Phytochemical study of the roots of Euphorbia phosphorea Mart. (Euphorbiaceae) was carried out through chromatographic techniques, resulting in the isolation of a new ent-abietane diterpene named 11β,12β-dihydroxy-ent-abieta-8(14),13(15)-dien-16,12α-olide (1), and of nine known ent-abietane diterpenes jolkinolide A (2), jolkinolide E (3), euphorin H (4), euphopilolide (5) jolkinolide F (6), ent-12-hydroxy-12[R]-abieta-8(14),13(15)-dien-16,12-olide (7), ent-11α-hydroxyabieta-8(14),13(15)-dien-16,12α-olide (8), 17-hydroxyjolkinolide B (9) and caudicifolin (10). The structures of all compounds were established using spectroscopic techniques such as 1D and 2D NMR, and the structure of the compound 1 was established also with MS, IR and ECD. All compounds were submitted to an in silico study through of a predictive model and then submitted to in vitro tests against Mycobacterium tuberculosis and M. smegmatis for evaluation of their antimycobacterial activity. Compounds 5 and 9 showed mycobacterial growth inhibition with MIC values of 62.5 μM against M. tuberculosis and M. smegmatis, respectively.

Abstract In this study, the potential antileukemic activity of grandisin, a lignan extracted from Piper solmsianum, was evaluated against the leukemic line K562. The cytotoxicity of grandisin (0.018 to 2.365 µM) was evaluated in K562 and normal peripheral blood lymphocytes by Trypan Blue Exclusion and MTT methods after 48h exposure to the drug. In both methods, cellular viability was concentration-dependent and the IC50 values were lower than 0.85µM. Analysis of K562 cells after treatment with grandisin showed that the cell cycle was arrested in the G1 phase with a 12.31% increase, while both S and G2 phases decreased. Morphological studies conducted after the exposure of K562 to grandisin revealed changes consistent with the apoptosis process, which was confirmed by anexin V stain and caspase activation. Thus, lignan grandisin showed antileukemic activities against the K562 cell line and the cell death process occurred via apoptosis.

The most studied phyto constituent isolated from Virola surinamensis (Rol. ex Rottb.) Warb., Myristicaceae, is the tetrahydrofuran neolignan grandisin, which exhibits a series of biological activities, including trypanocidal, larvicidal and antitumoral. Due to its extremely low solubility, additional studies, including in vivo investigations are challenged by the difficulties in the development of an effective drug delivery system for grandisin. The encapsulation in polymeric nanoparticles is a very attractive alternative for overcoming some of these limitations. In this work, PLGA nanocapsules loaded with grandisin were developed in an attempt to optimize the efficacy of grandisin as an antitumoral drug, with high drug loading and efficiency, prolonged drug release and increased physical-chemical stability. Mean diameter of the nanocapsules was lower than 200 nm, with very low polydispersity. Encapsulation efficiency was above 90%. A sustained in vitro drug release was achieved for up to twenty days and cytotoxicity was markedly increased (IC50 for grandisin-NC and grandisin were 0.005 µM and 0.078 µM, respectively), indicating that polymeric nanocapsules are a potential drug delivery system for grandisin allowing the preparation of formulations viable for further in vivo studies.

Se evaluó la actividad larvicida del "matico" neotropical Piper tuberculatum. Los compuestos secundarios fueron extraídos de hojas, tallos y espigas maduras (con frutos y semillas) de plantas silvestres y de plantas in vitro de P. tuberculatum. Fue evaluada la toxicidad aguda de extractos de espigas maduras con frutos y semillas y de plantas in vitro de P. tuberculatum sobre el "cogollero" Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) mediante bioen-sayos de contacto. Solamente extractos Diclorometano:metanol (CH2Cl2:MeOH) (2:1) y etanólicos (EtOH) de espigas maduras y el extracto CH2Cl2:MeOH (2:1) de plantas in vitro mostraron niveles significativos de mortalidad. Los extractos CH2Cl2:MeOH (2:1) y EtOH de espigas maduras causaron 90% de mortalidad cuando dosis de 0,1850 mg/µL se aplicaron sobre S. frugiperda en 24 y 48 h de exposición, respectivamente. El extracto CH2Cl2:MeOH (2:1) de plantas in vitro causó 95% de mortalidad cuando dosis de 0,1850 mg/µL también se aplicaron en 48 h de exposición. Los mejores resultados para las espigas maduras fueron: CL50 0,001 mg/µL con EtOH y 0,007 mg/µL con CH2Cl2:MeOH (2:1) y CL90 0,027 mg/µL con EtOH y 0,103 mg/µL con CH2Cl2:MeOH (2:1); y en el caso de las plantas in vitro, solamente el extracto CH2Cl2:MeOH (2:1) fue: CL50 0,003 mg/µL y CL90 0,060 mg/µL. Se discute el valor potencial de los extractos de P. tuberculatum como un eficiente insecticida sobre S. frugiperda.

