Cytotoxic activity of ethanolic extracts of Lippia graveolens HBK leaves and stem against lung cancer cell line SK-LU-1 / Actividad citotóxica de extractos etanólicos de hoja y tallos de Lippia graveolens HBK contra la línea celular de cáncer de pulmón SK-LU-1

Lippia graveolens HBK (Verbenaceae) is an aromatic herb of economic importance in Mexico, known as oregano. The leaves are used as condiments, and people use this species for respiratory and digestive disorders. The aim of this work was to evaluate the cytotoxic effect of ethanolic extracts obtained from free-oil leaves (L9) and stem (S15), against lung cancer cell line SK-LU-1, through tetrazolium salt (MTT) assay. Extracts concentrations of 0.3 to 300 µg/mL were used and HFF-1 as normal control cells. Both L9 and S15 extracts, showed cytotoxic effect, although stem was stronger than leaves and without damage to normal cell control. The phenolic compounds caffeic acid and acacetin werein higher concentration in L9, whereas naringenin, taxifolin, eriodictyol, luteolin, and apigenin had higher concentrations in S15. The ethanolic extracts of L. graveolens have excellent cytotoxic activity, and have a wide possibility of use in lung cancer treatment.

Lippia graveolens HBK. (Verbenaceae) es una hierba aromática de importancia económica en México, conocida como orégano. Las hojas se usan como condimento y en medicina tradicional se utiliza para aliviar malestares respiratorios y digestivos. El objetivo de este trabajo fue evaluar el efecto citotóxico de extractos etanólicos obtenidos de hojas sin aceite (L9) y tallo (S15), sobre la línea celular de cáncer de pulmón SK-LU-1, mediante el ensayo de la sal de tetrazolium (MTT). Los extractos se aplicaron a concentraciones de 0,3 a 300 µg/mL y se utilizaron células HFF-1 como control normal. Tanto los extractos L9 como S15 mostraron efecto citotóxico, aunque el efecto del tallo fue mayor al de las hojas y sin daño al control celular normal. Los compuestos fenólicos ácido cafeico y acacetina se encontraron en mayor concentración en L9, mientras que naringenina, taxifolina, eriodictyol, luteolina y apigenina tuvieron mayor concentración en S15. Los extractos etanólicos de L. graveolens tienen una excelente actividad citotóxica, con amplia posibilidad de utilizar en el tratamiento de cáncer de pulmón.

[Evalution of antileishmanial activity of the hydroalcoholic extract of medicago lupulina' leaves against leishmania major [MHOM/IR/75/ER] promastigotes by using MTT and microscopy method and GC/MS for hydroalcoholic extract of this plant' leaves]

Background: Leishmaniasis, has created enormous global health problem. Side effects, drug resistance and the lack of effective vaccines and to make the new compounds effective due to plant

Objective: The traditional medical plants such as black alfalfa can be a valuable source of new pharmaceutical agents against leishmaniasis

Methods: Alcoholic extracts were prepared by maceration method. L. major promastigotes [Leishmania major] in Schneider and then were cultured in RPMI- 1640. Then, using MTT [Methyl Thiazole Tetrazolium], the IC50 [Inhibitory Concentrations 50%] for extract and Glucantime was determined. MTT assay did for each sample, 3 times

Results: IC50 for alcoholic extract of alfalfa black against L. major promastigotes in vitro after 24, 48 and 72 hours, respectively 165, 98 and 45 micrograms per ml and for Glucantime also equal to 27, 12 and 8 mg l respectively. IC50 between Extract and Glucantime after 24, 48 and 72 hours there was a significant difference [P <0.05]. Morphological changes after challenge with meglumine and alcoholic extracts including cell shrinkage, round, dense cytoplasm and the cell was smaller. Presence of alkaloids and flavonoids in alcoholic extracts have been proved

Conclusion: As regards, plant extract had anti- leishmanial effects in vitro, further works are required to appraise the exact effect on Leishmania agent in animal models

