Metabolic profiling and correlation analysis for the determination of killer compounds of proliferating and clonogenic HRT-18 colon cancer cells from Lafoensia pacari.

ETHNOPHARMACOLOGICAL RELEVANCE: Lafoensia pacari A. St.-Hil., belonging to the family Lythraceae and popularly known as 'dedaleira' and 'mangava-brava,' is a native tree of the Brazilian Cerrado, and its barks have been traditionally used as a tonic to treat inflammatory conditions, particularly related to gastric ulcers, wounds or fevers and various types of cancer. AIM OF THE STUDY: We have previously demonstrated the apoptogenic effects of the methanolic extract of L. pacari using various cancer cell lines. In the present study, this extract has been partitioned into fractions to identify the components that might be responsible for the apoptogenic effects using HRT-18 cells, which have been previously demonstrated to be sensitive to this extract. MATERIALS AND METHODS: A standard methanolic extract was prepared and fractionated by centrifugal partition chromatography. The fractions were submitted to cytotoxicity and clonogenic assays to monitor the effects in parallel with LC-DAD-MS and statistical analyses to suggest the potential bioactive compounds. RESULTS: Besides ellagic acid, the primary constituent of the plant and also the biomarker of the species, punicalin, pedunculagin and punicalagin isomers, catechin and ellagic acid derivatives were putatively identified. CONCLUSIONS: The barks of L. pacari are rich in ellagic acid and various hydrolysable tannins, some of which were reported for the first time in this species, such as punicalagin and ellagitannins. This mixture of substances had the ability to kill proliferating cells and abrogate the growth of clonogenic cells in a similar manner shown by the methanolic extract of our previous study. The collective data reported herein suggest that the biological activities of the L. pacari barks used by population to treat cancer conditions are due to the apoptogenic effects promoted by a mixed content of ellagitannins.

Uleine Disrupts Key Enzymatic and Non-Enzymatic Biomarkers that Leads to Alzheimer's Disease.

BACKGROUND: Alzheimer´s disease, a progressive and degenerative disorder of the brain, is the most common cause of dementia among the elderly. To face its multifactorial nature, the use of single compounds that can simultaneously modulate different targets involved in the neurodegenerative cascade has emerged as an interesting therapeutic approach. OBJECTIVE: This work investigated the ability of uleine, the major indole alkaloid purified from stem barks of the Brazilian medicinal plant Himatanthus lancifolius, to interact with crucial Alzheimer´s disease disruptive targets associated with two of its major neurodegenerative pathways: acetylcholinesterase and butyrylcholinesterase (cholinergic pathway) and ß-secretase and ß-amyloid peptide (amyloidogenic pathway). METHODS: Uleine's capacity to inhibit human acetylcholinesterase and butyrylcholinesterase enzymes was determined measuring the difference between reaction rates with and without uleine monitored at 412 nm using 5,5'- dithiobis-(2- nitrobenzoic acid) as colorimetric agent. FRET based assay was used to evaluate ß-secretase inhibition using DABCYL- Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-EDANS as substrate and ß-amyloid peptide spontaneous aggregation assay was performed using the thioflavin T spectroscopy assay. Cell viability and toxicity experiments with PC12 and SH-SY5Y cell lines were performed using the MTT colorimetric assay. RESULTS: Uleine demonstrated strong inhibitory activities for both cholinesterases (IC50 279.0±4.5 and 24.0±1.5 µM, respectively) and ß-secretase (IC50 180±22 nM). Above all, uleine significantly inhibited the self-aggregation of amyloid- ß peptide and was not toxic for PC12 or SH-SY5Y neuronal cells. CONCLUSION: These data show for the first time that the natural compound uleine has a novel, multieffective ability to decelerate or even inhibit the development of Alzheimer´s disease.

Qualidade físico-química e atividades biológicas de caseinofosfopeptídeos obtidos por hidrólise tríptica de caseinato de sódio sob diferentes condições / Physico-chemical quality and biological activities of Casein phosphopeptides obtained by sodium caseinate tryptic hydrolysis under different conditions

