Grandisin induces apoptosis in leukemic K562 cells

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.

4-Nerolidylcatechol induces autophagy in human glioblastoma cells

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.

Induction and growth pattern of callus from Piper permucronatum leaves / Indução e padrão de crescimento de calos de folhas de Piper permucronatum

ABSTRACT Piper permucronatum is a perennial shrub, a medicinal plant native to the Amazon Rainforest. Traditionally, the tea of its leaves is used to combat menstrual and intestinal cramps, stomach pain, digestive problems, diarrhea, hemorrhage, and nausea. Its leaf’s essential oil is effective against Aedes aegypti larvae; its flavones and flavanones have a fungicidal effect against Clamidosporium cladosporioides and C. sphaerospermum; its hexanic extract is effective against Leishmania amazonensis. The objective of this study was to provide a protocol for callus induction from P. permucronatum leaves and an identification of the callus growth pattern, focusing on the deceleration phase, when the callus cells must be subcultured into liquid medium in order to produce a cell suspension cultures. Leaf explants were inoculated in a solid MS medium supplemented with factorial combinations of 2,4-D, BA, NAA and GA3. Callus formation was evaluated weekly until the 49th day. Subsequently, new explants were inoculated at the hormonal combination that resulted in the highest callus cell proliferation and, every seven days during a period of 70 days, samples were dried and weighed to determine the callus growth pattern. NAA and GA3 were not effective for callus induction. Combinations of 2,4-D and BA resulted in callus induction and proliferation. The highest percentage of callus induction was observed with the combination of 4.52 µM 2,4-D and 4.44 µM BA. The calluses thereby produced were friable and whitish. The callus growth pattern followed a sigmoid shape. The deceleration phase started on the 56th day of culture.

RESUMO Indução e padrão de crescimento de calos de folhas de Piper permucronatum. Piper permucronatum é um arbusto perene, uma planta medicinal native da Floresta Amazônica. Tradicionalmente, o chá de suas folhas é usado em casos de cólicas menstruais e intestinais, dores de estômago, problemas digestivos, diarreia, hemorragia e náusea. O óleo essencial das folhas é efetivo contra a larva de Aedes aegypti; suas flavonas e flavanonas têm efeito fungicida contra Clamidosporium cladosporioides e C. sphaerospermum; seu extrato hexânico é efetivo contra Leishmania amazonensis. O objetivo deste trabalho foi determinar um protocolo para indução de calos em folhas de P. permucronatum e identificar o padrão de crescimento dos calos, com foco na fase de desaceleração, quando as células de calo devem ser subcultivadas em meio líquido para produzir culturas de células em suspensão. Explantes foliares foram inoculados em meio MS sólido suplementado com combinações fatoriais de 2,4-D, BAP, ANA e GA3. A formação de calos foi avaliada semanalmente até o 49º dia. Posteriormente, novos explantes foram inoculados na combinação hormonal que resultou na maior proliferação de células de calo e, a cada sete dias durante 70 dias, amostras foram secas e pesadas para determinar o padrão de crescimento dos calos. ANA e GA3 não foram efetivas para a indução de calos. Combinações de 2,4-D e BAP resultaram em indução e proliferação de calos. A maior porcentagem de indução de calos foi observada com a combinação de 4,52 µM de 2,4-D e 4,44 µM de BAP. Os calos produzidos eram friáveis e esbranquiçados. O crescimento dos calos seguiu um padrão sigmoide. A fase de desaceleração iniciou no 56º dia de cultivo.

Propagação vegetativa de estacas de Piper hispidumSw. em diferentes substratos / Vegetative propagation of cuttings of Piper hispidum Sw. in different substrates

RESUMO Piper hispidum é uma espécie pioneira pertencente à família Piperaceae, com importância na medicina popular e na obtenção de óleo essencial. Assim como outras espécies da família, possui poucas informações sobre técnicas de cultivo. O objetivo foi avaliar a propagação via estaquia de Piper hispidum em função do tipo de substrato e estaca em Manaus, Amazonas, Brasil. O experimento foi realizado na Embrapa Amazônia Ocidental. O delineamento utilizado foi inteiramente casualizado em esquema fatorial 3 (estacas) x 5 (substratos), com três repetições de 12 estacas, sendo as estacas (apical, mediana e basal) e os substratos (areia lavada, substrato comercial, solo + esterco de aves, solo + casca de guaraná e fibra de coco). Foram avaliadas: enraizamento (%), número de brotações, comprimento da maior brotação (cm), número de folhas, comprimento da maior raiz (cm), massa seca da raiz (g) e massa seca das brotações (g). Foi realizada análise de variância pelo teste F a 5% de probabilidade e para as médias foi realizado o teste Tukey ao nível de 5% de probabilidade. Recomenda-se utilizar estacas apicais e basais, nesta ordem. Ocorreu interação entre os fatores substrato e tipos de estaca somente para variável número de brotações. Os substratos areia lavada e substrato comercial são indicados para maiores porcentagens de enraizamento com 81,56% e 81,33%, respectivamente. O enraizamento foi superior nas estacas apicais (85,67%) e basais (74,47%). Porém, para esta espécie os substratos solo + esterco de aves e/ou solo + casca de guaraná foram mais indicados quando o objetivo é obter estacas de qualidade.

