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a Universidade Regional do Cariri, URCA, Cel Ant^onio Luis, 1161, 63105-000, Pimenta, Crato, CE, Brazil b Universidade Federal do Paran a, UFPR, XV de Novembro, 1299, 80.060-000, Centro, Curitiba, PR, Brazil c Universidade Regional de Blumenau, FURB, Ant^onio da Veiga, 140, 89030-903, Itoupava Seca, Blumenau, SC, Brazil d Laborat orio de investiga¸c~ao em Gen etica e Hematologia Translacional, Instituto Gon¸calo Moniz (IGM), Funda¸c~ao Oswaldo Cruz (FIOCRUZ), Salvador, BA 40296- 710, Brazil e Centro Universit ario Doutor Le~ao Sampaio (Unile~ao), Av. Maria Leticia Leite Pereira s/n, Lagoa Seca, Juazeiro do Norte, Brazil

Aims This study aims to improve characteristics of Piper regnellii extract to make it applicable in formulations to treat dermatophytosis, also known as ringworm. Methods and results Microparticles were produced by spray-drying with gelatin, alginate and chitosan as encapsulating agents, characterized by scanning electron microscopy, encapsulation efficiency, thermal analyses, X-ray diffraction, and tested against Trichophyton rubrum by broth microdilution. Produced microparticles had a mean diameter less than 2 μm, an increase in stability and release of the extract and good results for encapsulation efficiency, being 85.6% to gelatin microparticle, 71.3% to chitosan microparticle and 60.6% to alginate. Microparticles preserved the antifungal activity of P. regnellii extract Trichophyton rubrum. Conclusion Microencapsulation provided a significant improvement in the stability of the P. regnellii extract e better solubilization of chemical compounds, maintaining the antifungal effect against T. rubrum. This article is protected by copyright. All rights reserved.

Many infections worldwide are associated with bacterial biofilms. The effects of isolated neolignans (conocarpan and eupomathenoid-5) and the dichloromethane extract of Piper regnellii (Miq.) C. DC., Piperaceae, were tested against isolates of methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus biofilms and S. aureus planktonic cells. The dichloromethane extract presented better results than isolated neolignans against all of the biofilms tested, with a minimum inhibitory concentration

This work combines the analytical capability of GC × GC/TOFMS, the use of retention indices and adequate software tools for the study of essential oils of Piper regnellii (Miq.) C. DC. (pariparoba) growing wild in “cerrado” landscape, Central-West region, Brazil. The leaves, stems and flowers of P. regnellii generated essential oils with 163, 119 and 110 compounds tentatively identified, respectively. The major compounds in each essential oil were approximately the same, except dill apiole, which was concentrated more highly in the stems. The major compounds were: myrcene, anethole E and bicyclogermacrene (22%, 19% and 5%, respectively) in leaves, anethole E , dill apiole and myrcene (20%, 19% and 16%, respectively) in stems and anethole E , myrcene and bicyclogermacrene (24%, 18% and 9%, respectively) in flowers. This is the first time that this plant was analyzed by GC × GC/TOFMS and this technique allows identification of a higher number of compounds when compared to traditional one-dimensional chromatography.

Piper regnellii (Piperaceae) contains high levels neolignans with diverse biological activities, including insecticidal. Despite the insecticidal activity of these neolignans, Naupactus bipes (Coleoptera: Curculionidae) larvae and adults were found feeding on the leaves (adults) and roots (larvae) of P. regnellii. The present study investigated the metabolic pathway of neolignans from P. regnellii leaves in the beetle N. bipes, focusing on possible biotransformation or sequestration of these compounds by the beetle. Two of the four plant neolignans could be recovered in the feces of adults feeding on P. regnellii leaves. In addition, four degradation products were detected, including the neolignan 3-[(2R,3R)-2,3-dihydro-3-(methyl)-2-(4-hydroxyphenyl)-5-benzofuranyl]-(2E)-propenal (7), an oxidation product of conocarpan, the major plant neolignan. The adult beetle sequesters the neolignan conocarpan selectively from leaves as well as accumulates neolignan 7. Neolignan 7 was also detected in the larvae of lab-reared beetles.

