ABSTRACT Gliomas account for the majority of primary malignant brain tumors and present invasive behavior into adjacent healthy tissue. While 4-NC had previously shown to induce apoptotic cell death in a melanoma model, for the glioma model described in this paper 4-NC is cytotoxic for the cells with the induction of the autophagic pathway. Trypan blue exclusion assay showed that 4-NC was cytotoxic in a dose-dependent manner for A172 and T98G cell lines. IC10 and IC50 values were at 32 µM and 41 µM for A172 and T98G respectively. Inhibition of cell proliferation was observed by total cell counts and by cell cycle analysis by flow cytometry, with cell cycle arrest of A172 and T98G cell lines respectively in the G1/G0 and S phases of the cell cycle. 4-NC induced up-regulation of autophagic pathways, as shown by immunoblotting for LC3-I/II, Real-Time PCR for ATG-7 and Beclin-1 genes, and by fluorescence microscopy observation of autophagic vacuoles in cells transfected with GFP-LC3 and electron microscopy. Glioma cells concomitantly treated with 4-NC and 3-MA, an inhibitor of the autophagic process, are more sensible to cell death, suggesting that autophagy protects the cells from the action of 4-NC.

The Passifloraceae family is extensively used in native Brazilian folk medicine to treat a wide variety of diseases. The problem of flavonoid extraction from Passiflora was treated by application of design of experiments (DOE), as an experiment with mixture including one categorical process variable. The components of the binary mixture were: ethanol (component A) and water (component B); the categorical process variable: extraction method (factor C) was varied at two levels: (+1) maceration and (-1) percolation. ANOVA suggested a cubic model for P. edulis extraction and a quadratic model for P. alata.These results indicate that the proportion of components A and B in the mixture is the main factor involved in significantly increasing flavonoid extraction. In regard to the extraction methods, no important differences were observed, which indicates that these two traditional extraction methods could be effectively used to extract flavonoids from both medicinal plants. The evaluation of antioxidant activity of the extract by ORAC method showed that P. edulis displays twice as much antioxidant activity as P. alata. Considering that maceration is a simple, rapid and environmentally friendly extraction method, in this study, the optimized conditions for flavonoid extraction from these Passiflora species is maceration with 75% ethanol for P. edulis and 50% ethanol for P. alata.

A maioria das pesquisas realizadas até hoje sobre os efeitos benéficos da pariparoba (Pothomorphe umbellata L. Miq) foi feita com o extrato da raíz desta espécie, e o seu emprego em larga escala comprometeria a exploração sustentável deste insumo natural. Neste sentido o uso das folhas da pariparoba, ao invés de raízes pela indústria cosmética, não põe em risco a existência da espécie. Neste trabalho foi determinada a concentração de 4-nerolidilcatecol (4-NC) de um extrato de folhas por metodologia analítica validada em nosso laboratório, cujo valor foi cerca de 30% menor que no extrato de raiz, obtido da mesma maneira. Na avaliação de fotoestabilidade do extrato de folhas, uma solução de 0,25 mg/mL não apresentou alterações significativas do perfil espectroscópico após 2 horas de exposição à radiação UVB, demonstrando sua estabilidade. Metaloproteinases (MMPs) são endopeptidases dependentes de zinco, envolvidas na remodelagem da matriz extracelular (MEC), e importantes na formação das rugas típicas do fotoenvelhecimento cutâneo. Também avaliamos, por técnica de zimografia, a capacidade do extrato de folhas de P. umbellata de inibir a atividade de MMPs 2 e 9 in vitro de pele de camundongos sem pêlo. O extrato de folhas (0,1 mg/mL) inibiu em 80% a atividade destas enzimas, conforme avaliação densitométrica.

Most researches that have been done until today about the beneficial effects of pariparoba (Pothomorphe umbellata L. Miq) have been done with root extract of this species, but the use in large scale would compromise the sustainable exploration of this natural resource. In this sense, the utilization of pariparoba leaves, substituting the roots, in the cosmetic industry does not put in risk the existence of the species. In this work the concentration of 4-nerolidylcathecol (4-NC) in leaf extract was determined by the analytical methodology validated in our laboratory. The concentration of 4-NC in leaf extract was around 30% less than that of root extract, obtained in the same way. Concerning the study of the photostability of a leaves extract solution containing 4-NC did not demonstrate meaningful alterations in the spectrometry profile after 2 hours of exposure under UVB radiation, showing its stability under this conditions. Metalloproteinases (MMPs) are endopeptidases that are zinc-dependent, involved in remodeling extracellular matrix (ECM), that are important in the appearance of typical photoaging wrinkles. In this work the capacity of leaf extract of P. umbellata to inhibit MMP-2 and 9 activities of hairless mouse skin in vitro by zymography gel was also evaluated. The leaf extract (0,1 mg/mL) inhibit in 80% activity of this enzymes, according to the densitometric zymography evaluation.