ABSTRACT Piper hispidum is a pioneer species belonging to the family Piperaceae, with relevance in popular medicine and in obtaining essential oil. As other species of this family, there is little information about cultivation techniques. The aim of this work was to evaluate the propagation through cutting from the Piper hispidum according to the type of substrate and cutting technique in Manaus, Amazonas, Brazil. The trial experiment was conducted at the Embrapa Western Amazon. The design was completely randomized factorial 3 x 5, with three replications of 12 cuttings with types of cuttings (apical, median and basal) and substrates (washed sand, commercial substrate, soil + poultry manure, soil + guarana shell and coconut fiber). After 60 days, the following characteristics were evaluated: rooting (%), number of shoots, length of the largest sprouting (cm), number of leaves, length of the longest root (cm), root dry weight (g) and dry weight of shoots (g). An analysis of variance was performed by the F test at 5% probability and for the averages` comparison the Tukey test was done at 5% level of probability. It is recommended to employ apical and basal cuttings, respectively. There was interaction between the factors and substrate types of cuttings only for the variable number of sprouts. These two substrates, washed sand and commercial substrate, are suggested for higher percentages of rooting with 81,56% and 81,33 %, respectively. The rooting was higher in the apical cuttings (85,67 %) and basal ones (74,47 % ). However, for this species, the substrates soil + poultry manure and/or soil + guaraná shell were most indicated when the goal was to obtain high-quality cuttings.

Evaluación de la actividad repelente de aceites esenciales de plantas Piperáceas del departamento de Chocó, Colombia / Evaluation of repellent activity of essential oils from plants Piperaceae Department of Chocó, Colombia

El objetivo de este trabajo fue determinar la composición química volátil del aceite esencial de varias especies de Piperáceas (Piper dilatatum, Piper aduncum, Piper divaricatum, Piper sp, Piper sanctifelicis) y la actividad repelente de ellos contra el gorgojo de granos almacenados Tribolium castaneum Herbst. Los aceites esenciales se obtuvieron por hidrodestilación, la separación e identificación de los componentes volátiles se realizó por cromatografía de gases acoplada a espectrometría de masas y para la evaluación de la actividad repelente se empleó el método de área de preferencia. Los compuestos mayoritarios encontrados en Piper dilatatum fueron el apiol 89,0% y trans-cariofileno (3,5%); en Piper divaricatum, eugenol (37,5%) y metil eugenol (36,3%); Piper sp, alpha-gurjuneno (o guaieno) (24,9%) y elemol (14,2%); Piper sanctifelicis, Delta-3-careno (35,3%) y limoneno (27,1%); Piper aduncum, dilapiol (48,2%) y 1,8 cineol (11,4%). Los porcentajes de repelencia de los aceites esenciales a una concentración de 1 µL/cm2, a un tiempo de exposición de 2 horas frente al T. castaneum fueron: Piper aduncum (99%), Piper sp (96%), Piper dilatatum (82%), Piper divaricatum (76%), Piper santifelicis (33%) (AU)

The objective of this study was to determine the volatile chemical composition of the essential oil of various species of Piperaceae (Piper dilatatum, Piper aduncum, Piper divaricatum, Piper sp, Piper sanctifelicis) and repellent activity of them against weevil stored grain Tribolium castaneum Herbst . Essential oils were obtained by hydrodistillation, the separation and identification of volatile components was performed by gas chromatography-mass spectrometry and to evaluate the repellent activity the area method was used preferably. The major compounds found in Piper dilatatum were 89.0% and apiol trans-caryophyllene (3.5%); Piper divaricatum, eugenol (37.5%) and methyl eugenol (36.3%); -gurjunene (or guaiene) (24.9%) and elemol (14.2%);Piper sp, Piper sanctifelicis, -(35.3%) and limonene (27.1%);3-carene Piper aduncum, dilapiol (48.2%) and 1,8 cineole (11.4%). Percent repellency of essential oils at a concentration of 1 uL / cm2, to an exposure time of 2 hours against T. castaneum were: Piper aduncum (99%), Piper sp (96%), Piper dilatatum (82 %), Piper divaricatum (76%), Piper santifelicis (33%)

Chemical composition of essential oils from four Vietnamese species of piper (piperaceae).

