Viscumalbum L. homeopathic mother tinctures: metabolome and antitumor activity

Viscum album L. is a semi-parasitic plant with antitumor activity attributed to theaqueous extracts. However, European V. album ethanolic extracts (VAE) have also demonstrated invitro activity in tumor models. Aims: Evaluate the metabolic profiles of fifty VAE harvested duringsummer and winter seasons and their antitumor activity through 2D and 3D models. Methodology:VAEwerepreparedbymacerationfrom:V.albumsubsp.albumgrowingonMalus domestica,Quercus sp.and Ulmus sp.; V. album subsp. austriacum from Pinus sylvestris; V. album subsp. abietis from Abies alba.Chemical analyses were performed through liquid chromatography coupled with high resolutionmass spectrometry and Partial Least Squares Discriminant Analysis (PLS-DA) was performed in theMetaboanalyst 4.0. The antitumor potential of the selected VAE was evaluated in 2D and 3D models(MDA-MB-231 cancer cells) by MTT, crystal violet and glycolytic pathway analysis. Results anddiscussion:Thefirst3principalcomponentsinPLS-DAexplained60%and40%ofdatavariationin positive andnegativemodesrespectively.Threegroupswereformedandshowedchemicalsimilarityamong V. album subspecies. The compounds responsible for group separation were tentativelyidentifiedas:pinobankasinornaringenin hexoside;isorhamnetin-3-hexoside,meglutolanddifferent aminoacids.ThesummerVAEat0.5%v/vinducedhighercytotoxicdamagethanthewinterpreparations, and Abies alba and Quercus sp. VAE promoted 49% and 42% reduction of tumorviability in 3D model (72h incubation), respectively. MDA-MB-231 glycolytic pathway in 2D modelshowed a decrease in the glucose consumption and extracellular lactate production. Also, PFK (6-phosphofructo-1-kinase)andPK(Pyruvatekinase)activitieswereinhibitedbyAbiesalbaandQuercus sp. VAE at 48h of incubation. Conclusion: VAE extracts showed different metabolomes andthe glycolytic pathway should be an important target involved in the inhibition of tumor growth bytheseextracts

Cell alterations induced by a biotherapic for influenza / Cell alterations induced by a biotherapic for influenza

Introduction: Influenza viruses have been responsible for highly contagious acute respiratory illnesses with high mortality, mainly in the elderly, which encourages the development of new drugs for the treatment of human flu. The biotherapics are medicines prepared from biological products, which are not chemically defined. They are compounded following the homeopathic procedures indicated for infectious diseases with known etiology [1]. Aim: The purpose of the present study is to verify cellular alterations induced by a biotherapic prepared from the infectious influenza A virus. Methodology: This biotherapic was prepared for this study in the homeopathic potency of 30X according to the Brazilian Homeopathic Pharmacopeia [2]. The concentration of 10% was not cytotoxic to cells, as verified by neutral red assay. The cellular alterations observed in MDCK cells were analyzed by optical microscopy for the quantification of mitosis, nucleoli and lipid bodies. The mitochondrial activity was assessed by MTT assay and the phosphosfructokinase-1 (PFK-1) enzyme activity was analyzed on the MDCK cells treated for 5, 10 and 30 days. Macrophages J778.G8 were treated with this biotherapic to evaluate the immunostimulatory cytokine release. Results: The cellular alterations observed in MDCK cells were verified by optical microscopy. The number of lipid bodies present in MDCK cells stimulated for 10 days was significantly lower (p <0.05) when compared to controls. The biotherapic significantly increased (p <0.05) the number of mitosis and the mitochondrial activity of MDCK cells stimulated for 10 and 30 days. These changes were confirmed by a significant reduction (p <0.05) on the PFK-1 activity. These results suggest that the biotherapic was able to activate the Krebs cycle and pentosephosphate metabolism to the generation of amino acids and nucleotides, situations common to cells whose rate of mitosis is increased. The quantification of immunostimulatory cytokines by macrophages J774.G8 indicated that the tumor necrosis factor (TNF-?) production was higher (p <0.05) in the supernatant of the macrophages pre-treated with this biotherapic and infected with influenza virus, suggesting an activation of the macrophages by this biotherapic. Conclusion: This biotherapic is able to induce some cellular alterations, which show strong evidence that it might be a promising option for the human flu. New experiments are being developed to understand the mechanisms of action of this biotherapic.

Crude ethanol extract from babassu (Orbignya speciosa): cytotoxicity on tumoral and non-tumoral cell lines

Plant-derived substances have been considered as important sources of drugs, including antineoplasic agents. Babassu mesocarp is popularly used in Brazil as a food additive, and in popular medicine against several conditions, such as inflammations, menstrual pains and leukaemia. From babassu Orbignya speciosa (Mart.) Barb. Rodr. [Arecaceae (Palmae)] epicarp/mesocarp, an ethanol extract was prepared and named OSEME, which was tested on the viability,morphology and metabolism of several cell lines, such as the leukaemic cell lines, HL-60, K562 and the latter multidrug resistant counterpart K562-Lucena 1, the human breast cancer cell line MCF-7, the mouse fibroblast cell line 3T3-L1 and fresh human lymphocytes. OSEME promoted a dose-dependent decrease on the viability of all cells. This effect was much more pronounced on the tumoral cell lines than on non-tumoral cells, a phenomenon revealed by the dose of OSEME which promotes half of maximal effect (ID50). The decrease on viability was followed by shrinkage of cells, alteration on their morphology, and a markedly nuclear condensation. Curiously, stimulation of 6-phosphofructokinase activity (6.6-times) was observed on HL-60 cells, treated with OSEME, when compared to control treated with ethanol (vehicle). These results support evidences to suggest OSEME as a promising source of novel antineoplasic agents.

