Atividade antioxidante, cito- e fototoxicidade do extrato das sementes de romã (Punica granatum L.)

In the present work, a hydroethanolic extract was prepared from the entire seeds of pomegranate [Punica granatum L. (Punicaceae)] with Cachaça, a distilled Brazilian alcoholic beverage, protected from light for an 80-hour period. The desorption curve of the seeds, presented an optimal time extraction of approximately 24 hours. The extract was divided into two samples: protected from light, (Extract 1), or not, (Extract 2). The extracts were characterized by UV-Visible absorption spectroscopy, quantification of total phenolics by the Folin-Ciocalteu method, and the antioxidant activity was determined by the DPPH quenching method. Extract 2 presented 9.8% less total polyphenols than Extract 1. The pomegranate seeds extract lost 79% of its antioxidant activity during light exposure. Extract 1 up to 3% (w/v) showed neither cyto nor phototoxicity in the Hela cells. In conclusion, Punica granatum L. seeds contain a significant total polyphenol and TEAC amount and they can be used in simple extractive process, by direct contact with Cachaça in up to 80 hours in the darkness, which gives it good coloration, taste, and smell. This extract showed neither cytotoxicity nor post-irradiation phototoxicity with solar simulator even though the extract proved photoinstable.Neste trabalho, foi realizada a extração de sementes inteiras de romã (Punica granatum L. - Punicaceae) com cachaça durante 80 horas no escuro. A curva de dessorção do extrato apresentou um tempo ótimo de extração das sementes de aproximadamente 24 horas. O extrato obtido foi dividido em duas amostras, uma delas foi mantida protegida da luz ambiente (extrato 1) e a outra não (extrato 2). As amostras foram caracterizadas por espectroscopia Ultravioleta-Visível, quantificação de polifenóis totais pelo método de Folin-Ciocalteu e pela atividade antioxidante utilizando o ensaio de inibição do radical DPPH. A exposição do extrato à luz ambiente, o fez perder 9,8% do seu teor de polifenóis totais (de 0,353 para 0,322 g.L-1). Por outro lado, a atividade antioxidante caiu a 20,7% (de 3,4 para 0,7 mM de trolox). A adição de até 3% do extrato 1 de sementes de romã não afetou significativamente a viabilidade de células epiteliais in vitro. As mesmas células tratadas com o extrato 1 e irradiadas com um simulador solar não apresentaram fototoxicidade na mesma faixa de concentração. Assim, foi possível concluir que os componentes das sementes de Punica granatum L. (romã) podem ser extraídos por contato direto com cachaça por um período de 80 horas no escuro, conferindo-lhe uma boa coloração e também propriedades organolépticas agradáveis. Tal extrato não apresenta citotoxicidade e nem fototoxicidade, embora o extrato tenha se mostrado fotoinstável.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

The Methylene Blue Self-aggregation in Water/Organic Solvent Mixtures: Relationship Between Solvatochromic Properties and Singlet Oxygen Production