Cytotoxicity assays as tools to assess water quality in the Sinos River basin / Ensaios de citotoxicidade como ferramentas para avaliar a qualidade da água da bacia do Rio do Sinos

Cytotoxicity assays using cell cultures may be an alternative to assess biological toxicity of surface waters and may help to improve the control of water quality. This study compared two methods to prepare culture media for the exposure of Hep-2 cells to water samples collected from the Rolante River, an important affluent of the Sinos River. The toxicity was evaluated using the MTT and neutral red assays. Two methods were used to prepare culture media. In method 1, the sample was diluted at 1:1, 1:10, 1:100, 1:1000, 1:10.000 (v/v, sample/medium) in a standard culture medium; in method 2, water samples were used as the solvent for the culture medium, which was prepared at concentrations of 100, 80, 60, 40 and 20%. Semi-confluent cultures were then exposed to the media test for 24 hours, and cytotoxicity was determined immediately using the MTT and NR assays. Mitochondrial activity (MTT) was significantly lower at all concentrations in both methods, except at 1:1000 in method 1. However, the lysosome viability (NR) results revealed cytotoxicity only in the 1:1 sample of method 1. Both culture preparation methods were efficient and sensitive to the MTT assay, but method 2 seemed to be more adequate for the NR assay. The Rolante River has cytotoxic contaminants to Hep-2 cells, which may be one of the explanations for the poor water quality of the Sinos River basin.

Os ensaios de citotoxicidade utilizando culturas de células constituem uma alternativa para avaliar a toxicidade biológica de águas de superfície e podem auxiliar no controle da qualidade da água. Este estudo comparou dois métodos de preparação dos meios de cultura com amostras de água coletadas no rio Rolante, um importante afluente do Rio dos Sinos, para a exposição de células Hep-2. A toxicidade foi avaliada usando os ensaios do MTT e do vermelho neutro (VN). Dois métodos foram utilizados para preparar os meios de cultura. No método 1, a amostra foi diluída a 1:1, 1:10, 1:100, 1:1000 e 1:10.000 (v/v, amostra/ meio de cultivo) em um meio de cultura padrão; no método 2, as amostras de água foram utilizados como solventes para o meio de cultura, o qual foi preparado em concentração de 100% e nas diluições de 80, 60, 40 e 20%. Culturas semi-confluentes foram então expostas aos meios teste durante 24 horas, e a citotoxicidade foi determinada imediatamente usando os ensaios MTT e VN. A atividade mitocondrial (MTT) foi significativamente menor em todas as concentrações em ambos os métodos, exceto na diluição 1:1000 do método 1. No entanto, os resultados de viabilidade lisossomal (VN) revelaram citotoxicidade apenas no na diluição 1:1 do método 1. Ambos os métodos de preparação do meio cultura foram eficientes e sensíveis para o ensaio do MTT, mas o método 2 foi mais adequado para o ensaio do VN. O rio Rolante possui contaminantes citotóxicos para as células Hep-2, o que pode ser uma das explicações para a baixa qualidade da água da Bacia do Rio dos Sinos.

Metodologia para teste de tetrazólio em sementes de Amburana cearensis (Allemão) A.C. Smith / Methodology for tetrazolium test in Amburana cearensis (Allemão) A.C. Smith seeds