The dermatophytes are filamentous fungi that cause cutaneous fungal infections because they use keratin as a nutrient source. For this study the antidermatophyte activity of the extracts and derivates from leaves of Piper regnellii was analyzed. From the dichloromethane extract (EBD) neolignans such as eupomatenoid-3 and eupomatenoid-5 were obtained, and it was submitted to fractionation to remove the green residue, designated as the chloroform fraction (FF). Extracts, chloroform fraction and compounds were tested against Trichophyton rubrum ATCC 28189 to determine the minimum inhibitory concentration (MIC). The chloroform fraction was incorporated to nail lacquer that was analyzed by photoacoustic spectroscopy, in vitro assay and scanning electronic microscopy. For antifungal activity in solid medium the dichloromethane extract and chloroform fraction were used. The compounds eupomatenoid-3 and eupomatenoid-5 were less active than the dichloromethane extract against T. rubrum. EBD and FF showed moderate activity in hyphal growth inhibition in solid medium and EBD did not link to ergosterol. Nail lacquer containing the chloroform fraction showed good penetration through the nail as determined by photoacoustic spectroscopy. From in vitro studies it was observed that nail lacquer concentrations above 20 mg/mL prevented the growth of fungi, but concentrations up to 2.5 inhibited the growth. Scanning electronic microscopy was used to confirm the in vitro nail lacquer activity results. The specie P. regnellii showed great antifungal activity against T. rubrum, and nail lacquer containing its chloroform fraction has great potential to treat onychomycosis caused by these microorganisms.

Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck (Piperaceae) is a medicinal plant traditionally used in Brazil to treat infectious diseases. The extracts obtained of the leaves from P. regnellii were investigated for their antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA). The ethyl acetate extract presented a good activity against MRSA, with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 16 microg/mL. Based on this finding, the ethyl acetate extract was fractionated by silica gel column chromatography into nine fractions. The hexane fraction was active against MRSA (MIC at 4 microg/mL). Further column chromatography separation of the hexane fraction afforded the pure compound eupomatenoid-5. The structure of the compound was established by spectral data (1H and 13C NMR HSQC, HMBC, gNOE, IR and MS). Eupomatenoid-5 was the only compound active on the bacterium. The antibacterial property of P. regnellii extract provides preliminary scientific validation for the traditional medicinal use of this plant. The active compound eupomatenoid-5 should be further studied in animal models to verify in vivo efficacy and toxicity.

A atividade biológica do extrato bruto e as várias frações obtidas de folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck foi avaliada em Leishmania amazonensis. Este estudo incluiu o processo de extração e o fracionamento biomonitorado pelo método de cromatografia de adsorção. Um aumento progressivo no efeito antileishmania foi observado durante o processo de purificação. O extrato hidroalcoólico solúvel em água (EBA) apresentou concentração inibitória 50% (IC50) igual a 167 µg/mL enquanto o extrato hidroalcoólico solúvel em acetato de etila (EBAcOEt) mostrou um IC50 de 30 mg/mL sobre o crescimento de formas promastigotas após 48 h de cultivo. A fração hexano (FHex) apresentou uma atividade antileishmania maior que o EBAcOEt com IC50 de 21,5 µg/mL. Análises de citotoxicidade indicaram que as concentrações tóxicas do EBA, EBAcOEt e das frações foram maiores para macrófagos J774G8 do que para os protozoários. Biological activity of the crude extract and several fractions obtained from Piper regnellii var. pallescens was assessed on Leishmania amazonensis. This study included the extraction process and bioassay-guided fractionation by the adsorption chromatography method. A progressive increase in the antileishmanial effect was observed in the course of the purification process. The hydroalcoholic extract water soluble (EBA) had a 50% inhibitory concentration (IC50) at 167 µg/mL whereas the hydroalcoholic extract acetate soluble (EBAcOEt) showed an IC50 of 30 µg/mL against the growth of promastigote forms after 48 h of culturing. The hexan fraction (FHex) showed an antileishmanial activity greater than EBAcOEt with IC50 at 21.5 µg/mL. Analysis of cytotoxicity indicated that the toxic concentrations of the EBA, EBAcOEt, and fractions were higher for J774G8 macrophages than for the protozoans.

Organ for the biosynthesis of secondary metabolite is not always a place for its biosynthesis, even the same compound was synthesized in the different organs in different plants. Neolignan is a secondary metabolite, known as an imunostimulant, synthesized through shikimic acid pathway with the important precursor is chorismic acid. The compound was known to be accumulated in the roots, stems and leaves of Piper regnellii with the concentration varies depending on the type of neolignan. It has been investigated the accumulation of two compound neolignans (Pc-1 and Pc-2) isolated from the methanol extract of red betel leaf (Piper crocatum Ruiz & Pav.) in the leaves, stems, and flowers of red betel. Chromatographic methods used was Gas Chromatography-Mass Spectrometry (GC-MS). Chromatogram of GC-MS showed that the Pc-1 with purity of 100%, m/z 460.3 could be detected at the minute of 29.986, while the Pc-2 with purity of 96.681%, m/z 418.3 was detected at the minute of 29.495. The research was then continued to investigate the existence and accumulation of both compounds in leaves, stems, and flowers of red betel. The GC-MS chromatogram shows that Pc-1 and Pc-2 could be detected in the leaves, stem and flower with various concentration among plant organs. Moreover, leaves contained the highest concentration of Pc-1 and Pc-2 compared to other plant organs.