Muitos métodos têm sido empregados na produção de peptídeos bioativos para a promoção da saúde. O objetivo deste estudo foi produzir caseinofosfopeptídeos por hidrólise tríptica do caseinato de sódio usando diferentes temperaturas (37 e 50 °C) e tempos de reação (2 e 4 h), caracterizá-los e analisar sua influência nas atividades citotóxica, antimicrobiana e antioxidante. Caseinofosfopeptídeos foram caracterizados através da composição centesimal, eletroforese em gel de poliacrilamida, Espectrometria de Massas e Cromatografia Líquida de Alta Eficiência. Toxicidade para leucócitos humanos, atividade antimicrobiana utilizando o teste de microdiluição em caldo e determinação da capacidade antioxidante pelo método de espécies reativas ao ácido tiobarbitúrico foram os ensaios biológicos realizados. Os resultados mostraram que as quatro frações peptídicas obtidas apresentaram-se com baixo peso molecular e elevados teores proteico e mineral; quanto ao perfil aminoacídico, apresentaram elevadas e diferenciadas quantidades de ácido glutâmico e serina, que pouco variaram de acordo com o processo de obtenção; não se mostraram tóxicos para leucócitos humanos; demonstraram atividade antimicrobiana para Escherichia coli e Salmonella Enteritidis e elevada capacidade antioxidante. Os resultados físico-químicos das frações de caseinofosfopeptídeos demonstraram elevada composição nutricional em termos de proteína e, principalmente, cálcio. O conjunto de dados indicou que alterações no tempo e na temperatura de reação para a obtenção dos hidrolisados não interferem nas suas qualidades biológicas, mostrando serem seguros para a promoção da saúde e para a aplicação em situações especiais, que envolvem pacientes desnutridos, imunossuprimidos, com comprometimento ósseo ou gastrintestinal decorrentes de inflamações e infecções.

Several methods have been employed for the production of bioactive peptides for health promotion. The aim of this study was to produce and characterize Casein phosphopeptides obtained by sodium caseinate tryptic hydrolysis under different temperatures (37 and 50 °C) and reaction times (2 and 4 h), and evaluate their biological capabilities. They have been characterized by assessing their centesimal composition, by polyacrylamide gel electrophoresis, Mass Spectrometry, and High-Performance Liquid Chromatography. The biological activities tested included toxicity for human leukocytes, antimicrobial assay using the microdilution test, and determination of the antioxidant capacity by the thiobarbituric acid reactive species method. The results showed that the four fractions obtained were of low molecular weight with high protein and mineral contents; their amino acid profile showed high and differentiated amounts of glutamic acid and serine independent of the methodological procedures. The results also showed no toxicity for human peripheral leukocytes, demonstrated antimicrobial activity against Escherichia coli and Salmonella Enteritidis as well as high antioxidant capacity. The results of the physicochemical Casein phosphopeptides' fractions showed high nutritional composition in terms of protein and, particularly, calcium. The biological assays indicated that time and temperature changes in the process for obtaining casein hydrolysates have not interfered with their biological qualities. In addition, they have proven safe in promoting health in special conditions involving malnourished and/or, immunocompromised patients or those with bone and/or gastrointestinal impairment due to inflammations and infections.

Cytotoxicity and apoptogenic effects of Lafoensia pacari.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem barks of Lafoensia pacari have been traditionally used not only by South Amerindians but also by Brazilian and Paraguayan populations for treating a variety of unhealthy conditions to which their biological potential has been scientifically documented in several reports over the last decade. Although its anticancer usage is also popular, no scientific support for such activity has been found. AIM: To provide scientific evidence for the anticancer popularity of Lafoensia pacari. MATERIALS AND METHODS: Extracts prepared according to the popular use along with a methanol extract and its four fractions were produced from Lafoensia pacari stem barks. The chromatogram profile of each one was obtained by HPLC. Several tumor cell lines were exposed to these solutions in in vitro assays and the effects evaluated by morphological, growth, and cell cycle status changes. RESULTS: High toxicity determined by the lactate dehydrogenase levels with a significant drop in the cell proliferation index were found for all cell lines included in this study after exposition to Lafoensia pacari extract and fractions. The morphological features along with the expression of annexin V have strongly suggested apoptosis induction, which has been confirmed by G0/G1 cell cycle arrest. CONCLUSIONS: The data have clearly shown that exposition of human tumor cell lines to Lafoensia pacari stem barks extract leads to apoptosis induction due to cell cycle arrest in G0/G1 phases, supporting its anticancer use.

Malva sylvestris L. extract suppresses desferrioxamine-induced PGE2 and PGD2 release in differentiated U937 cells: the development and validation of an LC-MS/MS method for prostaglandin quantification.

