Recebido em 21/6/10; aceito em 17/8/10; publicado na web em 20/10/10 METHODS FOR DETERMINATION OF IN VITRO ANTIOXIDANT ACTIVITY FOR EXTRACTS AND ORGANIC COMPOUNDS. In the literature there are a considerable number of chemical and biochemical tests for evaluation of in vitro antioxidant activities of pure compounds or fractions and organic extracts. These tests are important tools for screening of synthetic and natural bioactive compound as well as they can be employed in food chemistry. This work is a critical review of the main methods employed for in vitro antioxidant determination.Keywords: antioxidant activity; oxygen reactive species; radicalar quenching. INTRODUÇÃOO termo oxidação de uma substância era comumente definido como a incorporação de oxigênio em sua estrutura. Atualmente, pode ser mais precisamente definido como sendo a conversão de uma substância química em um derivado com menor número de elétrons. Oxidação, portanto, é a perda de um ou mais elétrons para outra substância e o procedimento inverso pode ser considerado como redução. 1 A transferência de elétrons é um dos processos químicos mais fundamentais para a sobrevivência das células. O efeito colateral dessa dependência é a produção de radicais livres e outras espécies reativas de oxigênio (ERO) que podem causar dano oxidativo. Radicais livres são átomos ou moléculas produzidos continuamente durante os processos metabólicos e atuam como mediadores para a transferência de elétrons em várias reações bioquímicas, desempenhando funções relevantes ao metabolismo.2 As principais fontes de radicais livres são as organelas citoplasmáticas que metabolizam oxigênio, nitrogênio e cloro, gerando grande quantidade de metabólitos. Os radicais livres possuem diferentes papéis no organismo e encontram-se envolvidos na produção de energia, fagocitose, regulação do crescimento celular, sinalização intercelular e síntese de substâncias biológicas importantes. Entretanto, seu excesso apresenta efeitos deletérios, tais como danos ao DNA, proteínas e organelas celulares, como mitocôndrias e membranas, provocando alterações na estrutura e funções celulares e, dessa forma, se encontram envolvidos em diversas patologias a exemplo de câncer, envelhecimento precoce, doenças cardiovasculares, degenerativas e neurológicas, choque hemorrágico, catarata, disfunções cognitivas, etc.4 Para combater os radicais livres os organismos vivos produzem substâncias que são capazes de regenerar ou prevenir os danos oxidativos, exercendo seu papel como antioxidante. Além destes, substâncias com habilidade de sequestrar radicais livres podem ser obtidas de fontes externas, como alimentos e bebidas. Quando os antioxidantes produzidos pelo corpo são insuficientes para combater os radicais livres produzidos pelo organismo, este sofre ações degenerativas através do distúrbio conhecido como estresse oxidativo.Os estudos sobre radicais livres e o desenvolvimento de novos métodos para avaliação de atividade antioxidante (AA) têm aumentado consideravelmente nos últimos anos. As descobertas d...

Flavonoids have key functions in the regulation of multiple cellular processes; however, their effects have been poorly examined in pluripotent stem cells. Here, we tested the hypothesis that neurogenesis induced by all-trans retinoic acid (RA) is enhanced by agathisflavone (FAB, Caesalpinia pyramidalis Tull). Mouse embryonic stem (mES) cells and induced pluripotent stem (miPS) cells growing as embryoid bodies (EBs) for 4 days were treated with FAB (60 μM) and/or RA (2 μM) for additional 4 days. FAB did not interfere with the EB mitotic rate of mES cells, as evidenced by similar percentages of mitotic figures labeled by phospho-histone H3 in control (3.4% ± 0.4%) and FAB-treated groups (3.5% ± 1.1%). Nevertheless, the biflavonoid reduced cell death in both control and RA-treated EBs from mES cells by almost 2-fold compared with untreated EBs. FAB was unable, by itself, to induce neuronal differentiation in EBs after 4 days of treatment. On the other hand, FAB enhanced neuronal differentiation induced by RA in both EBs of mES and miPS. FAB increased the percentage of nestin-labeled cells by 2.7-fold (mES) and 2.4 (miPS) and β-tubulin III-positive cells by 2-fold (mES) and 2.7 (miPS) in comparison to RA-treated EBs only. FAB increased the expression of RA receptors α and β in mES EBs, suggesting that the availability of RA receptors is limiting RA-induced neurogenesis in pluripotent stem cells. This is the first report to describe that naturally occurring biflavonoids regulate apoptosis and neuronal differentiation in pluripotent stem cells.