Orientadores: João Ernesto de Carvalho, Mary Ann Foglio Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: O câncer é a segunda causa de morte da população nos países desenvolvidos e em desenvolvimento. Caracteriza-se pelo desenvolvimento de células instáveis que tendem a não responder aos estímulos internos e externos que controlam a proliferação, diferenciação e morte celular. O crescente entendimento da biologia tumoral tem fornecido alvos moleculares para a triagem orientada de quimioterápicos e de agentes quimiopreventivos, geralmente de origem natural ou sintetizados com base em produtos naturais. A partir do extrato bruto diclorometânico das folhas de Piper regnellii foram isolados dois compostos, o eupomatenóide-5 e o apiol. O eupomatenóide-5 é uma neolignana cuja potencial atividade anticâncer foi demonstrada in vitro (cultura de células tumorais humanas) e in vivo (tumor sólido de Ehrlich) em trabalhos anteriores publicados pelo nosso grupo. Dentre as linhagens mais sensíveis, MCF-7 (mama) e 786-0 (rim) foram selecionadas para dar continuidade aos estudos de mecanismo de ação deste composto. Os resultados obtidos no presente estudo sugerem que o eupomatenóide-5 possui ação multialvo, uma vez que demonstraram claramente que existe um mecanismo de ação diferente para cada linhagem celular. Sugere-se que o efeito antiproliferativo do eupomatenóide-5 esteja associado com indução de apoptose em linhagem MCF-7 e necrose programada em linhagem 786-0. O apiol, outro composto isolado, é um fenilpropanóide com conhecida ação bloqueadora dos canais de cálcio. Sabe-se que a inibição de canais de cálcio tem relação com a reversão da resistência a múltiplas drogas (MDR) e, por este motivo, o efeito do apiol sobre a glicoproteína-P, uma das principais proteínas moduladoras dessa resistência, foi avaliado; entretanto o apiol não inibiu sua ação. Por outro lado, neste estudo foi possível evidenciar que embora tenha apresentado atividade citostática baixa para as linhagens tumorais, ele potencializou a atividade específica dos quimioterápicos doxorrubicina e vincristina quando associado a estes em linhagens tumorais com e sem fenótipo de resistência, sugerindo um efeito sinérgico. Além disso, o apiol potencializou a atividade antiproliferativa do eupomatenóide-5 e essa associação reduziu a quantidade de células tumorais de Ehrlich em modelo in vivo, sem sinais de toxicidade evidentes. Os resultados encorajam o aprofundamento desses estudos para a determinação dos mecanismos de ação envolvidos nas atividades anticâncer do eupomatenóide-5 e de reversão da MDR do apiol Abstract: Cancer is the second leading cause of mortality worldwide. The loss of normal cell growth control is the main event in the development of cancer and includes specific steps known as cell proliferation, differentiation and apoptosis. Natural compounds are good source of new anticancer agents, being the investigation into cell death mechanisms involved in this activity a significant endeavor. From the crude dichloromethane extract of Piper regnellii leaves two compounds were obtained: eupomatenoid-5 and apiole. Eupomatenoid-5 is a neolignan whose in vitro (tumor cell line culture) and in vivo (Ehrlich solid tumor) anticancer activity was previously reported by our group. Among the cancer cell lines most sensible to eupomatenoid-5 treatment, MCF-7 (breast) and 786-0 (kidney) were chosen to continue the cell death mechanism of action studies. The results obtained suggest that eupomatenoid-5 is a multiple-target compound. Our findings support the possibility that eupomatenoid-5 might be acting in both cell death via: apoptosis in MCF-7 cell line and programmed necroptosis in 786-0 cell line. Apiole, the other compound isolated, is a phenylpropanoid and a calcium channel blocker. Recent pharmacological studies have reported the ability of several calcium channel blockers to reverse the multidrug resistance (MDR) and, therefore, the effect of apiole on P-glycoprotein (P-gp), the main protein involved in MDR, was evaluated. Apiole did not inhibit P-gp and, moreover, presented low cytotoxic activity for the most tumor cell lines. Nonetheless, the combined therapy with chemotherapies (doxorubicin and vincristine) was efficient in potentializing their antiproliferative action in tumor cell lines with or without resistance phenotype, suggesting synergic effect. Furthermore, apiole potentiated the antiproliferative activity of eupomatenoid-5 and this association reduced the in vivo tumor cell quantity with no side effects. The results encourage further studies to be developed with the aim to determine the mechanisms of action involved in the anticancer activity of eupomatenoid-5 and in the MDR reversion by apiole Doutorado

The aim of this paper is to present a procedure based on analyses of 13 C NMR data for identification of known and new chemical constituents in essential oils. A novel program developed to analyze complex mixtures of terpenoid compound was evaluated for the identification of components in the essential oils from leaves of Piper cernuum and Piper regnellii .