The chemical composition of essential oils from four Piper species, Piper retrofractum Vahl., P. boehmeriaefolium (Miq.) C. DC., P. sarmentosum Roxb., and P. maclurei Merr., were analysed by gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Nineteen to sixty-four compounds representing 92.0%-98.4% of the total contents were identified in the oil samples. The major constituents identified in P. retrofractum leaf oil were benzyl benzoate (14.4%), myrcene (14.4%), bicycloelemene (9.9%), bicyclogermacrene (7.0%) and ß-caryophyllene (5.3%). On the other hand, the main constituents of P. boehmeriaefolium were α-copaene (28.3%), α-pinene (7.4%) and 1, 8-cineole (5.7%). P. sarmentosum showed a very different chemical profile characterized mainly by aromatic compounds and devoid of monoterpene hydrocarbons. The major constituents were benzyl benzoate (49.1%), benzyl alcohol (17.9%), 2-hydroxy-benzoic acid phenylmethyl ester (10.0%) and 2-butenyl-benzene (7.9%). The leaf of P. maclurei was characterized by higher amount of (E)-cinnamic acid (37.4%) and (E)-nerolidol (19.4%). Moreover, (Z)-9-octadecanoic acid methyl ester (28.0%), (E)-cinnamyl acetate (17.2%), phytol (12.2%) and (E)-cinnamaldehyde (8.8%) were the major compounds identified in the stem oil.

OVICIDAL EFFECT OF PIPERACEAE SPECIES ON Biomphalaria glabrata, Schistosoma mansoni HOST / Efeito ovicida de espécies de Piperaceae em Biomphalaria glabrata, hospedeiro do Schistosoma mansoni

SUMMARY Schistosomiasis is a neglected disease with public health importance in tropical and subtropical regions. An alternative to the disease control is the use of molluscicides to eliminate or reduce the intermediate host snail population causing a reduction of transmission in endemic regions. In this study nine extracts from eight Piperaceae species were evaluated against Biomphalaria glabrata embryos at blastula stage. The extracts were evaluated in concentrations ranging from 100 to 10 mg/L. Piper crassinervium and Piper tuberculatum extracts were the most active (100% of mortality at 20 mg/L and 30 mg/L respectively). .

RESUMO A esquistossomose é uma doença negligenciada de importância para a saúde pública em regiões tropicais e subtropicais. Uma alternativa para o controle da doença é o uso de moluscicidas para eliminar ou reduzir a população de caramujo hospedeiro, acarretando uma redução da transmissão da doença nas regiões endemicas. Neste estudo, nove extratos vegetais provenientes de oito espécies de Piperaceae foram expostos a embriões de Biomphalaria glabrata no estágio de blástula. Os extratos foram avaliados em concentrações que variaram entre 100 e 10 mg/L, sendo Piper crassinervium e Piper tuberculatum os extratos mais ativos (100% de mortalidade a 20 mg/L e 30 mg/L respectivamente). .

Ovicidal effect of Piperaceae species on Biomphalaria glabrata, Schistosoma mansoni host.

Schistosomiasis is a neglected disease with public health importance in tropical and subtropical regions. An alternative to the disease control is the use of molluscicides to eliminate or reduce the intermediate host snail population causing a reduction of transmission in endemic regions. In this study nine extracts from eight Piperaceae species were evaluated against Biomphalaria glabrata embryos at blastula stage. The extracts were evaluated in concentrations ranging from 100 to 10 mg/L. Piper crassinervium and Piper tuberculatum extracts were the most active (100% of mortality at 20 mg/L and 30 mg/L respectively).

Molluscicidal and ovicidal activities of plant extracts of the Piperaceae on Biomphalaria glabrata (Say, 1818).