Objetivou-se com este trabalho estudar a metodologia do teste de tetrazólio para sementes de Amburana cearensis (Allemão) A.C. Smith, determinando as melhores condições de pré-umedecimento, bem como temperatura, período de coloração e concentração da solução de tetrazólio. Avaliou-se a coloração das sementes intactas e escarificadas (lixadas), ambas com tegumento, diretamente na solução de tetrazólio nas concentrações de 0,025; 0,050; 0,075 e 1 por cento a 35 e 40ºC, por 12 e 24h no escuro. Verificou-se o pré-umedecimento para retirar o tegumento utilizando-se sementes intactas e escarificadas, as quais foram imersas diretamente em água e submetidas a embebição em papel toalha e por 12, 24, 36 e 48 horas em temperaturas de 35 e 40ºC. Após a determinação do método para retirar o tegumento os embriões foram submetidas à coloração em solução de tetrazólio nas concentrações de 0,025; 0,050; 0,075 e 1 por cento. Com os resultados obtidos verificou-se a necessidade da retirada total do tegumento para que ocorra a coloração e que a temperatura de 40ºC é prejudicial para a embebição no pré-umedecimento. O teste de tetrazólio nas sementes de A. cearensis deve ser realizado com o pré-umedecimento das sementes escarificadas (lixadas) e imersas diretamente em água por 24 horas, na temperatura de 35ºC, para posterior retirada total do tegumento. E para atingir a coloração ideal os embriões devem ser imersos em solução de tetrazólio a 0,05 por cento por 3 horas, a 40ºC.

This work aimed to evaluate the methodology of tetrazolium test in Amburana cearensis (Allemão) A.C. Smith seeds, establishing the best pre-moistening conditions, as well as temperature, coloration period and tetrazolium solution concentration. The coloration of intact and scarified (sanded) seeds, both with tegument, was directly evaluated in tetrazolium solution at the concentrations of 0.025; 0.050; 0.075; and 1 percent, at 35 and 40ºC, for 12 and 24 h in the dark. Pre-moistening to remove the tegument was evaluated by using intact and scarified seeds, which were directly immersed in water and subjected to imbibition in paper towel for 12, 24, 36, and 48 h at 35 and 40ºC. After establishing the method to remove the tegument, embryos were subjected to coloration in tetrazolium solution at the concentrations of 0.025; 0.050; 0.075; and 1 percent. Based on the obtained results, the complete tegument removal is needed for coloration; besides, the temperature of 40ºC is harmful for imbibition during pre-moistening. The tetrazolium test in A. cearensis seeds should be performed including pre-moistening through direct immersion of scarified (sanded) seeds in water for 24h at 35ºC for later complete tegument removal. To achieve the ideal coloration, embryos must be immersed in tetrazolium solution at 0.05 percent for 3h at 40ºC.

Antimutagenic and anticarcinogenic effect of methanol extracts of Petasites japonicus Maxim leaves

The methanol extract from the leaves of Petasites japonicus Maxim (PJ) was studied for its (anti-)mutagenic effect with the SOS chromotest and reverse mutation assay. The (anti-)carcinogenic effects were evaluated by the cytotoxicity on human cancer line cells and by the function and the expression of gap junctions in rat liver epithelial cell. PJ extracts significantly decreased spontaneous beta-galactosidase activity and beta-galactosidase activity induced by a mutagen, ICR, in Salmonella (S.) typhimurium TA 1535/pSK 1002. All doses of the extract (0.08-100 mg/plate) decreased the reversion frequency induced by benzo (alpha)pyrene (BaP) in S. typhimurium TA 98. It decreased not only the spontaneous reversion frequency but also that induced by BaP in S. typhimurium TA 100. PJ extract showed greater cytotoxic effects on human stomach, colon and uterus cancer cells than on other cancer cell types and normal rat liver epithelial cells. Dye transfers though gap junctions were significantly increased by PJ extracts at concentrations greater than 200 microg/mL and the inhibition of dye transfer by 12-O-tetradecanoylphorobol-13-acetate (TPA) was obstructed in all concentrations of PJ. PJ significantly increased the numbers of gap junction protein connexin 43, and increased the protein expression decreased by TPA in a dose-dependent manner. Based on these findings, PJ is suggested to contain antimutagenic and anticarcionogenic compounds.

Clarithromycin Susceptibility Testing of Mycobacterium avium Complex Using 2,3-Diphenyl-5-thienyl-(2)-tetrazolium Chloride Microplate Assay with Middlebrook 7H9 Broth

A series of 119 Mycobacterium avium complex isolates were subjected to clarithromycin susceptibility testing using microplates containing 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC). Among 119 isolates, 114 (95.8%) were susceptible to clarithromycin and 5 were resistant according to the new and the standard method. STC counts the low cost and reduces the number of procedures needed for susceptibility testing.