Roots of Piper regnellii (Piperaceae) contain three phenylpropanoids: apiol, dillapiol and myristicin; seven 4′,7-epoxy-8,3′-neolignans; and three 8′,9′-dinor-4′,7-epoxy-8,3′-neolignans. The structures and absolute configurations of the isolates (four new neolignans including regnelline) were established based on analysis of spectroscopic data.

Leishmaniaspp. are protozoa responsible for leishmaniasis, a neglected disease that kills up to 50,000 people every year. Current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy and safety and increased drug resistance. An urgent need exists to find new and more affordable drugs. Our previous study demonstrated the antileishmanial activity of eupomatenoid-5, a neolignan obtained from leaves ofPiper regnelliivar.pallescens. The aim of the present study was to clarify the mode of action of eupomatenoid-5 againstL. amazonensis. We used biochemical and morphological techniques and demonstrated that eupomatenoid-5 induced cell death inL. amazonensispromastigotes, sharing some phenotypic features observed in metazoan apoptosis, including increased reactive oxygen species production, hypopolarization of mitochondrial potential, phosphatidylserine exposure, decreased cell volume, and G0/G1 phase cell cycle arrest.

The present study determined the anti-Mycobacterium tuberculosis activities of supercritical CO2 extracts, neolignans eupomatenoid-5 (1), conocarpan (4) and eupomatenoid-3 (7) and their derivatives (2, 3, 5, 6, and 8) from Piper regnellii, as well as their cytotoxicities. The supercritical CO2 extract from leaves was purified by chromatographic methods, yielding compounds (1), (4) and (7), which were identified by (1)H NMR and comparison with literature data. Anti-M. tuberculosis activity (H37Rv and clinical isolates) was evaluated using a resazurin microtiter assay plate (REMA) to determine the MIC. The cytotoxicity assay was carried out in macrophages J774G.8 by sulforhodamine B colorimetric assay. The supercritical CO2 extracts from leaves and stems, and compound (4) showed activity against M. tuberculosis (MIC 15.6 μg/ml). Compound (1) showed the best activity (MIC 1.9 μg/ml), with good SI. Compounds (7) and (8) showed low activity against M. tuberculosis H37Rv. The derivative compounds did not show increased anti-M. tuberculosis activity. This is the first report, to our knowledge, to describe neolignans from P. regnellii with activity against M. tuberculosis, and compound (1) is a potential candidate for future antituberculosis drugs.