In this work is presented a spectrophotometric investigation focused on the solvatochromic effects upon Methylene Blue (MB). Measurements were carried out in four different water/organic solvent mixtures: low polarity protic solvent (ethanol), polar non-protic solvent (acetonitrile), highly polar protic solvent (glycerol), and non-polar solvent (dioxane). The results showed that the photophysical behavior of MB is highly affected by self-aggregates formation at 80% of water/organic solvent blends. Besides polarity, the protic character and the coordinating properties of the solvent molecules are the key parameters for its photophysical behavior, since the sulfur atom of the cationic structure can act as a coordination center due to its Lewis acid character. In fact, water and acetonitrile coordinating properties have proved to be important to determine MB interactions intensity and its spectroscopic properties as singlet oxygen emission. It was observed that an increase of the amount of the organic solvent resulted in an enhancement of the singlet oxygen emission intensity. The presence of the water increases the dielectric constant of the medium and favors the self-aggregation process. Besides, the water molecules can act as a quencher and it decreases the quantum yield of the fluorescence of MB.FAPESPCNPqFarma Service BioExtract LtdaUniv Fed Sao Joao del Rei, Dept Zootecnia DEZOO, Campus Dom Bosco, BR-36301160 Sao Joao Del Rei, MG, BrazilUniv Vale Paraiba, Ave Shishima Hifumi 2911, BR-12244000 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Quim, Rua Prof Arthur Riedel 275, BR-09972270 Sao Paulo, BrazilUniv Sao Paulo, Dept Bioquim, Inst Quim, Ave Prof Lineu Prestes 748, BR-05508900 Sao Paulo, SP, BrazilUniv Tecnol Fed Parana, Campus Apucarana,Rua Marcilio Dias 635, BR-86812460 Apucarana, Parana, BrazilUniv Fed Santa Catarina, BR-88040900 Florianopolis, SC, BrazilUniv Estadual Maringa, Dept Quim, Av Colombo 5790,Zona 07, BR-87020900 Maringa, Parana, BrazilUniv Fed Rio de Janeiro, Campus Macae,Rua Aloisio da Silva Gomes 50, BR-27930560 Rio De Janeiro, BrazilUniv Estadual Campinas, Inst Quim, BR-13083970 Sao Paulo, BrazilUniv Fed Rio Grande, Escola Quim & Alimentos, Campus Carreiros Pavilhao Quim, BR-96201900 Rio Grande, RS, BrazilUniv Fed ABC, Ctr Engn Modelagem & Ciencias Sociais Aplicadas, Ave Estados 5001, BR-09210580 Sao Paulo, BrazilUniv Fed ABC, Ctr Ciencias Nat & Humanas, Ave Estados 5001, BR-09210580 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Quim, Rua Prof Arthur Riedel 275, BR-09972270 Sao Paulo, BrazilFAPESP: JP 04/02193-1FAPESP: 02/00272-6FAPESP: 06/56701-3FAPESP: 2005/51597-8CNPq: 474019/2012-8]CNPqFarma Service BioExtract LtdaWeb of Scienc

Metallochlorophylls of magnesium, copper and zinc: evaluation of the influence of the first coordination sphere on their solvatochromism and aggregation properties

In this study the role of different metal centers (magnesium, zinc and copper) on the enhancement of the hydrophilic character of metallochlorophylls, was evaluated. The solvatochromism as well as the aggregation process for these compounds in water/ethanol mixtures at different volume ratios were evaluated using Fluorescence, and Resonant Light Scattering (RLS) measurements, aiming to characterize the behavior of these compounds. Independently on the studied metallochlorophyll, the presence of at least 60% of water results in a considerable increase in the fluorescence emission, probably a direct consequence of a lower aggregation of these compounds, which is confirmed by the results from RLS measurements. Additionally, the results suggest that magnesium and zinc chlorophyll should be promising phototherapeutic agents for Photodynamic Therapy.No presente estudo, o papel de diferentes centros metálicos (magnésio, zinco e cobre) sobre o caráter hidrofílico de metaloclorofilas foi avaliado. O solvatocromismo, assim como o processo de agregação para esses compostos, em misturas água/etanol foram avaliados usando medidas de fluorescência e de espalhamento de luz ressonante (ELR), visando caracterizar o comportamento desses compostos. Independente da metaloclorofila estudada, a presença de ao menos 60% de água resulta em incremento considerável na emissão de fluorescência apresentada por esses compostos. Isso, provavelmente, é uma conseqüência direta de uma menor agregação, o que é confirmado pelos resultados de ELR. Adicionalmente, os resultados sugerem que as clorofilas de magnésio e de zinco devem ser promissores agentes fototerapêuticos para emprego em Terapia Fotodinâmica.FAPESPFundação AraucáriaFAPEMIGCNPqNanobra

A Comparative Study of Head-Mounted and Table-Mounted Augmented Vision Systems for Assembly Error Detection