The larvicidal activity of the neotropical "matico" Piper tuberculatum was evaluated. The secondary compounds were extracted of leaves, stems and mature spikes with fruits and seeds from wild plants and in vitro plants of Piper tuberculatum. The acute toxicities to the fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), of extracts of spikes with fruits and seeds and in vitro plants of P. tuberculatum were evaluated by means of contact bioassays. Only CH2Cl2:MeOH (2:1) and EtOH extracts of mature spikes and CH2Cl2:MeOH (2:1) extract from in vitro plants showed significant levels of larval mortality. The CH2Cl2:MeOH (2:1) and EtOH extracts of mature spikes caused 90% mortality when doses of 0.1850 mg/µL were applied to the S. frugiperda in 24 and 48 h of exposure, respectively. The CH2Cl2:MeOH (2:1) extract from in vitro plants caused 95% mortality when doses of 0.1850 mg/mg/µL were too applied in 48 h of exposure. The mature spikes test best results were: LD50 0.001 mg/µL with EtOH and 0.007 mg/mg/µL with CH2Cl2:MeOH (2:1) and LD90 0.027 mg/µL with EtOH and 0.103 mg/µL with CH2Cl2MeOH (2:1); and, in the case of in vitro plants, only CH2Cl2:MeOH (2:1) extract was: LD50 0.003 mg/µL and LD90 0.060 mg/µL. The potential value of extracts derived from P. tuberculatum as efficient insecticides against S. frugiperda is discussed.

Piper solmsianum é uma espécie herbácea do sudeste brasileiro onde vários compostos biologicamente ativos já foram identificados. O objetivo deste trabalho foi estabelecer suspensões celulares nesta espécie. Para tanto, foram utilizados explantes de pecíolos e folhas, retirados de plântulas cultivadas in vitro, os quais foram submetidos a diferentes combinações de reguladores de crescimento (AIA, ANA, 2,4-D e BAP). Foi obtida a neo-formação de raízes e brotos, estes últimos através do processo de organogênese indireta evidenciada por estudos histológicos. Para a indução e crescimento dos calos, foram avaliados, além das diferentes combinações de reguladores de crescimento, a suplementação ao meio de cultura de carvão ativado (1,5 mg.l-1) e o regime de luz. Culturas mantidas na luz, em meio de cultura suplementado com 0,2 mg.l-1 2,4-D e 2 mg.l-1 BAP e sem carvão ativado, resultaram em maior crescimento (massa fresca) dos calos. A partir destes calos, foram obtidas suspensões celulares, cuja dinâmica de crescimento e acúmulo de metabólitos foi estudado. Os resultados obtidos deverão ser utilizados para a caracterização de rotas biosintéticas em culturas in vitro em P. solmsianum.

Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA). Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l-1) were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l-1 2,4-D and 2 mg.l-1 BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.

No escopo de nossas pesquisas sobre agentes bioativos da flora brasileira, vinte e quatro extratos e frações de Piper arboreum Aub. e Piper tuberculatum Jacq. (Piperaceae) tiveram sua atividade tripanocida avaliada através do ensaio colorimétrico com MTT. As atividades mais potentes foram manifestadas pelas frações hexânicas das folhas de P. arboreum (CI50 = 13,3 µg/mL) e P. tuberculatum (CI50 = 17,2 µg/mL). As frações hexânicas dos frutos de P. tuberculatum e P. arboreum também apresentaram efeito tóxico potente contra as formas epimastigotas de Trypanosoma cruzi, com valores de CI50 (µg/mL) de 32,2 e 31,3, respectivamente. Adicionalmente, o estudo fitoquímico da fração hexânica das folhas de P. arboreum forneceu duas amidas pirrolidínicas, piperilina (1) e 4,5-diidropiperilina (2), que podem ser responsáveis pela atividade antiprotozoária desta fração.

In the scope of our ongoing research on bioactive agents from Brazilian flora, twenty-four extracts and fractions obtained from Piper arboreum Aub. and Piper tuberculatum Jacq. (Piperaceae) were screened for trypanocidal activity by using MTT colorimetric assay. The strongest activity was found in hexane fractions from the leaves of P. arboreum (IC50= 13.3 µg/ mL) and P. tuberculatum (IC50 = 17.2 µg/mL). Hexane fractions of the fruits of P. tuberculatum and P. arboreum showed potent toxic effects on epimastigote forms of Trypanosoma cruzi, with values of IC50 (µg/mL) of 32.2 and 31.3, respectively. Additionally, the phytochemical study of the hexane fraction of P. arboreum leaves furnished two pyrrolidine amides, piperyline (1) and 4,5-dihydropiperyline (2), which could be responsible, at least in part for the observed antiprotozoal activity.