Piper regnellii var.pallescens is popularly known as pariparoba caapeba and capeba.In folk medicine, its roots are used in the treatment of obstructions of the liver and spleen, wound healing, it reduces swelling and skin irritation.It also gives broad spectrum of antimicrobial activity.The application of supercritical technology for the extraction of bioactive compounds of this kind can be attractive due to low temperature operation and high purity of the extracts.In this context, the objective of this study was to evaluate the biological quality of supercritical extracts of Piper regnellii obtained under variation of pressure and temperature parameters and compare them with the extracts obtained by conventional method.For this task extracts from the leaves and stems of pariparoba were obtained in various ways to extract and subsequently characterized according to their composition and neolignans conocarpan and eupomatenoid-5.The supercritical extraction occurred at temperatures of 40 and 60 °C and at pressures ranging from 109.2 to 250 bar.Tests of antifungal, antimicrobial, antioxidant and cytotoxic activity were performed.Despite the low yield in mass of extract obtained in supercritical extraction, they have higher levels of neolignans and conocarpan eupomatenoid-5.Due to the high quality of these extracts, the biological activities tested proved best in the soxhlet extraction method.The extracts obtained by supercritical extraction showed MICs between 1.95 and 7.81 μg/mL against S.aureus and MICs from 0.98 to 7.81 μg/mL against MRSA.In tests with different species of the genus Candida, these same extracts showed MIC ranging from 7.81 to 250 μg/mL.In the analysis of cytotoxicity extracts were more toxic to the pathogens than for animal cell tested.In addition, the test material showed high antioxidant exhibiting EC50 values between 41.23 and 85.41 μg/mL. To represent the kinetics of extraction in a fixed-bed column a second-order mathematical model was employed and provedsatisfactory, based on the comparison between the experimental and theoretical results.Thus, the supercritical extraction is presented as the best method for obtaining bioactive compounds promising for the development of drugs based on natural products. A Piper regnellii var. pallescens é conhecida popularmente como pariparoba, caapeba e capeba. Na medicina popular, suas raízes são utilizadas no tratamento das obstruções do fígado e do baço, para cicatrização de feridas, redução de inchaços e irritações na pele. Além disso, apresenta amplo espectro de atividade antimicrobiana. A aplicação de tecnologia supercrítica para a extração de compostos bioativos desta espécie pode ser atrativa, devido à baixa temperatura de operação e alta pureza dos extratos. Neste contexto, o objetivo deste trabalho foi avaliar a qualidade biológica de extratos supercríticos de Piper regnellii obtidos sob variação dos parâmetros pressão e temperatura e compará-las com os extratos obtidos por método convencional. Para tal tarefa extratos das folhas e caules de pariparoba foram obtidos sob diversas formas de extração e posteriormente caracterizados segundo sua composição das neolignanas conocarpano e eupomatenóide-5. Aextração supercrítica ocorreu nas temperaturas de 40 e 60 oC e em pressões que variaram de 109,2 à 250 bar. Testes de atividade antifúngica, antimicrobiana, citotóxica e antioxidante foram realizados. Apesar do baixo rendimento em massa de extrato obtido na extração supercrítica, estes apresentam maiores teores das neolignanas conocarpano e eupomatenóide-5. Devido à alta qualidade destes extratos, as atividades biológicas testadas se mostraram melhores no método de extração por soxhlet. Os extratos obtidos por extração supercrítica apresentaram CIMs entre 1,95 e 7,81 μg/mL frente a S. aureus e CIMs de 0,98 a 7,81 μg/mL frente à MRSA. Em testes com diferentes espécies do gênero Candida estes mesmos extratos apresentaram CIM variando de 7,81 à 250 μg/mL. Na análise da citotoxicidade os extratos se mostraram mais tóxicos para os patógenos do que para a célula animal testada. Além disso, o material testado apresentou elevado poder antioxidante exibindo valores de CE50entre41,23 e 85,41 μg/mL.Para representar a cinética de extração em uma coluna de leito fixo, foi empregado um modelo matemático de segunda ordem que se mostrou satisfatório, com base na comparação entre o resultado experimental e teórico. Sendo assim, a extração supercrítica apresenta-se como o melhor método para a obtenção de compostos bioativos promissores para o desenvolvimento de medicamentos a base de produtos naturais. xviii, 89 f

Orientadores: João Ernesto de Carvalho, Mary Ann Foglio Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: O câncer é um conjunto de doenças que atinge milhões de pessoas, sendo a segunda causa de morte da população mundial. Caracteriza-se pelo desenvolvimento de células instáveis que passam a não responder aos estímulos internos e externos que controlam a proliferação, diferenciação e morte celular. Apesar da grande quantidade de estudos relacionados ao gênero Piper e suas espécies, não há descrição sobre a atividade antiproliferativa in vitro e antitumoral in vivo da espécie P. regnellii (Miq.) C. DC. var. regnellii. O presente estudo avaliou a atividade antiproliferativa in vitro desta espécie, coletada em três épocas diferentes: junho e outubro de 2008 e fevereiro de 2009. Dentre as coletas realizadas, o extrato bruto diclorometânico de junho e suas frações de média polaridade apresentaram atividade citotóxica in vitro com boa correlação entre concentração e efeito e também seletividade para as linhagens hormônio-dependentes de ovário (OVCAR-3) e mama (MCF7), bem como para as linhagens de melanoma (UACC- 62) e próstata (PC-3). De acordo com os nossos estudos, uma das substâncias responsáveis por esta atividade é uma neolignana denominada eupomatenóide-5. Os efeitos observados in vitro pela fração rica em lignanas foram comprovados em modelo in vivo de tumor sólido de Ehrlich. Além disso, o extrato bruto diclorometânico de P. regnellii reduziu o edema de pata produzido por carragenina sugerindo uma correlação entre câncer e inflamação. Este é o primeiro trabalho que relata o efeito antiproliferativo e antiinflamatório de P. regnellii e os resultados obtidos incentivam a continuação dos estudos visando a elucidação do mecanismo de ação e identificando outros princípios ativos que, em sinergismo com o composto eupomatenóide-5, potencializem a atividade de P. regnellii. Abstract: Cancer is a pool of many diseases diagnosed in thousands of people all over the world and represents the second major cause of death worldwide. The loss of normal cell growth control is the main event in the development of cancer and includes specific steps known as cell proliferation, differentiation and apoptosis. Despite numerous studies related to Piper genus, there is no description of any antiproliferative (in vitro) or antitumoral activity (in vivo) of Piper regnellii (Miq.) C. DC. var. regnellii. The present study evaluated the antiproliferative activity of this species, collected in three different periods: June and October, 2008 and February 2009. Among the plants evaluated, the medium polarity fractions obtained from the dichloromethanic crude extract of the plants collected in June presented concentration-effect correlation and also selectivity towards hormonedependent ovary (OVCAR-3) and breast (MCF7) cell lines, as well as melanoma (UACC- 62) and prostate (PC-3). According to our studies, one of the compounds responsible for this activity is classified as eupomatenoid-5, that is a neolignan. Effects observed in vitro for the lignans enriched fractions were confirmed through the Ehrlich solid tumor model in mice. In addition, preliminary results with dichloromethane crude extract of P. regnellii in anti-inflammatory experimental model of paw edema induced by carrageenan suggest the connection between cancer and inflammation. The present study represents the first report of both antiproliferative and anti-inflammatory promising activities of this species indicating the benefits of further studies in the search of more purified compounds and active principles that in synergism with eupomatenoid-5 can potentiate anticancer activity's result, as well as their mechanisms of action. Mestrado Biologia Celular Mestre em Biologia Celular e Estrutural