Substâncias derivadas de plantas têm sido usadas como importante fonte de agentes antineoplásicos. O mesocarpo do babaçu é popularmente usado no Brasil como suplemento alimentar e na medicina popular para o tratamento de várias afecções, tais como: inflamações, cólicas menstruais e leucemia. A partir do epicarpo/mesocarpo do babaçu Orbignya speciosa (Mart.) Barb. Rodr. [Arecaceae (Palmae)] foi preparado um extrato etanólico, denominado OSEME, o qual foi incubado com as seguintes linhagens humanas leucêmicas: HL-60, K562 e a sua derivada resistente a múltiplas drogas, K562-Lucena 1; além destas, foram testadas a linhagem humana de câncer de mama, MCF-7; a linhagem de fibroblastos de camundongo, 3T3-L1 e linfócitos humanos de sangue periférico. OSEME promoveu diminuição da viabilidade em todas as linhagens celulares testadas de maneira dose-dependente. Este efeito foi mais pronunciado sobre as linhagens celulares tumorais quando comparado às não tumorais, o que foi revelado pela dose de OSEME capaz de promover metade do efeito máximo (ID50). A diminuição da viabilidade foi acompanhada por danos sobre a morfologia celular com pronunciada condensação citoplasmática e nuclear. Curiosamente, quando a linhagem HL-60 foi tratada com OSEME, foi detectado um aumento de 6,6 vezes da atividade da enzima 6-fosfofrutoquinase, quando comparado ao grupo controle (células tratadas com o veículo etanol). Esses resultados sugerem que OSEME pode ser uma promissora fonte de novos agentes antineoplásicos.

High dilutions of Euphorbia tirucalli L. (aveloz) modify the viability and glycolytic metabolism of cell lines / Altas diluições de Euphorbia tirucalli L. (aveloz) modificam a viabilidade e o metabolismo glicolítico de linhas celulares

The latex of Euphorbia tirucalli L. (Aveloz) is popularly used to treat cancers from diverse sources such as: breast, prostate, lung and kidney. Furthermore, high dilutions of this latex (latex-HDs) have been successfully used in the treatment of tumors, although the mechanisms involved in this antitumoral activity are not yet known [...] Latex-HDs were obtained through the interaction of two procedures: 1:100 dilution in mass and agitation, using 2 solvents, water and 70% ethanol in homeopathic preparations 5, 15 and 30cH, following the Brazilian Homeopathic Pharmacopeia. Cell lines were incubated with 1 % and 10 % of latex-HDs for 24 hours. Controls treated with similar preparations without latex were incubated under the same conditions. Cell viability was analyzed by MTT assay and morphological features were assessed by May-Grunwald-Giemsa method. Cell ultrastructure was analyzed by transmission electron microscopy. Metabolic alterations were detected by spectrophotometric assay for the enzyme 6-phosphofructo-1-kinase (PFK-1) activity. 1% of ethanolic latex-HDs induced no alterations in MelanA cells viability, however Aveloz 15cH induced an increase in MCF7 cells viability. After aqueous treatment, MelanA cells viability decreased in almost all systems, whereas in MCF7 cells, an increase was detected in dynamized water systems and Aveloz 15CH. However, only water 15cH increased cell viability in comparison to control and non-dynamized water. No alterations in MCF7 morphology and ultrastructure were visualized through light microscopy or electronic microscopy, respectively. Interesting results were detected when we studied the glycolytic metabolism of both cells, giving support to evidences showing that HDs interfere in the metabolism of cell lines. Further studies are needed to better understand the mechanisms involved in this in vitro biological response to HDs.

O látex de Ephorbia tirucalli L. (aveloz) é usado popularmente no tratamento de diversos tipos de câncer incluindo mama, próstata, pulmão e rim. Além do mais, altas diluições desse látex (latex-HDs) têm sido utilizadas com sucesso no tratamento de tumores, embora o mecanismo dessa ação ainda seja desconhecido [...] As látex-HDs foram obtidas através de 2 procedimentos: diluição 1:100 em massa e agitação, com 2 solventes, água e etanol 70%, nas diluições homeopáticas 5, 15cH e 30cH de acordo com a Farmacopéia Homeopática Brasileira. As linhas celulares foram incubadas com látex-HDs 1% e 10% por 24 horas. Foram preparados controles similares sem o látex e incubados sob as mesmas condições. A viabilidade celular foi analisada através do método May-grunwald-Giemsa. A ultrastrutura celular foi analisada através de microscopia eletrônica de transmissão. As alterações metabólicas foram detectadas por ensaio de espectrofotometria para a atividade da enzima 6-fosfofructo-1-quinase (PFK-1). Látex-Hds em etanol 1% não induziram qualquer alteração na viabilidade das células MelanA, mas Aveloz 15cH induziu aumento da viabilidade das células MCF7. Após tratamento, a viabilidade das células MelanA diminuiu em quase todos os sistemas, enquanto que nas células MCF7 foi detectado aumento nos sistemas aquosos e Aveloz 15cH, mas só água 15cH aumentou a viabilidade celular por comparação ao controle e água não dinamizada. Não foram detectadas alterações na morfologia celular por microscopis de luz quando se estudou o metabolismo glicolítico em ambas as células, demonstrando que os padrões de resposta celular dependem do tipo de solvente, metabolismo celular e procedimento de diluição. Esta é a primeira vez que se mostra que as altas diluições interferem no metabolismo de linhas celulares. Outros estudos são necessários para se compreender melhor os mecanismos envolvidos nesta resposta in vitro a altas diluições de aveloz.