José Luiz Mazzei

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.

Análise química quantitativa para a padronização do óleo de copaíba por cromatografia em fase gasosa de alta resolução

Quantitative GC-FID was evaluated for analysis of methylated copaiba oils, using trans-(-)-caryophyllene or methyl copalate as external standards. Analytical curves showed good linearity and reproducibility in terms of correlation coefficients (0.9992 and 0.996, respectively) and relative standard deviation (< 3%). Quantification of sesquiterpenes and diterpenic acids were performed with each standard, separately. When compared with the integrator response normalization, the standardization was statistically similar for the case of methyl copalate, but the response of trans-(-)-caryophyllene was statistically (P < 0.05) different. This method showed to be suitable for classification and quality control of commercial samples of the oils.

Genotoxicity of polycyclic aromatic hydrocarbons and nitro-derived in respirable airborne particulate matter collected from urban areas of Rio de Janeiro (Brazil).

Air pollution toxic effects are mainly attributed to small inhalable particulates with an aerodynamic diameter of less than 2.5 µm (PM 2.5). Our objective was to investigate mutagenic and clastogenic activity in PM samples collected in Rio de Janeiro. Samples were collected using a high-volume sampler at three sites: with low traffic and (2) and (3) with a heavy traffic. Six polycyclic aromatic hydrocarbons (PAHs) were quantified by gas chromatography/mass spectrometry (GC/MS). Salmonella typhimurium TA98 and the derivative strains YG1021 and YG1024 were used in mutagenicity assays in the presence of organic extracts (10–50 µg/ plate) with and without exogenous metabolization. Allium cepa test was performed to evaluate possible cytotoxic and clastogenic activities. The highest PM 2.5 µm (132.73 µm/m3) and PAH values (1.22 ng/m3 for benzo(a)pyrene) were detected at site 3. High mutagenic frameshift responses in absence and presence of metabolic activation were detected at site 3. The participation of nitroarenes and dinitroarenes was detected in the total mutagenicity of the extracts studied. The cytotoxic effect and the abnormalities detected by Allium cepa test can be attributed to the PAH nitroderivatives in the organic extracts. Evaluation of the genotoxicity of urban airborne particulate matter is important as a basis for decision making by regulatory authorities.

Toxicological evaluation of Euterpe edulis: a potential superfruit to be considered.

Superfruits have a high nutritional value due to their richness in nutrients, antioxidants, proven or potential health benefits and taste appeal. However, there are no scientific criteria for defining which fruits are superfruits. In Brazil, several palms have an edible palm heart, the best known and most widely appreciated of which is called Acai (Euterpe oleracea). Euterpe edulis Mart., commonly called jussara, is an evergreen species that grows in the rainforest. Having initially been consumed in the form of juice and pulp, they have since been incorporated as an ingredient in many foods. A risk assessment to identify adverse health effects is a prerequisite for taking forward the development of new drugs, cosmetics and foods. To make a toxicological evaluation of E. edulis, in the present work this prerequisite was met by an interdisciplinary network that performed mass spectroscopy analyses, blood biochemistry, genotoxicity, bacterial reverse mutation and cytotoxicity assays. Positive mutagenicity results were detected for Salmonella typhimurium TA97 at low doses, and positive results were also obtained for the mammalian erythrocyte micronucleus assay, indicating that the pulp of E. edulis contains compounds with the capacity to induce mutagenicity and clastogenic/aneugenic effects.

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.

Mutagenic risks induced by homemade hair straightening creams with high formaldehyde content.

