Schiefer counter: An alternative method for clonogenic assay evaluation.

INTRODUCTION: Clonogenic assay evaluates the potential of cells to undergo division or generate clones following treatment with a chemical or other agent, thereby allowing the evaluation of cytotoxic and/or antiproliferative effects. Clonogenic assay analysis using traditional methods tends to be time-consuming and yield inconsistent results, whereas results from analyses conducted using automated image processing methods may be misleading or subject to misinterpretation. Thus, the aim of this work was to validate and demonstrate the applicability of a recently developed software. METHODS: Repeatability of measurements was evaluated by comparing results from 10 replicate images from a single well. To evaluate the viability of the software, results were compared with those obtained from manual counting, crystal violet optical density, and up-to-date automated methods. A clonogenic index was experimentally developed using the individual area occupied by colonies, while clone stratification was used to differentiate between antiproliferative and cytotoxic effects. RESULTS: The developed software showed to be a reliable and consistent tool for clonogenic assay evaluation, presenting a repeatability mean error of 0.79% for the number of colonies and 0.89% for the total area of colonies, as well as exhibiting a significant correlation (p < 0.05) with results obtained from widely adopted gold standard methods. The software was also able to detect an appropriate dose-dependent effect as well as a predominant cytotoxic effect of vincristine on MCF-7 cells and calculate the clonogenic index. DISCUSSION: Therefore, this software is adequate for the analysis of clonogenic assay images, differentiating between cytotoxic and antiproliferative trends.

In vitro evaluation of the efficacy of commercial green tea extracts in UV protection.

Plants with antioxidant properties are beneficial for preventing the ageing events evoked by UV light, and numerous products based on Camellila sinensis (green tea) are commercially available, many of which claiming to contain bioactive compounds that would prevent UV-induced skin damage. In this study, we tested the efficacy of five commercial green tea extracts used to enrich cosmetic formulations for protecting human and mouse fibroblasts against UV radiation effects and compared with a fluid one prepared according to the Brazilian Pharmacopoeia recommendations. Taking into consideration that the ageing process can be accelerated by solar radiation by excessive free radical generation, leading to depletion of skin antioxidant defences, and its collapse caused by disruption of the metalloproteinase metabolism, we have used their individual (-)-epigallocathechin-3-gallate (EGCG) content, the catalase and SOD status and the matrix-degrading metalloproteases (MMP)-1, MMP-9 and MMP-13 levels as comparative parameters. The EGCG content of the commercial products showed wide variability, ranging from undetectable levels to 58.65 ± 1.12 µg mL(-1) , in contrast with the fluid extract (87.82 ± 1.35 µg mL(-1) ). Moreover, only the pharmacopoeic extract was able to significantly reduce MMP degradation while enhancing the levels of SOD and catalase. These results indicate, for the first time, that the methodologies for preparing herbal mixtures can interfere significantly with compounds endowed with photoprotective effects, and the efficacy of products containing C. sinensis extracts thought to act against effects of solar radiation can be compromised.

Influência de extratos hidroetanólicos de plantas medicinais sobre a quimiotaxia de leucócitos humanos / Influence of some medicinal plant hydroethanolic extracts on human leukocyte chemotaxis

Vários métodos in vitro têm sido empregados para a investigação das atividades biológicas de plantas usadas na medicina popular para o tratamento de processos inflamatórios. Neste trabalho, investigou-se a influência dos extratos hidroetanólicos de Rauvolfia sellowii Muell. Arg, Hybanthus bigibbosus (St.-Hil) Hassler e Anchieta pyrifolia (Mart.) G. Don, conhecidas popularmente como pau-pra-tudo, canela-de-veado e cipó-suma, respectivamente, sobre a quimiotaxia de leucócitos humanos, estimulados a migrar contra um gradiente de caseína, utilizando-se o método de Boyden. A dexametasona foi utilizada como substância de referência da inibição da quimiotaxia leucocitária. Os resultados demonstraram efeito inibitório significativo de todos os extratos das plantas testadas, sobre a migração de polimorfonucleares, induzida por caseína. Entretanto, essa atividade variou de intensidade conforme a concentração e a espécie estudada. Efeitos máximos foram observados, nas concentrações de 1000, 10 e 1µg/ml com os extratos de pau-pra-tudo, canela-de-veado e cipó-suma, respectivamente, com migração de 81,6±3,9 por cento; 85,4±2,4 por cento e 91,7±2,2 por cento dos polimorfonucleares, enquanto que, com a dexametasona, este efeito foi de 70,3±5,9 por cento. Embora estudos mais aprofundados sejam necessários, os resultados apresentados podem servir como base preliminar de dados, contribuindo para esclarecer o mecanismo da atividade antiinflamatória atribuída às essas plantas na medicina caseira.

Several in vitro methods have been used for the investigation of the biological activities of plants used in folk medicine for the treatment of inflammatory conditions. In this study, we have investigated the ability of the hydroethanolic extracts from Rauvolfia sellowii Muell. Arg, Hybanthus bigibbosus (St.-Hil) Hassler, and Anchieta pyrifolia (Mart.) G. Don, locally known as pau-pra-tudo, canela-de-veado, and cipó-suma, respectively, in interfering with the human leukocytes migration induced by casein, using the Boyden chamber method. Dexamethasone has been used as a positive control for leukocyte inhibition in the same experimental approach. The data herein presented showed a significant inhibition of the casein-induced polymorphonuclear leukocytes migration for all plants studied. However, the intensity of such activity was variable according to the dose and plant tested. For pau-pra-tudo, canela-de-veado, and cipó-suma extracts the average number of migrated polymorphonuclear leukocytes was 81.6±3.9 percent, 85.4±2.4 percent and 91.7±2.2 percent of the input for the doses of 1000, 10, and 1mg/ml, respectively, while for dexamethasone, the value found was 70.3±5.9 percent. Although further studies are needed, the results presented in this study may be useful to clarify the anti-inflammatory properties of these herbal medicines, supporting their ethnobothanical use for the treatment of inflammatory diseases.

Effect of vitamin E on red blood cell preservation.

The effect of exogenous vitamin E (2.1 mg/100 ml) was determined on the preservation of red blood cell integrity (PCV and RBC counts) during long-term storage. Cells from healthy human blood donors, normal rats and rats raised on a vitamin E-deficient diet were stored in CE medium at 4 degrees C for 40-60 days. The addition of vitamin E to the CE medium protected normal rat RBC and those from vitamin E-deficient animals to even a larger extent. Neither the PVC nor RBC count in human blood changed during storage for up to 60 days and no effect of exogenous vitamin E was demonstrable.