Schistosomiasis is a tropical disease caused by Schistosoma and occurs in 54 countries, mainly in South America, the Caribbean region, Africa and the eastern Mediterranean. Currently, 5 to 6 million Brazilian people are infected and 30,000 are under infection risk. Typical of poor regions, this disease is associated with the lack of basic sanitation and very frequently to the use of contaminated water in agriculture, housework and leisure. One of the most efficient methods of controlling the disease is application of molluscicides to eliminate or to reduce the population of the intermediate host snail Biomphalaria glabrata. Studies on molluscicidal activity of plant extracts have been stimulated by issues such as environmental preservation, high cost and recurrent resistance of snails to synthetic molluscicides. The aim of this study was to determine the molluscicide action of extracts from Piperaceae species on adult and embryonic stages of B. glabrata. Fifteen extracts from 13 Piperaceae species were obtained from stems, leaves and roots. Toxicity of extracts was evaluated against snails at two different concentrations (500 and 100 ppm) and those causing 100% mortality at 100 ppm concentration were selected to obtain the LC90 (lethal concentration of 90% mortality). Piper aduncum, P. crassinervium, P. cuyabanum, P. diospyrifolium and P. hostmannianum gave 100% mortality of adult snails at concentrations ranging from 10 to 60 ppm. These extracts were also assayed on embryonic stages of B. glabrata and those from P. cuyabanum and P. hostmannianum showed 100% ovicidal action at 20 ppm.

Verhuellia is a segregate lineage in Piperaceae: more evidence from flower, fruit and pollen morphology, anatomy and development.

BACKGROUND AND AIMS: The perianthless Piperales, i.e. Saururaceae and Piperaceae, have simple reduced flowers strikingly different from the other families of the order (e.g. Aristolochiaceae). Recent molecular phylogenies proved Verhuellia to be the first branch in Piperaceae, making it a promising subject to study the detailed structure and development of the flowers. Based on recently collected material, the first detailed study since 1872 was conducted with respect to morphology, anatomy and development of the inflorescence, pollen ultrastructure and fruit anatomy. METHODS: Original scanning electron microscopy (SEM), transmission electron microscopy (TEM) and light microscopy (LM) observations on Verhuellia lunaria were compared with those of Piperaceae, Saururaceae and fossils. KEY RESULTS: The inflorescence is an indeterminate spike with sessile flowers, each in the axil of a bract, developing in acropetal, helical succession. Flowers consist of two (occasionally three) stamens with basifixed tetrasporangiate anthers and latrorse dehiscence by a longitudinal slit. The gynoecium lacks a style but has 3-4 stigma branches and a single, basal orthotropous and unitegmic ovule. The fruit is a drupe with large multicellular epidermal protuberances. The pollen is very small, inaperturate and areolate, with hemispherical microechinate exine elements. CONCLUSIONS: Despite the superficial similarities with different genera of Piperaceae and Saururaceae, the segregate position of Verhuellia revealed by molecular phylogenetics is supported by morphological, developmental and anatomical data presented here. Unitegmic ovules and inaperturate pollen, which are synapomorphies for the genus Peperomia, are also present in Verhuellia.

From forgotten taxon to a missing link? The position of the genus Verhuellia (piperaceae) revealed by molecules.

BACKGROUND AND AIMS: The species-poor and little-studied genus Verhuellia has often been treated as a synonym of the genus Peperomia, downplaying its significance in the relationships and evolutionary aspects in Piperaceae and Piperales. The lack of knowledge concerning Verhuellia is largely due to its restricted distribution, poorly known collection localities, limited availability in herbaria and absence in botanical gardens and lack of material suitable for molecular phylogenetic studies until recently. Because Verhuellia has some of the most reduced flowers in Piperales, the reconstruction of floral evolution which shows strong trends towards reduction in all lineages needs to be revised. METHODS: Verhuellia is included in a molecular phylogenetic analysis of Piperales (trnT-trnL-trnF and trnK/matK), based on nearly 6000 aligned characters and more than 1400 potentially parsimony-informative sites which were partly generated for the present study. Character states for stamen and carpel number are mapped on the combined molecular tree to reconstruct the ancestral states. KEY RESULTS: The genus Peperomia is generally considered to have the most reduced flowers in Piperales but this study shows that this is only partially true. Verhuellia, with almost equally reduced flowers, is not part of or sister to Peperomia as expected, but is revealed as sister to all other Piperaceae in all analyses, putting character evolution in this family and in the perianthless Piperales in a different light. A robust phylogenetic analysis including all relevant taxa is presented as a framework for inferring patterns and processes of evolution in Piperales and Piperaceae. CONCLUSIONS: Verhuellia is a further example of how a molecular phylogenetic study can elucidate the relationships of an unplaced taxon. When more material becomes available, it will be possible to investigate character evolution in Piperales more thoroughly and to answer some evolutionary questions concerning Piperaceae.