It is proposed a simple and inexpensive strategy to determine singlet oxygen (¹O2) quantum yields (ΦΔ) of photosensitizers (PS) in water using beetroot extract containing betacyanin (Bc) and a set of light emitting diodes (LEDs) for excitation. Bc, a cationic natural dye, was obtained by flash chromatography purification from the red beet extract (Beta vulgaris) and employed as a convenient probe for ¹O2 detection. Solutions of Bc and PS were illuminated with an array of LEDs adapted in the cuvette compartment of a commercial spectrophotometer, and the decrease in Bc absorbance was followed as a function of time. Bc photobleaching decreased in de-aerated solution and increased in D2O, indicating the involvement of ¹O2. The observed photobleaching rate constant (k obs) was proportional to the LED intensity, concentration and ΦΔ of the PS. By keeping the light source constant we could estimate the overlap integral (R) between the LED emission and PS absorbance for different PS concentrations. The slope of R versus k obs is the value of the photobleaching rate constant (k), which was shown to be proportional to ΦΔ. Values of ΦΔ obtained by this method were compared with those obtained by measuring NIR (near infrared) emission for a series phenothiazine dyes.É proposta uma estratégia simples e barata para determinar rendimentos quânticos (ΦΔ) de oxigênio singlete (¹O2) de fotossensibilizadores (PS) em água utilizando extrato de beterraba contendo betacianina (Bc) e um conjunto de diodos emissores de luz (LEDs) para excitação. Bc, um corante catiônico natural, foi obtida por purificação através de cromatografia a partir do extrato de beterraba vermelha (Beta vulgaris), e utilizada como uma sonda para detecção de ¹O2. Soluções do Bc e PS foram iluminadas com um arranjo de LEDs adaptado no compartimento de um espectrofotômetro comercial e a diminuição da absorvância de Bc foi seguida em função do tempo. O fotobranqueamento de Bc diminuiu em solução purgada com nitrogênio e aumentou em D2O, indicando o envolvimento de ¹O2. A constante de velocidade de fotobranqueamento observada (k obs) foi proporcional à intensidade do LED, à concentração e ao ΦΔ do PS. Mantendo a fonte de luz constante pudemos estimar a integral de sobreposição (R) entre a absorção do PS e a emissão do LED para diferentes concentrações de PS. A inclinação da curva de R em função de k obs é o valor da constante de velocidade de fotobranqueamento (k), que foi mostrada ser proporcional a ΦΔ. Valores de ΦΔ obtidos por este método foram comparados com aqueles obtidos através da medição da emissão no NIR (infravermelho próximo) para uma série de corantes fenotiazínicos.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Rede de Nanotecnologia Molecular e InterfacesInstituto do Milênio de Materiais Complexo

Generation and suppression of singlet oxygen in hair by photosensitization of melanin

We have studied the spectroscopic properties of hair (white, blond, red, brown, and black) under illumination with visible light, giving special emphasis to the photoinduced generation of singlet oxygen ((1)O(2)). Irradiation of hair shafts (lambda(ex)>400 nm) changed their properties by degrading the melanin. Formation of C3 hydroperoxides in the melanin indol groups was proven by (1)H NMR. After 532-nm excitation, all hair shafts presented the characteristic (1)O(2) emission (lambda(em) = 1270 nm), whose intensity varied inversely with the melanin content. (1)O(2) lifetime was also shown to vary with hair type, being five times shorter in black hair than in blond hair, indicating the role of melanin as a (1)O(2) suppressor. Lifetime ranged from tenths of a nanosecond to a few microseconds, which is much shorter than the lifetime expected for (1)O(2) in the solvents in which the hair shafts were suspended, indicating that (1)O(2) is generated and suppressed inside the hair structure. Both eumelanin and pheomelanin were shown to produce and to suppress (1)O(2), with similar efficiencies. The higher amount of (1)O(2) generated in blond hair and its longer lifetime is compatible with the stronger damage that light exposure causes in blond hair. We propose a model to explain the formation and suppression of (1)O(2) in hair by photosensitization of melanin with visible light and the deleterious effects that an excess of visible light may cause in hair and skin. 2011 Published by Elsevier Inc.FAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Farma Service BioextractFarma Service Bioextrac

Rapid screening of potential autophagic inductor agents using mammalian cell lines

Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability - Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) - to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.CAPES PNPD/FINEP 02533/09-