Um novo método de cromatografia líquida de alta eficiência foi desenvolvido para a determinação simultânea de ácido clorogênico, ácido caféico e cafeína no extrato hidroalcoólico e aquoso de Ilex paraguariensis. As curvas de calibração mostraram boa regressão linear nas faixas de concentração 0,49-7,8 µg/mL para o ácido clorogênico, 0,25-3,9 µg/mL para ácido caféico e 0,244-7,8 µg/mL para cafeína. A redução do radical DPPH· foi usada para determinar a capacidade antioxidante dos extratos. Nosso método para a determinação simultânea de ácido caféico, ácido clorogênico e cafeína foi altamente sensível, possuindo limites de detecção e de quantificação menores do que em outros trabalhos que empregaram metodologias semelhantes.

A new high performance liquid chromatographic method has been established for simultaneous determination of chlorogenic acid, caffeic acid and caffeine in hydroalcoholic and aqueous extracts of Ilex paraguariensis. Analytical curves showed good linear regression in the concentration ranges 0.49-7.8 µg/mL for chlorogenic acid, 0.25-3.9 µg/mL for caffeic acid and 0.244-7.8 µg/mL for caffeine. Reduction of the DPPH· radical was used to determine the antioxidant capacity of the extracts. Our method for the simultaneous determination of chlorogenic acid, caffeic acid and caffeine was highly sensitive, having lower detection and quantitation limits than other papers that used similar methodology.

Foi avaliada a influência dos fatores (1) tempo: 10 e 40 minutos; (2) tamanho de partícula: 840 e 420 mm; (3) hidromódulo: 1:50 e 1:100 e (4) temperatura: 40 e 60 °C na extração do 4-nerolidilcatecol, (4-NC) mediante planejamento fatorial "2(4)" em que quatro variáveis foram estudadas em dois níveis (máximo e mínimo). O método de extração foi maceração e a quantificação do 4-nerolidilcatecol foi realizada por cromatografia liquida de alta eficiência (CLAE) com detetor eletroquímico. Os resultados da análise fatorial indicam que o fator principal que favorece a extração do princípio ativo é o tamanho de partícula [efeito (2): 12,2086]. Diminuindo o tamanho de partícula aumenta três vezes a quantidade de 4-nerolidilcatecol extraída, enquanto o tempo de maceração [efeito (1): -0,64198], hidromódulo [efeito (3): 1,069804] e temperatura [efeito (4): -0,64198] não influenciam de forma significativa a extração. As interações de dois fatores: (2:3) tamanho:hidromódulo: 1,181142; (2:1) tamanho:tempo: 0,9435065 e (2:4) tamanho:temperatura: 0,0817575 mostraram que apesar do fator tamanho(2) ter favorecido o processo, quando combinado com os demais fatores, não aumenta a eficiência da extração. A metodologia de otimização mediante análise fatorial aplicada ao processo de extração do 4-nerolidilcatecol demonstra a importância do estudo das interações e não de cada fator isolado.

The influence of factors: (1) time, 10 and 40 minutes, (2) particle size, 840 and 420 mm; (3) hydromodule, 1:50 and 1:100, and (4) temperature, 40 and 60 °C, in the extraction of 4-nerolidylcathecol (4-NC) from the roots of P. umbellata using factorial design "2(4)" was studied in two levels (maximal and minimal). The extraction method was maceration and the measurement of 4-NC was by HPLC with electrochemical detection. The results of the factorial analysis indicated that the main factor that increases the extraction of the active principle is particle size [effect (2): 12.2086]. The reduction of the particle size (mesh 60) increases threefold the amount of 4-nerolidylchatecol in the extract, while the time of maceration [effect (1): -0.64198], hydromodule [effect (3): 1.069804] and temperature [effect (4): -0.64198] practically do not influence the extraction. Interaction between two factors (2:3) size-hydromodule 1.181142, (2:1) size-time 0.9435065 and (2:4) size-temperature 0.0817575, showed that although the main factor size (2) increases the efficiency of the process, when one of the other three factors was taken together the amount of 4-NC extracted was not significantly increased. The technique of optimization using factorial analysis to investigate the extraction of 4-nerolylilchatecol showed to be useful to notice the interactions between factors and not only the effect of each isolated factor.