Clarithromycin Susceptibility Testing of Mycobacterium avium Complex Using 2,3-Diphenyl-5-thienyl-(2)-tetrazolium Chloride Microplate Assay with Middlebrook 7H9 Broth

A series of 119 Mycobacterium avium complex isolates were subjected to clarithromycin susceptibility testing using microplates containing 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC). Among 119 isolates, 114 (95.8%) were susceptible to clarithromycin and 5 were resistant according to the new and the standard method. STC counts the low cost and reduces the number of procedures needed for susceptibility testing.

Improvement of XTT assay performance for studies involving Candida albicans biofilms

2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay has been used to study Candida biofilm formation. However, considering that the XTT reduction assay is dependent on cell activity, its use for evaluating mature biofilms may lead to inaccuracies since biofilm bottom cell layers tend to be relatively quiescent at later stages of biofilm formation. The aim of this study was to improve XTT reduction assay by adding glucose supplements to the standard XTT formulation. Candida albicans ATCC 90028 was used to form 24-, 48- and 72-h biofilms. The oxidative activity at 90, 180 and 270 min of incubation was evaluated. The control consisted of standard XTT formulation without glucose supplements, and was modified by the addition of 50, 100 and 200 mM of glucose. The XTT assay with 200 mM glucose showed more accurate and consistent readings correlating with biofilm development at 24, 48 and 72 h. Biofilm growth yield after 180 min incubation, when evaluated with the 200 mM glucose supplemented XTT, produced the most consistent readings on repetitive testing. It may be concluded that glucose supplementation of XTT could minimize variation and produce more accurate data for the XTT assay.

O teste de redução do 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) tem sido utilizado para mensurar o desenvolvimento de biofilmes de Candida. Contudo, a reação de XTT é dependente da atividade celular e o seu uso para biofilmes maduros pode ser questionado, considerando que diferentes camadas celulares têm atividade metabólica diferenciadas. O objetivo deste estudo foi avaliar se a adição de glicose à formula de XTT diminuiria a variabilidade na mensuração da atividade metabólica. Biofilmes de Candida albicans ATCC 90028 com tempos de crescimento de 24, 48 e 72 h foram utilizados. Para avaliar o melhor tempo de incubação do XTT, este foi mantido a temperatura de 37 °C, em tempos de 90 180 e 270 min. A fórmula padrão do teste XTT (controle) foi modificada com a adição de 50, 100 e 200 mM de glicose para os grupos experimentais. Os melhores resultados para a incubação foi observado com tempo de 180 min e para a suplementação de glicose à concentração de 200 mM (p<0.001). Concluiu-se que a incubação de 180 min utilizando a suplementação de 200 mM de glicose apresenta resultados de atividade metabólica celular com a menor variação para o estudo de biofilmes de Candida albicans.

The use of MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide]-reduction as an indicator of the effects of strain-specific, polyclonal rabbit antisera on Candida albicans and C. krusei.

There is only scanty data on the effects of specific antibody, with or without complement, on Candida albicans or Candida krusei in cell-free systems in vitro, although previously published work has shown that specific antibody mediates anti- Candida immunity in vivo by inhibition of adherence to host cells or surfaces and by the promotion of phagocytosis and intra-phagocytic killing. The MTT (3-[4, 5-dimethyl-2-thiazolyl] -2, 5-diphenyl -2H- tetrazolium bromide)-reduction method as a test of the viability of fungi was used to investigate the effect of complement, normal serum and immune serum on these two species of Candida that are of increasing importance as opportunistic pathogens. We report that normal rabbit serum or strain-specific, polyclonal anti- Candida rabbit antibody, with or without guinea pig complement, did not cause the reduction of total cell-mass or of the viability of either C. albicans or C. krusei, in vitro as determined by the MTT-reduction test. Complement alone without specific antibody, also, had no such effect on these two Candida species.