The essential oils of leaves of Piper gaudichaudianum (samples A and B) and P. regnellii collected at campus of Sao Paulo University, in Southeast Brazil, were analyzed by GC/MS. The major constituents identified in sample-A of P. gaudichaudianum were β-caryophyllene (12.1%), α-humulene (13.3%), β-selinene (15.7%) and α-selinene (16.6%). The sample-B was dominated by β-caryophyllene (19.3%), α-humulene (29.2%) and β-selinene (8.9%). The main constituents found in the oil of P. regnelii were β-caryophyllene (23.4%), (E)-nerolidol (13.7%) and spathulenol (11.1%).

The main remedies recommended to the treatment of cutaneus and visceral leishmaniasis are derived from the pentavalents antimonials such as sodium estibogluconate (Pentostan®) and meglumine antimonial (Glucantime®), however they are far to be ideal drugs because they are not active when administered orally and need a long period for the treatment. Besides, the side effects can reveal the patients intolerance for this medicaments. Considering that leishmaniasis is endemic in various regions of Brazil, the quest for herbal remedies that are effective for the treatment of this illness is very important, thus the availability of this products could contribute to the its control. The aim of the present work was to invetigate the activity of eupomatenoid-5, isolated from leaves of Piper regnelli (Miq.) C. DC. var. pallescens (C. DC.) Yunck (Piperaceae), over promastigote and amastigote forms of Leishmania amazonensis strain MHOM/BR/75/Josefa. Toxicity of eupomatenoid-5 on J774G8 macrophages and its effect on host-parasite interaction was also evaluated The extraction process and fractionation of Piper regnellii var. pallescens leaves was realized by adsorption chromatography and the compound dihidrobenzofuranic neolignan (eupomatenoid-5) was isolated and characterized by RMN 1H, 13C spectroscopy, comparing to literature data. At µg/ml eupomatenoid-5 presented an inhibitory growth concentration of 50% against promastigotes after 72 h at 28ºC. Under these conditions, significative ultrastructural alterations of the protozoa were visualized through transmission electron microscopy. The toxicity of eupomatenoid-5 against macrophages cell cultures was observed at concentrations higher than the necessary to inhibit the protozoa growth. The effect of eupomatenoid-5 in the leishmania-macrophage interaction is higher when the parasites are treated before the interaction. Os principais medicamentos recomendados para o tratamento das leishmanioses cutâneas e viscerais são os derivados dos antimoniais pentavalentes, como o estibogluconato de sódio (Pentostan®) e o antimoniato de meglumina (Glucantime®), entretanto, estão longe de serem as drogas ideais, pois não são ativos quando administrados oralmente e necessitam de um longo período de tratamento. Além disso, os efeitos tóxicos podem revelar a intolerância destes medicamentos por muitos pacientes. Considerando que a leishmaniose é endêmica em várias regiões do Brasil, a busca de produtos naturais que sejam efetivos no tratamento da leishmaniose é muito importante, pois a disponibilidade de tais produtos poderá contribuir para o controle da doença. O presente trabalho teve como objetivo investigar a atividade do eupomatenóide-5, substância isolada das folhas de Piper regnellii (Miq.) C. DC. var. pailescens (C. DC.) Yunck., (Piperaceae), sobre formas promastigotas e amastigotas de Leishmania amazonensis cepa MHOM/BR175/Josefa. Foi avaliada também a toxicidade da substância sobre cultura de macrófagos J774G8 e o efeito na interação parasita-hospedeiro. O processo de extração e fracionamento das folhas de Piper regnellii var. pailescens foi realizado através de métodos cromatográficos de adsorção chegando ao isolamento da neolignana dihidrobenzofurânica: o eupomatenóide-5. Esta substância foi identificada por análise espectral de RMN 1H, '3C, comparada com dados da literatura. Na concentração de 9pg/ml, apresentou 50% de inibição do crescimento de formas promastigotas de L. amazonensis após 72 h de incubação a 28°C. Nessas condições, significativas alterações ultraestruturais do protozoário foram visualizadas através da microscopia eletrônica de transmissão. A toxicidade do eupomatenóide-5 sobre cultura de macrófagos só foi observada em concentração acima da necessária para inibir o crescimento do protozoário. O efeito do eupomatenóide-5 na interação leishmânia-macrófago é maior quando os parasitas são tratados antes da interação. 87 f

Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck (Piperaceae) is a medicinal plant traditionally used in Brazil to treat infectious diseases. The extracts obtained from the leaves of P. regnellii were investigated for their antifungal activities against the yeasts Candida albicans, C. krusei, C. parapsilosis, and C. tropicalis. The EtOAc extract presented a significant activity against Candida albicans with MIC at 125 µg mL-1, and a moderate activity against both C. krusei and C. parapsilosis with MIC at 500 µg mL-1. Candida tropicalis was not inhibited by this extract at concentrations as high as 1000 µg mL-1. Based on these findings, the EtOAc extract was fractionated by silica gel column chromatography into nine fractions. The hexane and CHCl3 fractions showed varied levels of antifungal activity against all test yeasts. Further column chromatography separation of the hexane fraction afforded the pure compounds eupomatenoid-6, eupomatenoid-5, eupomatenoid-3 and conocarpan. The structure of the compounds was based on spectral data (¹H and 13C NMR, HSQC, HMBC, gNOE, IR and MS). Conocarpan was the only active compound on the yeasts. The antifungal property of P. regnellii extract provides preliminary scientific validation for the traditional medicinal use of this plant. Extratos de folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck (Piperaceae), uma planta medicinal utilizada no Brasil para tratar doenças infecciosas, foram testados para a atividade antifúngica sobre as leveduras Candida albicans, C. krusei, C. parapsilosis e C. tropicalis. O extrato em acetato de etila apresentou boa atividade contra C. albicans com uma CIM de 125 mg µL-1, moderada atividade contra C. krusei e C. parapsilosis (CIM de 500 mg µL-1) e foi inativo contra C. tropicalis (CMI> 1000 mg µL-1). Com base nestes resultados, o extrato em acetato de etila foi fracionado em nove frações em cromatografia de coluna de sílica gel. As frações hexano e clorofórmio mostraram variados níveis de atividade antifúngica contra as leveduras testadas. Posterior separação da fração hexânica em cromatografia de coluna resultou nas substâncias puras eupomatenóide-6, eupomatenóide-5, eupomatenóide-3 e conocarpano. A elucidação estrutural das substâncias foi baseada em dados espectrais (RMN de ¹H e de 13C, HSQC, HMBC, gNOE, IV e EM). O conocarpano foi a única substância isolada, com atividade contra as leveduras. As propriedades antifúngicas do extrato de P. regnellii demonstraram preliminar validação científica do uso da planta na medicina popular.

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extract displayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 micrograms/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 micrograms/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrationsor = 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 micrograms/ml) and B. subtilis (MIC at 3.9 and 7.8 micrograms/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6 identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 micrograms/ml and 3.12 micrograms/ml, respectively. Both compounds presented MIC of 3.12 micrograms/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 micrograms/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.