Existe uma grande preocupação quanto ao uso seguro de extratos vegetais e, por esta razão, a necessidade de estudos toxicológicos pré-clínicos e clínicos destes extratos. O objetivo deste trabalho foi o de avaliar a toxicidade aguda e subcrônica do extrato hidroalcoólico liofilizado de Pothomorphe umbellata L. Miq., administrado por via oral para animais de laboratório. O potencial mutagênico do extrato foi também avaliado pelo teste do micronúcleo. Os resultados dos estudos a curto e médio prazo demonstraram que o extrato não apresenta propriedades tóxicas.

There is a high degree of concern regarding the secure use of plant extracts and, for this very reason, preclinical and clinic toxicological evaluation of these extracts are needed. With the aim to assure the quality and the safety of the extract and due to the scarcity of literature information about Pariparoba extract toxicity, our purpose was to investigate the acute and subchronic toxicity of the standardized ethanolic dried root extract of Pothomorphe umbellata L. Miq. This extract was administered orally to adult swiss mice and wistar rats and the mutagenic potencial of the extract was also evaluated. The extract showed to be non toxic.

O 4-nerolidilcatecol representa o metabólito secundário mais abundante de Pothomorphe umbellata (Piperaceae), cujo uso terapêutico deve ser fundamentado após a caracterização farmacocinética do mesmo (4-nerolidicatecol). Os níveis séricos de animais tratados com 10 mg/kg do fármaco i.v. foram avaliados por validação analítica em CLAE-UV. As condições cromatográficas empregadas foram fase móvel de acetonitrila:metanol:água (62:20:18), coluna RP-18, fluxo de 1,6 mL/min e detector UV em 282 nm, com tempo total de corrida inferior a 15 minutos. Observou-se linearidade do 4-NRC em plasmas na faixa de 1,0 a 80,0 mcg/mL, com valores de precisão e exatidão entre 14,4-3,0% e 24,0-0,2%, respectivamente. O procedimento de extração líquido-líquido apresentou reprodutível e com recuperação quantitativa (106,5%±3,0%).

The oral dosages formulations from Pothomorphe umbellata (Piperaceae) can only be therapeutically evaluated after establishing 4-nerolidylcatechol (4-NRC) pharmacokinetic profile in plasma. For that purpose, a unique analytical method validation using HPLC-UV detection was developed for analysis of rat plasma samples. The animals received 10 mg/kg b.w. of 4-NRC i.v.. Analytical conditions were set with C18 column, methanol: acetonitrile: water (62:20:18) as mobile phase, and a flow rate of 1.6 mL/min. The assay was linear from 1.0 - 80.0 mcg/mL. The recovery procedure was performed by liquid-liquid extraction showing quantitative extraction (106.4±8.7%). Limit of Quantification (LOQ) was found to be 1.0 mcg/mL. Interassay precision and accuracy ranged from values of 14.4-3.0% and 24.0-0.2%, respectively. Recovery after liquid-liquid extraction was found to be 106.5 ± 3%. The method is simple and reliable with total run time of less than 15 min.

Este trabalho avaliou os níveis de ácido delta-aminolevulínico (ALA) em fígado de ratos expostos a diferentes doses de hexaclorobenzeno (HCB) (25, 50 e 100 mg/kg de peso corpóreo) durante 4 semanas e correlacionou com os parâmetros de peroxidação lipídica. Os níveis de ALA foram determinados por cromatografia líquida de alta eficiência após derivatização com acetilacetona e formaldeído, seguida de detecção de fluorescência. A metodologia foi cuidadosamente validada, apesar disso, os níveis hepáticos de ALA em todos os animais tratados ou não foram abaixo do limite de detecção do método (2,27 mg de ALA/ g de fígado). Por outro lado, para a peroxidação lipídica, avaliada como produção de reagentes ao ácido tiobarbitúrico e quimiluminescência, os resultados foram significativamente elevados em todos os animais tratados em comparação com os do grupo controle (p <0,05). Estes resultados sugerem que o estresse oxidativo hepático observado nos animais após tratamento com HCB não deve necessariamente estar associado com o aumento das concentrações de ALA no fígado. Uma outra possibilidade seria o aumento nos níveis de ALA no fígado, mesmo que abaixo do limite de detecção do método, sendo suficiente para induzir estresse oxidativo hepático.