Regardless of official recommendations, the inappropriate use of homemade hair creams has became a popular practice in Brazil and high formaldehyde content in the ‘progressive straightening’ creams has been reported. In the present work, three of these creams were analyzed by spectrophotometric, chromatographic and genotoxic assays in order to evaluate mutagenic risks associated with the uncontrolled addition of formaldehyde at contents higher than those allowed by regulation. The ultraviolet and Fourier‐transformed infrared absorption spectra showed characteristic signals that can be assigned to formaldehyde, although with different relative intensities, revealing distinct compositions. Using high‐performance liquid chromatography 1.6–10.5% w/v formaldehyde was quantified. Antibacterial activity was detected in all creams. At 0.10 μg per plate, one of them showed positive mutagenicity induction (P < 0.05) in the Salmonella/microsome assay using the TA100 strain. The measurement of β‐galactosidase induction in the SOS chromotest by this cream, at dosages of 10–100 μg per assay, was positive (P < 0.05) in Escherichia coli PQ37 and OG100 strains. Our data show a more intense genotoxic response than those reported before for formaldehyde, suggesting that this compound may be acting synergistically with any unknown components in the creams or perhaps these unspecified components by themselves might have significant genotoxic potential. We call attention to the popular use of homemade formulations of cosmetics, such as hair straightening creams, because they can contain mutagens that could increase the incidence of neoplasia in those people who use them. Copyright

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.

Perfil cromatográfico de duas espécies de Plumbaginaceae: Plumbago scandens L. E Plumbago auriculata Lam

The genus Plumbago belongs to the family Plumbaginaceae, order Plumbaginales. Comparative chemical profile of P. scandens (native) and P. auriculata (cultivated) was obtained by normal and reversed-phase high performance liquid chromatography with photodiode array detector. Comparison of the ultraviolet espectra and the retention times for the compounds allowed to find similar metabolic patterns in roots, stems and leaves. Four flavonoids, one phenolic acid or derivative and the naphtoquinone plumbagin were comparatively identified to standards.

Anti-inflammatory effect of Schinus terebinthifolius Raddi hydroalcoholic extract on neutrophil migration in zymosan-induced arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE Schinus terebinthifolius is a species of plant from the Anacardiaceae family, which can be found in different regions of Brazil. Schinus is popularly known as aroeirinha, aroeira-vermelha, or Brazilian pepper. In folk medicine, S. terebinthifolius is used for several disorders, including inflammatory conditions, skin wounds, mucosal membrane ulcers, respiratory problems, gout, tumors, diarrhea and arthritis. According to chemical analyses, gallic acid, methyl gallate and pentagalloylglucose are the main components of hydroalcoholic extracts from S. terebinthifolius leaves. In the present study, we demonstrated the ability of a hydroalcoholic extract to inhibit cell migration in arthritis and investigated the mechanisms underlying this phenomenon. MATERIALS AND METHODS The anti-inflammatory effect of S. terebinthifolius hydroalcoholic leaf extract (ST-70) was investigated in a zymosan-induced experimental model of inflammation. Male Swiss and C57Bl/6 mice received zymosan (100 µg/cavity) via intra-thoracic (i.t.) or intra-articular (i.a.) injection after oral pre-treatment with ST-70. The direct action of ST-70 on neutrophils was evaluated via chemotaxis. RESULTS ST-70 exhibited a dose-dependent effect in the pleurisy model. The median effective dose (ED50) was 100mg/kg, which inhibited 70% of neutrophil accumulation when compared with the control group. ST-70 reduced joint diameter and neutrophil influx for synovial tissues at 6h and 24h in zymosan-induced arthritis. Additionally, ST-70 inhibited synovial interleukin (IL)-6, IL-1β, keratinocyte-derived chemokine (CXCL1/KC) and Tumor Necrosis Factor (TNF)-α production at 6h and CXCL1/KC and IL-1β production at 24h. The direct activity of ST-70 on neutrophils was observed via the impairment of CXCL1/KC-induced chemotaxis in neutrophils. Oral administration of ST-70 did not induce gastric damage. Daily administration for twenty days did not kill any animals. In contrast, similar administrations of diclofenac induced gastric damage and killed all animals by the fifth day. CONCLUSIONS Our results demonstrated significant anti-inflammatory effects of ST-70, suggesting a putative use of this herb for the development of phytomedicines to treat inflammatory diseases, such as joint inflammation.