Nesta espécie ocorrem dois tipos de tricomas secretores: glândula perolada e tricoma saculiforme. Ambos originam-se na protoderme do primórdio foliar e atingem sua maturidade nas folhas jovens presentes no ápice caulinar. A densidade dos dois tipos de tricomas diminui durante a expansão do limbo, sendo raros na folha adulta. As glândulas peroladas são constituídas por uma célula basal e por uma célula apical grande cuja forma varia de semi-globóide a espatulada. A liberação da secreção ocorre após a ruptura da cutícula distendida. Os tricomas saculiformes são constituídos por uma célula basal, 1-2 células colares e uma célula apical cônica, de posição inclinada à superfície foliar. A secreção acumula-se em espaços subcuticulares diminutos, não tendo sido observada ruptura da cutícula. Piper regnellii has two types of trichomes: pearl glands and sac-like trichomes. Both secretory trichomes originate from the protoderm of the leaf primordium and reach maturity in the young leaves found in the shoot apex. The pearl glands are more abundant on the abaxial surface, while the sac-like trichomes cover completely both young leaf blade. The density of both trichomes decreases during blade expansion, being scarce in the adult leaf. The pearl glands are composed of a basal cell and an apical cell whose shape ranges from semi-globular to spatula-like. Secretion release occurs after rupture of the distended cuticle. The sac-like trichomes are composed of a basal cell, 1-2 stalked cells and an apical conical cell parallel to the leaf surface. The secretion is accumulated in minute subcuticular spaces. No cuticular ruptures have been observed.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:52:16Z No. of bitstreams: 1 S0100-84041999000200002.pdf: 551555 bytes, checksum: 8392a8b4e548b792085e20ec2b9f33c5 (MD5) Made available in DSpace on 2013-08-22T18:52:16Z (GMT). No. of bitstreams: 1 S0100-84041999000200002.pdf: 551555 bytes, checksum: 8392a8b4e548b792085e20ec2b9f33c5 (MD5) Previous issue date: 1999-08-01 Made available in DSpace on 2013-09-30T19:43:18Z (GMT). No. of bitstreams: 2 S0100-84041999000200002.pdf: 551555 bytes, checksum: 8392a8b4e548b792085e20ec2b9f33c5 (MD5) S0100-84041999000200002.pdf.txt: 26027 bytes, checksum: 7036179ed6cecb6a03603166dd66fbb9 (MD5) Previous issue date: 1999-08-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:11:18Z No. of bitstreams: 2 S0100-84041999000200002.pdf: 551555 bytes, checksum: 8392a8b4e548b792085e20ec2b9f33c5 (MD5) S0100-84041999000200002.pdf.txt: 26027 bytes, checksum: 7036179ed6cecb6a03603166dd66fbb9 (MD5) Made available in DSpace on 2014-05-20T15:11:18Z (GMT). No. of bitstreams: 2 S0100-84041999000200002.pdf: 551555 bytes, checksum: 8392a8b4e548b792085e20ec2b9f33c5 (MD5) S0100-84041999000200002.pdf.txt: 26027 bytes, checksum: 7036179ed6cecb6a03603166dd66fbb9 (MD5) Previous issue date: 1999-08-01 Nesta espécie ocorrem dois tipos de tricomas secretores: glândula perolada e tricoma saculiforme. Ambos originam-se na protoderme do primórdio foliar e atingem sua maturidade nas folhas jovens presentes no ápice caulinar. A densidade dos dois tipos de tricomas diminui durante a expansão do limbo, sendo raros na folha adulta. As glândulas peroladas são constituídas por uma célula basal e por uma célula apical grande cuja forma varia de semi-globóide a espatulada. A liberação da secreção ocorre após a ruptura da cutícula distendida. Os tricomas saculiformes são constituídos por uma célula basal, 1-2 células colares e uma célula apical cônica, de posição inclinada à superfície foliar. A secreção acumula-se em espaços subcuticulares diminutos, não tendo sido observada ruptura da cutícula. Piper regnellii has two types of trichomes: pearl glands and sac-like trichomes. Both secretory trichomes originate from the protoderm of the leaf primordium and reach maturity in the young leaves found in the shoot apex. The pearl glands are more abundant on the abaxial surface, while the sac-like trichomes cover completely both young leaf blade. The density of both trichomes decreases during blade expansion, being scarce in the adult leaf. The pearl glands are composed of a basal cell and an apical cell whose shape ranges from semi-globular to spatula-like. Secretion release occurs after rupture of the distended cuticle. The sac-like trichomes are composed of a basal cell, 1-2 stalked cells and an apical conical cell parallel to the leaf surface. The secretion is accumulated in minute subcuticular spaces. No cuticular ruptures have been observed. Universidade Estadual Paulista Universidade Estadual Paulista

O óleo essencial das folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck Piparaceae, coletadas no Horto de Plantas Medicinais da Universidade Estadual de Maringá, foi obtido por hidrodestilação. Uma análise preliminar por CG/EM e RMN 13C foi realizada. O b-mirceno (70%) foi identificado como componente majoritário através da comparação dos espectros de massa e RMN 13C com dados da literatura. Quatro neolignanas foram isoladas do extrato hidroetanólico das folhas e identificadas: eupomatenóide-6, eupomatenóide-5, eupomatenóide-3 e conocarpano. As estruturas dessas substâncias foram estabelecidas por meio de estudos de RMN ¹H e 13C, ¹H x ¹H - COSY, HETCOR, HMBC, gNOE e EM. The essential oil of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck Piparaceae leaves, which were collected at a tree farm named Horto de Plantas Medicinais of the Universidade Estadual de Maringá, was obtained by hydrodistillation. A preliminary analysis by GC/MS was carried out. b-mirceno (70%) was identified as the main constituent by comparing MS and 13C NMR with the literature data. Four neolignans were isolated from the leaves and identified: eupomatenoid-6, eupomatenoid-5, eupomatenoid-3 and conocarpan. Their structures were established by extensive ¹H and 13C NMR, ¹H x ¹H - COSY, HETCOR, HMBC, gNOE and MS spectral studies.