Laboratory and greenhouse experiments were conducted to evaluate the phytotoxic effect of black mustard extracts and root exudates on two crops: Trifolium alexandrinum and Triticum aestivum, and two weeds: Phalaris paradoxa and Sisymbrium irio. The seeds were treated with aqueous and ethanolic extracts and chloroform for eight days, or subjected to root exudates of just harvested mustard in a greenhouse for five weeks. High-performance liquid chromatography (HPLC) was used to quantify phytotoxins from plant tissues. Seed germination of P. paradoxa was reduced with the lowest concentration of the different extracts. However, the aqueous extract at 4% completely curtailed the germination of all the target species. In general, plant extracts had a concentration-dependent reduction of seedling growth of the target species. However, the ethanolic extract, at the lowest concentration, has stimulated the shoot length of both T. alexandrinum and T. aestivum, and the root length of the former. Mustard root exudates inhibited emergence and growth of the target species throughout the experiment. Ferulic and syringic acids were the dominant allelochemicals found when HPLC was used.

Chemical composition, acetylcholinesterase inhibitory and antifungal activities of Pera glabrata (Schott) Baill. (Euphorbiaceae)

Pera glabrata (Schott) Baill. was selected for this study after showing a preliminary positive result in a screening of Atlantic Forest plant species in the search for acetylcholinesterase inhibitors and antifungal compounds. The bioassays were conducted with crude ethanol extract of the leaves using direct bioautography method for acetylcholinesterase and antifungal activities. This extract was partitioned with hexane, chloroform and ethyl acetate solvents. The active chloroform fraction was submitted to silica gel chromatography column affording 12 groups. Caffeine, an alkaloid, which showed detection limits of 0.1 and 1.0 µg for anticholinesterasic and antifungal activities, respectively, was isolated from group nine. After microplate analyses, only groups four, nine, 10, 11 and 12 showed acetylcholinesterase inhibitory activity of 40% or higher. The group 12 was purified by preparative layer chromatography affording four sub-fractions. Two sub-fractions from this group were analyzed by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector. The first sub-fraction showed anticholinesterasic activity and contained two major compounds: 9-hydroxy-4-megastigmen-3-one (84%) and caffeine (6%). The second sub-fraction presented five major compounds identified as 9-hydroxy-4-megastigmen-3-one, isololiolide, (-) loliolide, palmitic acid and lupeol and did not show activity.

Lack of mutations of exon 2 of the MEN1 gene in endocrine and nonendocrine sporadic tumors

In addition to the mutations that underlie most cases of the multiple endocrine neoplasia type 1 (MEN1) syndrome, somatic mutations of the MEN1 gene have also been described in sporadic tumors like gastrinomas, insulinomas and bronchial carcinoid neoplasm. We examined exon 2 of this gene, where most of the mutations have been described, in 148 endocrine and nonendocrine sporadic tumors. DNA was obtained by phenol/chloroform extraction and ethanol precipitation from 92 formalin-fixed, paraffin-embedded samples, and from 40 fresh tumor tissue samples. We used 5 pairs of primers to encompass the complete coding sequence of exon 2 of the MEN1 gene that was screened by the polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) technique in 78 sporadic thyroid cancers: 28 follicular adenomas, 35 papillary carcinomas, 14 follicular carcinomas, and 1 anaplastic thyroid carcinoma. We also examined 46 adrenal lesions (3 hyperplasias, 3 adenomas and 35 adrenocortical carcinomas, 2 pheochromocytomas, 2 ganglioneuroblastomas, and 1 lymphoma) and 24 breast cancers (6 noninvasive, 16 infiltrating ductal, and 2 invasive lobular tumors). The PCR product of 5 tumors suspected to present band shifts by SSCP was cloned. Direct sense and antisense sequencing did not identify mutations. These results suggest that the MEN1 gene is not important in breast, thyroid or adrenal sporadic tumorigenesis. Because the frequency of mutations varies significantly among tumor subgroups and allelic deletions are frequently observed at 11q13 in thyroid and adrenal cancers, another tumor suppressor gene residing in this region is likely to be involved in the tumorigenesis of these neoplasms.

Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or = 99% purity, a yield of 25.0 ± 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60°C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37°C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.

Inotropic effects of extracts of Psidium guajava L. (guava) leaves on the guinea pig atrium