The biocid action of DCM:MeOH (2:1), EtOH and aqueous extracts of leaves, stems and mature spikes (with fruits and seeds) of field plants and DCM:MeOH (2:1) extract of in vitro plants of Piper tuberculatum on III larval stage of Diatraea saccharalis was evaluated. The method was by inoculating the previously eluted extract with distillate water as topic applications on the larval mesothorax. Only DCM:MeOH and EtOH extracts of mature spikes and DCM:MeOH extract of in vitro plants showed significant levels of larval mortality. The corresponding highest toxic effect was (a) for mature spikes respect to in vitro plants and (b) EtOH extract respect to DCM:MeOH extract, according to the results showed for the lethal concentration to 50% (LC50) and 90% (LC90), in 72 hours of exposure. Thus, in the case of mature spikes was: LC50 0,11 mg/mL with EtOH and 0,16 mg/mL with DCM:MeOH) and LC90 (0,35 mg/mL with EtOH and 0,55 mg/mL with DCM:MeOH); and, in the case of in vitro plants, only with DCM:MeOH extract, was: LC50 0,39 mg/mL and LC90 2,62 mg/mL. The results of probit values-mortality lines showed the same tendency.

En el presente trabajo evaluamos la acción biocida sobre larvas del III estadío de Diatraea saccharalis, usando extractos acuoso, diclorometano-metanol (DCM:MeOH, 2:1) y alcohólico (EtOH, 96%) de hojas, tallos y espigas maduras (con frutos y semillas) de Piper tuberculatum, en larvas del III estadío. El método de inoculación del extracto, previamente eluido con agua destilada, fue de aplicación tópica en el mesotorax de las larvas. Solamente los extractos DCM:MeOH y EtOH de espigas maduras y extracto DCM:MeOH de plantas in vitro mostraron niveles significativos de mortalidad larval. El mayor efecto tóxico correspondió a extractos de espigas maduras respecto a plantas in vitro y a extracto EtOH respecto a extracto DCM:MeOH, tal como lo expresan los resultados de las concentraciones letales a 50% (CL50) y 90% (CL90), en 72 h de exposición. Así tenemos que en el caso de espigas maduras fue: CL50 (0,11 mg/mL con EtOH y 0,16 mg/mL con DCM:MeOH) y CL90 (0,35 mg/mL con EtOH y 0,55 mg/mL con DCM:MeOH); en el caso de plantas in vitro, unicamente con DCM:MeOH, fue: CL50 0,39 mg/mL y CL90 2,62 mg/mL. Los resultados de las rectas valores probitos-mortalidad expresaron la misma tendencia.

A origem biossintética das unidades isoprênicas do 4-nerolidilcatecol (1), principal metabólito secundário de Potomorphe umbellata, foi investigada através da incorporação de glicose marcada com [14C] e [13C], e com precursores das vias mevalonoídica e triose/piruvato, incluindo mevalonolactona-[2-14C] e 3-fosfato de gliceraldeído-[U-14C], respectivamente. O padrão de incorporação de glicose-[1-13C] em 1 foi determinado por experimentos de RMN 13C quantitativos. O padrão de marcação revelou que o precursor foi especificamente incorporado, e que as unidades isoprênicas presentes no 4-nerolidilcatecol são derivadas tanto do ácido mevalônico, quanto da via triose/piruvato. Os resultados também indicaram que tanto a via plastídica quanto citoplasmática são capazes de gerar unidades de difosfato de isopentenila para a biossíntese de 1.

The biosynthetic origins of the isoprene units of 4-nerolidylcatechol (1), the major constituent of Potomorphe umbellata, have been studied through feeding experiments with [14C]- and [13C]-glucose, and with precursors of the mevalonic acid and triose/pyruvate pathways, namely, [2-14C]-mevalonolactone and [U-14C]-glyceraldehyde-3-phosphate, respectively. The pattern of incorporation of label from [1-13C]-glucose into 1 was determined by quantitative 13C NMR spectroscopy. The labelling pattern revealed that the additive was specifically incorporated, and that the isoprene units of the sesquiterpenoid moiety of 4-nerolidylcatechol were derived from both the mevalonic acid and the triose/pyruvate pathways. The results indicate that both plastidic and cytoplasmic pathways are able to provide isopentenyl diphosphate units for the biosynthesis of 1.

Através de experimentos de administração in vivo em plântulas de Virola surinamensis, observaram-se as incorporações de fenilalanina e do fenilpropanóide E-isoeugenol no 4-O-metil-5-metoxi-E-isoeugenol (E-isoelemicina) e na lignana tetraidrofurânica verrucosina.

The labelled substrates phenylalanine and phenylpropanoid E-isoeugenol were incorporated to 5-methoxy-4-O-methyl-E-isoeugenol (E-isoelemicin) and to the tetrahydrofuran lignan verrucosin in plantlets of Virola surinamensis (Myristicaceae).