Luiz Antonio d'Avila

Immunomodulating and antiviral activities of Uncaria tomentosa on human monocytes infected with Dengue Virus-2

Uncaria tomentosa (Willd.) DC., a large woody vine native to the Amazon and Central American rainforests has been used medicinally by indigenous peoples since ancient times and has scientifically proven immunomodulating, anti-inflammatory, cytotoxic and antioxidant activities. Several inflammatory mediators that are implicated in vascular permeability and shock are produced after Dengue Virus (DENV) infection by monocytes, the primary targets for virus replication. Here we assessed the immunoregulatory and antiviral activities from U. tomentosa-derived samples, which were tested in an in vitro DENV infection model. DENV-2 infected human monocytes were incubated with U. tomentosa hydro-alcoholic extract or either its pentacyclic oxindole alkaloid-enriched or non-alkaloid fractions. The antiviral activity was determined by viral antigen (DENV-Ag) detection in monocytes by flow cytometry. Our results demonstrated an in vitro inhibitory activity by both extract and alkaloidal fraction, reducing DENV-Ag+ cell rates in treated monocytes. A multiple microbead immunoassay was applied for cytokine determination (TNF-alpha, IFN-alpha, IL-6 and IL-10) in infected monocyte culture supernatants. The alkaloidal fraction induced a strong immunomodulation: TNF-alpha and IFN-alpha levels were significantly decreased and there was a tendency towards IL-10 modulation. We conclude that the alkaloidal fraction was the most effective in reducing monocyte infection rates and cytokine levels. The antiviral and immunomodulating in vitro effects from U. tomentosa pentacyclic oxindole alkaloids displayed novel properties regarding therapeutic procedures in Dengue Fever and might be further investigated as a promising candidate for clinical application.

Brazilian gasoline quality: study of adulteration by statistical analysis and gas chromatography

The addition of organic solvents (heavy aliphatic, light aliphatic and aromatic hydrocarbons) in Brazilian gasoline is unfortunately very frequent, and this illicit practice does not guarantee gasoline quality. Organic solvent adulterations of gasoline samples have been investigated. For characterization and comparison of these samples, physico-chemical parameters were selected as the factor for hierarchic multivariate analysis (Cluster Analysis). However, these parameters are not efficient to detected all kinds of adulteration. Gas Chromatographic (GC) analysis can be used as a procedure to improve the detection of adulterated gasoline. More detailed information of their compositions was revealed. The use of physico-chemical properties of gasoline samples for hierarchic multivariate analysis and gas chromatographic fingerprints is a practicable method to adulteration detection.

Chromatographic models as tools for scale-up of isolation of natural products by semi-preparative HPLC

Abstract Scale‐up of high performance liquid chromatography (HPLC) using mathematical models has been found in the literature for separation of binary mixtures or monocomponent materials. This procedure provides graphical representation, in contrast to direct scale‐up, which has been usually employed for separation of natural fraction components. In this report, the application of models to the scale‐up of isocratic separations, from literature data, of carotenoids, vitamins, ginsenosides, and monoterpenes fractions, in terms of use of larger columns and sample overloads, is discussed. Statistical moment analysis and an ideal rate model were applied using a computer spreadsheet to estimate parameters from analytical data and predict semi‐preparative separations. Non‐competitive effects between the components were assumed, due to the few available data. The predicted chromatograms showed good agreement with the experimentals, demonstrating the applicability of scale‐up using models on separation of natural fractions.

In vitro cultivated Uncaria tomentosa and Uncaria guianensis with determination of the pentacyclic oxindole alkaloid contents and profiles

Uncaria tomentosa (Willd.) DC. and Uncaria guianensis (Aubl.) Gmel., commercially known as cats claw, are large woody vines native of the Amazon and Central America rainforests. These Rubiaceae species face nowadays an imminent risk of extinction due to indiscriminate harvesting in the wild as well as to increasing deforestation of their natural habitat. This work describes in vitro cultivation methods for both species with determination, by HPLC, of the pentacyclic oxindole alkaloid contents and profiles of the plant material. The results have proved that the methods we developed were able to produce plants with alkaloid profiles and contents similar to the wild and in vivo cultivated species, and with the additional advantage of producing suitable young plants in a shorter period of time. The data showed that the in vitro technique can be a feasible tool for the growth of the species, and may thus be important for their commercialization and for their conservation as a forest resource.