Malva sylvestris is a species used worldwide as an alternative to anti-inflammatory therapies; however, its mechanism of action remains unknown. In this paper, the anti-inflammatory effects of M. sylvestris alcoholic extracts were evaluated by measuring the pro-inflammatory mediators PGE2 and PGD2 in desferrioxamine-stimulated phorbol 12-myristate 13-acetate-differentiated U937 cells. An HPLC-DAD fingerprint of the M. sylvestris extract was performed and caffeic acid, ferulic acid and scopoletin were identified and quantified. An HPLC-MS/MS method was developed and validated to separate and measure the prostaglandins. The lower limits of detection (~0.5 ng/mL for PGE2 and PGD2) and quantification (1.0 ng/mL for PGE2 and PGD2) indicated that the method is highly sensitive. The calibration curves showed excellent coefficients of correlation (r > 0.99) over the range of 1.0-500.0 ng/mL, and at different levels, the accuracy ranged from 96.4 to 106.4% with an RSD < 10.0% for the precision study. This method was successfully applied using U937-d cells. A significant dose-dependent reduction of PGE2 and PGD2 levels occurred using 10 µg/mL (10.74 ± 2.86 and 9.60 ± 6.89%) and 50 µg/mL of extract (48.37 ± 3.24 and 53.06 ± 6.15%), suggesting that the anti-inflammatory mechanisms evoked by M. sylvestris may be related to modulation of these mediators.

Alkaloid-rich fraction of Himatanthus lancifolius contains anti-tumor agents against leukemic cells

The effects of the alkaloid-rich fraction of Himatanthus lancifolius (Müll. Arg) Woodson on normal marrow cells and leukemic cell lines were investigated. After 48 h exposure, the proliferation assay showed significant cell growth inhibition for Daudi (0.1-10 µg/mL), K-562 (1-10 µg/mL), and REH cells (10-100 µg/mL), yet was inert for normal marrow cells. A similar inhibition profile was observed in clonogenic assays. This alkaloid-rich fraction, in which uleine is the main compound, showed no signs of toxicity to any cells up to 10 µg/mL. Cell feature analyses after induction of differentiation showed maintenance of the initial phenotype. Flow cytometric expression of Annexin-V and 7-AAD in K-562 and Daudi cells has indicated that the cells were not undergoing apoptosis or necrosis, suggesting cytostatic activity for tumor cells.

Os efeitos da fração rica em alcalóides indólicos de Himatanthus lancifolius (Müll. Arg) Woodson sobre células normais de medula óssea e linhagens celulares leucêmicas foram investigados. Após 48 horas de exposição, os ensaios de proliferação demonstraram efeitos inibitórios significativos para as linhagens Daudi (0,1-10 µg/mL), K-562 (1-10 µg/mL) e REH (10-100 µg/mL), enquanto mostrou-se inerte sobre células normais de medula óssea. Os perfis de inibição se repetiram nos ensaios clonogênicos. A fração rica em alcalóides, na qual a uleína é a substância majoritária, não demonstrou toxicidade até a dose de 10 µg/mL para nenhuma das células incluídas no estudo. Da mesma forma, não se observou influência dessa fração sobre a diferenciação celular dessas linhagens, mas manutenção de seu estado maturacional inicial. O conjunto de dados descritos associado à baixa co-expressão de anexina-V e 7-AAD sugerem que esta fração exerce atividade citostática para células tumorais.

The uleine-rich fraction of Himatanthus lancifolius blocks proliferative responses of human lymphoid cells.

The purpose of the present work is to report the effects shown by the uleine-rich fraction extracted from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson (Apocynaceae) on human peripheral blood mononuclear cells in which we were able to inhibit the proliferation of phytohemagglutinin-stimulated lymphocytes by blocking their transformation into blast-dividing cells. Furthermore, it inhibited the proliferation of Daudi and Reh cells, two leukemic cell lines of lymphoid origin. The present study widens the biological applications of H. lancifolius' alkaloids to include its possible use as a modulator of the immune system.

Effects of Himatanthus lancifolius on human leukocyte chemotaxis and their adhesion to integrins.

The aim of this work was to investigate the anti-inflammatory activities of the uleine-rich fraction extracted from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson (Apocynaceae). To achieve this, we focused on its in vitro effects on some steps of the inflammatory response using peripheral human leukocytes. The results presented herein show that the uleine-rich fraction significantly inhibits the migration of casein-induced granulocytes and their adhesion to fibronectin and vitronectin, along with mononuclear cells, by down-regulating the expression of alpha 4beta1 and alpha5beta1 integrins. The data suggest that H. LANCIFOLIUS has the potential of interferring with leukocyte trafficking through its uleine-rich fraction, emphasizing its usefulness in inflammatory conditions. DEXA:dexamethasone disodium phosphate FN:fibronectin PMN:polymorphonuclear URF:uleine-rich fraction VN:vitronectin.

Comparison of chemical constituents of Chamomilla recutita (L) rauschert essential oil and its anti-chemotactic activity

Several essential oil samples from dried flower-heads of Chamomilla recutita (L.) Rauschert, grown and commercially available around Curitiba metropolitan area, South of Brazil, were analyzed by GC-MS and the chemical constituents were compared with an Egyptian sample obtained under similar conditions and used as a control. The local grown herbs showed levels of essential oil below the standards recommended by the Brazilian Pharmacopoeia. Also, differences in their composition as well as in the quantity of several components were found such as the unexpected inversion of the relative constitution of the A and B alpha-bisabolol oxides. Of particular interest was the striking effect of the chamomile extracts upon human leukocyte chemotaxis, a biological anti-inflammatory activity not reported before, in which cell migration was in vitro inhibited at the same level as showed by dexamethasone.