Many pharmacological effects have been ascribed to extracts of Psidium guajava L. (guava) leaves. However, in spite of its widespread use in Brazilian folk medicine and a reasonable number of scientific reports about it, we could not find any study dealing with its action on the mammalian myocardium. In the present study, by measuring isometric force, we observed that the crude extract of P. guajava (water-alcohol extract obtained by macerating dry leaves) depresses the guinea pig atrial contractility in a concentration-dependent fashion (N = 8 hearts, 15 trials). The compound with cardiac activity was concentrated by extraction in a Soxhlet apparatus using 17 M glacial acetic acid after removing the less polar fractions (hexane, chloroform, acetone, ethanol and methanol), suggesting that this compound is a highly polar substance. In the isolated guinea pig left atrium the acetic acid fraction (10-800 mg/l) of P. guajava 1) reversibly decreased myocardial force in a concentration-dependent fashion (EC50 = 0.07g/l, N = 5 hearts, 9 trials, P<0.05), 2) increased the atrial relaxation time measured at 20% of the force amplitude up to 35% (91 ± 15 to 123 ± 30 ms, N = 3 hearts, 6 trials, P<0.05), 3) abolished the positive staircase effect (Bowditch phenomenon) in a concentration-dependent fashion suggesting a decrease of the cellular inward calcium current (N = 4 hearts, 8 trials, P<0.05), and 4) its inotropic effect was abolished by cholinergic receptor blockade with 1.5 mM atropine sulfate, indicating a cholinergic involvement in the mechanism of action of the extract (N = 7 hearts, 15 trials, P<0.05). The acetic acid extract was 20 times more potent than crude extract (EC50 = 1.4 g/l). The results showed that extracts from P. guajava leaves depress myocardial inotropism.

Cholesterol-dependent hemolytic activity of Passiflora quadrangularis leaves

Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.

Cardiovascular activity of the n-butanol fraction of the methanol extract of Loranthus ferrugineus Roxb.

We investigated the vascular responses and the blood pressure reducing effects of different fractions obtained from the methanol extract of Loranthus ferrugineus Roxb. (F. Loranthaceae). By means of solvent-solvent extraction, L. ferrugineus methanol extract (LFME) was successively fractionated with chloroform, ethyl acetate and n-butanol. The ability of these LFME fractions to relax vascular smooth muscle against phenylephrine (PE)- and KCl-induced contractions in isolated rat aortic rings was determined. In another set of experiments, LFME fractions were tested for blood pressure lowering activity in anesthetized adult male Sprague-Dawley rats (250-300 g, 14-18 weeks). The n-butanol fraction of LFME (NBF-LFME) produced a significant concentration-dependent inhibition of PE- and KCl-induced aortic ring contractions compared to other fractions. Moreover, NBF-LFME had a significantly higher relaxant effect against PE- than against high K+-induced contractions. In anesthetized Sprague-Dawley rats, NBF-LFME significantly lowered blood pressure in a dose-dependent manner and with a relatively longer duration of action compared to the other fractions. HPLC, UV and IR spectra suggested the presence of terpenoid constituents in both LFME and NBF-LFME. Accordingly, we conclude that NBF-LFME is the most potent fraction producing a concentration-dependent relaxation in vascular smooth muscle in vitro and a dose-dependent blood pressure lowering activity in vivo. The cardiovascular effects of NBF-LFME are most likely attributable to its terpenoid content.

Alternative chromatographic system for the quality control of lipophilic technetium-99m radiopharmaceuticals such as [99mTc(MIBI)6]+

Knowledge of the radiochemical purity of radiopharmaceuticals is mandatory and can be evaluated by several methods and techniques. Planar chromatography is the technique normally employed in nuclear medicine since it is simple, rapid and usually of low cost. There is no standard system for the chromatographic technique, but price, separation efficiency and short time for execution must be considered. We have studied an alternative system using common chromatographic stationary phase and alcohol or alcohol:chloroform mixtures as the mobile phase, using the lipophilic radiopharmaceutical [99mTc(MIBI)6]+ as a model. Whatman 1 modified phase paper and absolute ethanol, Whatman 1 paper and methanol:chloroform (25:75), Whatman 3MM paper and ethanol:chloroform (25:75), and the more expensive ITLC-SG and 1-propanol:chloroform (10:90) were suitable systems for the direct determination of radiochemical purity of [99mTc(MIBI)6]+ since impurities such as99mTc-reduced-hydrolyzed (RH),99mTcO4- and [99mTc(cysteine)2]-complex were completely separated from the radiopharmaceutical, which moved toward the front of chromatographic systems while impurities were retained at the origin. The time required for analysis was 4 to 15 min, which is appropriate for nuclear medicine routines.

Enhanced production and organic solvent stability of a protease fromBrevibacillus laterosporus strain PAP04

A bacterial strain (PAP04) isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99%) to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source), skim milk (nitrogen source), pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74%) followed by acetone (63%) and chloroform (54.8%). In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50%) concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.

Toxigenic potential of Aspergillus flavus tested in different culture conditions

The objective of the present work was to evaluate the capacity of three isolates of Aspergillus flavus to produce aflatoxin under different culture conditions. This experiment was based on a 2³ factorial design, in which the independent variables were temperature (20-40 °C), incubation time (7-21 days), and the pH (2.0-6.0) in two different synthetic media. The optimal conditions were applied to non-aflatoxigenic isolates previously tested in coconut agar. Aflatoxin B1 was extracted directly from the synthetic cultures with chloroform. Thin Layer Chromatography (TLC) and Photographic Photometry were utilized to identify and quantify the compounds. Preliminary results showed that YES agar was an alternative medium for detecting the toxigenic potential of Aspergillus flavus in the following conditions: pH of 5.2, temperature of 25 °C, and incubation time of 11 days producing 206.05 ng.CFU-1 of aflatoxin B1. Of the 30 non-aflatoxigenic isolates, 12 presented a positive result in the optimal media and conditions tested.