Desenvolvimento e aplicação de metodologia por cromatografia em camada delgada para determinação do perfil de alcalóides oxindólicos pentacíclicos nas espécies sul-americanas do gênero Uncaria

The species Uncaria tomentosa (Willd.) DC. and U. guianensis Gmel. (Rubiaceae), known as cats claw, are large woody vines occurring in the Amazon rain forest and other tropical areas of South and Central America. It has been used medicinally by indigenous peoples for at least 2,000 years for several diseases. Tetra- and pentacyclic oxindole alkaloids, triterpenoid glycosides, sterols and flavonoids are found in these plants. Among these metabolites, six pentacyclic oxindole alkaloids, speciophylline, mitraphylline, pteropodine, uncarine F, isopteropodine and isomitraphylline, are considered to be the biochemical markers and are used to standardize commercial herbal medicines. The present study describes the development of an analytical methodology to determine the profile of these alkaloid markers through thin layer chromatography (TLC). This development has also included a comparison among the use of the crude methanol extract and fractions obtained through the classical acid-base partition or by using the basic resin Polyamide 6. Silica gel was used as stationary phase with the variation of some parameters such as solvent systems, sample concentration, distance of development and detection method. The TLC method developed was shown to be reliable, reproducible and selective for the target alkaloids. It has been applied to the analysis of leaves and stems from both species as well as phytopharmaceutical derivatives based on U. tomentosa.

Alternative extraction of alkaloid anticarcinogens from Brazilian "vinca rosea" using Ion exchange chromatography

Extracts in ethanol and ethanol-ammonia of dried leaves from Catharanthus roseus, gathered at Rio de Janeiro state, were adsorbed in a strongly acidic cation exchange resin with sulfonic acid group, using the finite bath method, resulting in an alkaloid retained fraction and an acidic and neutral unretained fraction. High Performance Liquid Chromatography showed the isolation of the alkaloid fraction to be highly selective and with good performance, with an absence of alkaloids in the unretained fraction, while the retained fraction presented 1,54-6,35 mg/g of vindoline and 0,12-0,91 mg/g of vinblastine, common for an alkaloid-rich concentrate, usually obtained by classic extraction with several steps using solvents.

In-Column Derivatization for the Separation of Heavy Aza-Arenes and Olefins by Liquid Chromatography

Abstract The formation of complexes with mercury ions (Hg ++) and protons is used to improve the separation of heavy olefins and aza-arenes. The complexation process is irreversible and similar to precolumn derivatization. Complexation makes possible to use mobile phases with high water contents thus providing high hydrophobic selectivities.

DETERMINAÇÃO DO TEOR DE BIODIESEL EM DIESEL EMPREGANDO O ENSAIO COLORIMÉTRICO DO ÁCIDO HIDROXÂMICO

The aim of this paper was to use colorimetric assays for hydroxamic acid to quantify the biodiesel content in diesel and compare it with the traditional method (infrared spectroscopy, using the EN 14078 method). Samples were prepared from B2 to B10 with two kinds of diesel - S500 (red) and S50 (yellow) - to obtain two calibration curves. Through statistical methods it was shown that the slopes of the straight lines obtained for the different types of diesel were the same. Thus, the type of diesel did not influence the results of the colorimetric assay for hydroxamic acid. Real samples collected from gas stations were analyzed by both methods (colorimetry and EN 14078). By applying Students t-test it was concluded that the methods could be considered statistically equivalent. Therefore, it was confirmed that the colorimetric assay for hydroxamic acid is suitable for detecting and quantifying the content of biodiesel in biodiesel/diesel blends and can also be easily adapted to field analyses.