Oleos essenciais extraídos de amostras de Chamomilla recutita (l.) Rauschert cultivadas e comercializadas na região metropolitana de Curitiba, sul do Brasil, foram analisadas por cromatografia com fase gasosa acoplada à espectrometria de massa e seus constituintes químicos comparados com uma amostra egípcia, usada como controle. A quantidade de óleo essencial obtida das amostras locais foi abaixo do padrão recomendado pela Farmacopéia Brasileira IV. Observou-se, também, diferenças na composição química desses óleos, assim como na quantidade relativa dos constituintes, como os óxidos A e B do alfa-bisabolol. De particular interesse foram os efeitos inibidores dos extratos de camomila sobre a quimiotaxia de leucócitos humanos induzida por caseína, efeito biológico descrito pela primeira vez e semelhante ao promovido pela dexametasona.

Cultura de células primordiais de medula óssea tipo Dexter: um procedimento laboratorial que mimetiza a hematopoese / Dexter long-term bone marrow culture: a laboratorial procedure that mimics hemopoiesis

Várias técnicas de cultivo têm sido utilizadas na investigação de fatores que influenciam a biologia das células primordiais hemotopoéticas, tornando-se fundamentais na elucidação dos mecanismos que envolvem a hematopoiese. Este trabalho descreve o sistema de cultura idealizado por Dexter e colaboradores, no qual proliferação e diferenciação de células primitivas de medula óssea normal ocorrem quando cultivadas em meio sintético enriquecido com soros de animais e antibióticos. Células do sobrenadante removidas durante a manutença das culturas foram quantificadas e ensaiadas para estimativa de seu conteúdo em progenitores hemotopoéticos. Simultaneamente, desenvolveu-se uma camada de células aderentes, que foram também contadas e ensaiadas para estimativa de seu conteúdo em progenitores. A fração celular do sobrenadante decaiu de 1,65 x 10(elevado a potência 7) para uma média de 1,59 +- 0,42 x 10(elevado a potência 5)células mononucleares/frasco de cultura após a quinta semana de cultivocom um plantôestabelecendo-se entre esta e a décima semanas. Colônias de GM-CFC de ambas frações foram detectadas em todas as semanas ensaiadas e mostraram-se em maior número na camada aderente. Os resultados demonstram que hematopoiese pode ser mantida nas condições experimentais testadas devido ao desenvolvimento de um estroma, o qual demonstrou capacidade de sustentar uma população de células primordiais hematopoéticas em contínuas proliferação e diferenciação por 10 semanas.

Long-Term Bone Marrow Culture: a laboratorial procedure that mimics hemopoiesis

Várias técnicas de cultivo celular têm sido utilizadas na investigaçäo de fatores que influenciam a proliferaçäo e a diferenciaçäo das células tronco hemopoéticas, tornando-se fundamentais na elucidaçäo dos mecanismos que envolvem a produçäo de células sanguíneas.Este trabalho descreve um sistema de cultura onde ocorre proliferaçäo e diferenciaçäo de células hemopoiéticas primitivas de medula óssea normal. Usou-se células de medula óssea de 18 doadores que foram cultivadas, por 10 semanas, em meio sintético com antibióticos, enriquecido com soros fetal bovino e de cavalo. Após serem as culturas alimentadas semanalmente, as células do sobrenadante foram quantificadas e ensaiadas para estimativa de seu conteúdo em progenitores hemopoéticos através de ensaios clonogênicos de Unidades Formadoras de Colônias (CFU). Simulteamente, desenvolveu-se, na base do frasco de cultura, uma camada estromal contendo uma populaçäo de células aderentes, que também foram contadas e ensaiadas para estimativa de seu conteúdo em progenitores hemopoéticos. A fraçäo celular do sobrenadante decaiu de 1,65 x 107 para uma média de 1,25 x 105 células mononucleares/frasco de cultura após a sétima semana de cultivo. Um plateau pôde ser observado entre a quinta semana e o final do experimento. Colônias de GM-CFC de ambas fraçôes foram detectadas em todas as semanas ensaiadas e mostraram-se em maior número na camada aderente. Hemopoese, em especial granulomono-poese, pôde ser mantida in vitro nas condiçöes experimentais testadas e a camada estromal demonstrou capacidade de sustentar uma populaçäo de células tronco em proliferaçäo e diferenciaçäo por pelo menos 10 semanas.