Two new glycosyl phenylpropenoid acids, 4-O-beta-glucopyranosyloxy-(Z)-7-hydroxycinnamic acid (1) and 4-O-beta-glucopyranosyloxy-(Z)-8-hydroxycinnamic acid (2), besides lupeol and aghatisflavone, were isolated from the leaves of Caesalpinia pyramidalis.

O reestudo do extrato clorofórmico das folhas de Caesalpinia pyramidalis (Caesalpinioidea, Fabaceae) forneceu, além do novo biflavonóide denominado caesalflavona, podocarpusflavona A, agathisflavona, apigenina, kaempferol, sitosterol e lupeol. Por outro lado, a partir do extrato clorofórmico do caule foram obtidos 4, 4'-diidroxi-2'-metoxi-chalcona, (-)-siringaresinol e galato de metila. Não foram encontrados biflavonóides nesta parte da planta. Até o presente, C. pyramidalis é a primeira espécie do gênero que contém biflavonóides. As estruturas das substâncias foram estabelecidas através da análise dos seus dados espectrométricos utilizandose técnicas de EM, IV, UV, RMN 1D e 2D.The chloroform extract of the leaves of Caesalpinia pyramidalis (Caesalpinioidea, Fabaceae) yielded the new biflavonoid named caesalflavone, as well as podocarpusflavone A, agathisflavone, apigenin and kaempferol. The chloroform extract of the trunk wood gave 4,4'-dihydroxy-2'-methoxychalcone, (-)-syringaresinol, and methyl gallate. Biflavonoids were not found in trunk wood. Until now, C. pyramidalis is the first species in the genus to present biflavonoids. The structural elucidation of the isolated compounds and their derivatives were based on MS, IR, UV, 1D and 2D NMR spectral analyses.

Recebido em 17/9/09; aceito em 2/2/10; publicado na web em 18/6/10The chloroform partition of methanol extract of leaves of Caesalpinia pyramidalis was submitted to different chromatographic procedures which afforded besides agathisflavone and taxifolin, the minor biflavones loniflavone, amentoflavone, 5'-hydroxyamentoflavone and podocarpusflavone A. The structures of the compounds were established on the basis of NMR and MS data analysis. Besides, the content of biflavones of different specimens of C. pyramidalis, which are collected in different habitats of the Brazilian semi-arid region, was determinated by LC-APCI-MS analysis. These analysis demonstrated that only the specimens harvested in Bahia state showed collectively the presence of agathisflavone, amentoflavone, sequoiaflavone and podocarpusflavone A.Keywords: Caesalpinia pyramidalis; biflavones; flavonoid analysis. INTRODUCTIONCaesalpinia pyramidalis Tul. is a tree belonging to the family Leguminosae-Caesalpinoideae, which is endemic of Brazilian northeastern, especially in the caatinga. It is popularly known as "catingueira" or "pau-de-rato" and its leaves are used in the preparation of infusions and decocts, which are used by the local population as diuretic, antidispeptic, stomach aches and for fever. 1 Species of this genus are known to present different biological activities. For instance, the extract of seeds of C. bonducella showed antimicrobial activities 2 and they are also employed in the treatment of diabetes. 3 Extracts of C. volkensii and C. pluviosa presents antimalarial activity 4 and C. pulcherrima antiviral activity. 5 The extract of C. pyramidalis is responsible for antibacterial, 6 larvicidal and moluscicidal 7 activities.The presence of diterpenes and flavonoids besides other phenolics is characteristic of this genus as well as the subfamily. 8 From the previous studies regarding with C. pyramidalis were isolated phenylpropanoids, biflavonoids, lignan, flavonoids besides gallic acid. 9 In the present study it was reported the isolation and characterization of the minor biflavonoids loniflavone (1), amentoflavone (2), 5¢-hydroxyamentoflavone (3) and podocarpusflavone A (5), besides agathisflavone (4) and taxifolin (6) from the leaves of C. pyramidalis (Figure 1). It was also evaluated by LC-MS the composition of biflavonoids present in leaves of specimens of C. pyramidalis collected in different habitats of Brazilian semi-arid region. EXPERIMENTAL General proceduresThe NMR spectra were obtained in Varian Gemini 2000, Inova 500 and Bruker AMX500 spectrometers operating at 300 and 500 MHz ( 1 H) and, 75 and 125 MHz ( 13 C), employing CD 3 OD, C 5 D 5 N and (CD 3 ) 2 CO as solvents and TMS as internal standard. The MS and the LC-MS analysis were carried out in a Shimadzu chromatographer mod. Bahia et al. 1298 Quim. Nova LCMS-2310, with autosampler 5 mL loop. The detection of biflavonoids was obtained in positive and negative APCI mode. The chromatograms were obtained using a VP-ODS (RP18 -5 mm; 3.9 x 150 mm) column and as mobile pha...