This work evaluated the levels of 5-aminolevulinic acid (ALA) in the liver of rats exposed to different doses of HCB (25,50, and 100 mg/kg b.w. for 4 weeks) and correlated them with lipid peroxidation parameters. Levels of ALA were determined by high-pressure liquid chromatography after derivatization with acetylacetone and formaldehyde, followed by fluorescence detection. The methodology was carefully validated, nonetheless hepatic levels of ALA in all animals treated or not were below the detection limit of the method (2.27mg of ALA/g liver). On the other hand, lipid peroxidation, evaluated as thiobarbituric acid reactants production and chemiluminescence was found significantly increased in the livers of all treated rats, in comparison with control values (p<0.05). These results suggest that the hepatic oxidative stress observed in animals following HCB treatment may not necessarily be associated with increased levels of ALA in the liver. Another possibility is that increased levels of ALA in the liver, even below the detection limit of the method are sufficient to induce hepatic oxidative stress.

O objetivo do nosso trabalho foi desenvolver um método para avaliação da concentração do ±-tocoferol, considerado o antioxidante lipofílico de maior importância, e do 4-nerolidilcatecol (4-NC), uma substância natural com comprovada ação antioxidante in vitro e in vivo, em matriz biológica (homogeneizado de pele). Utilizamos a cromatografia de alta eficiência acoplada a um detector eletroquímico, sendo que o método apresentou linearidade para as concentrações de 0,025 µg/mL a 0,1 µg/mL para o ±-T (tempo de retenção 3, 4 min) e de 0,15 µg/mL a 2,5 µg/mL para o 4-NC (tempo de retenção 2,06 min), dissolvidos em etanol e etanol:água (1:1). A taxa de recuperação do ±-T adicionado nas concentrações de 0,5; 0,1 e 0,025 µg/mL aos homogeneizados de pele foi de 94,03; 111,2 e 80,7%, respectivamente. A taxa de recuperação de 4-NC adicionado nas concentrações de 2,5; 0,625 e 0,156 µg/mL foi de 103,7; 91,7 e 91,7%. Este método analítico foi e está sendo empregado, com sucesso devido à sua precisão e rapidez, em diversas análises do laboratório.

Topical administration of antioxidants, such as ±-tocopherol (±-T), provides an efficient manner of enriching the endogenous cutaneous protection system, and it constitutes a successful strategy for diminishing the ultraviolet radiation-mediated oxidative damage. Besides ±-tocopherol the use of other natural occurring compounds with antioxidant activity has been proposed for the same purpose. The aim of this study was to develop a validated analytical method for the determination of a-tocopherol and 4-nerolidylcathecol (4-NC) concentrations in skin homogenates in a pharmaceutical formulations. We employed liquid chromatography with electrochemical detection. Chromatography was performed on a Supelcosil LC-8, 3 mm, 75x4.6 mm column (Supelco, Bellefonte, PA, USA) with a mobile phase of methanol:water (9:1) for 4-NC and (95:5) for a-T, both containing 20 mM LiClO4 and 2 mM KCl. The flow rate was set at 1.0 ml/min. We established validation parameters including sensitivity, precision, accuracy, stability and found a linear relationship between the concentrations ranges of 0.025 µg/mL to 0.1 µg/mL of ±-T and 0.15 mg/mL to 2.5 mg/mL of 4-NC. The recovery of ±-T from skin homogenates was 94.03, 111.2 and 80.7% for the concentrations of 0.5, 0.1 and 0.025 µg/mL respectively. The recovery for the following concentrations of 4-NC: 2.5, 0.625 and 0.156 µg/mL was 103.7, 91.7 and 91.7%. This analytical procedure has been successfully employed in cutaneous permeation studies, antioxidant activity studies and determinations of 4-NC in Pothomorphe umbellata root extracts.