Química y actividad biológica en Huperzia saururus. Chemistry and biological activity in Huperzia saururus.

El presente proyecto apunta a buscar una posible solución a dos problemáticas de salud que afectan a un importante grupo de la población mundial y son los asociados a la enfermedad de Alzheimer (EA) y a la disfunción eréctil. La EA es una enfermedad neurodegenerativa progresiva caracterizada por la pérdida de memoria a corto plazo, entre otras manifestaciones. En la actualidad la terapia más utilizada son los inhibidores de la acetilcolinesterasa (AChE), ya que acetilcolina es el principal neurotransmisor afectado. No obstante, los agentes terapéuticos utilizados presentan efectos no deseados a nivel gastrointestinal, lo que motiva la búsqueda de nuevos agentes. A partir del estudio de la especie autóctona Huperzia saururus aislamos hasta el presente 10 alcaloides, algunos de los cuales resultaron activos sobre la retención de memoria, investigación realizada mediante ensayos tanto in vitro como in vivo. A partir de los promisorios resultados obtenidos se continuarán profundizando estos estudios y también se buscará el farmacóforo, la parte de la estructura responsable de la actividad. Para ello, se realizarán modificaciones químicas de los grupos funcionales y los productos obtenidos serán igualmente evaluados por su actividad sobre memoria. Los que resulten activos serán investigados en relación a su toxicidad, en la búsqueda de potenciales agentes terapéuticos. Paralelamente se estudiarán las diferentes familias de compuestos presentes en la especie, dado que el uso popular proclama su efecto afrodisíaco. La búsqueda de un afrodisíaco perfecto que incremente el deseo sexual, el placer y el desempeño, ha sido una constante desde los tiempos remotos. En todos los casos, se ha buscado mejorar el rendimiento sexual o combatir la disfunción eréctil (DE). Este término se describe como “la incapacidad constante para alcanzar o mantener una erección que es satisfactoria para el rendimiento sexual”. Para investigar la actividad biológica de H. saururus, los extractos y compuestos aislados serán evaluados por la generación de NO que produzcan, la que será estimada mediante la acumulación de nitrito, metabolito estable del NO, en sobrenadantes de cultivo de células endoteliales (HUVEC). Los que resulten efectivos serán evaluados mediante ensayos in vivo tales como, el de conducta sexual en machos y de comportamiento de monta. Del mismo modo que para los alcaloides, se estudiará la toxicidad de los compuestos que resulten activos a los fines de proseguir la metodología en la investigación preclínica de potenciales medicamentos. The present Project aims to look for a possible solution for two health problems that affect an important group of world population and they are those associated to Alzheimer Disease (AD) and Erectile Dysfunction. AD is a neurodegenerative progressive disease characterized by short term memory loss, among other signs. Nowadays the most used therapy are the acethylcholinesterase inhibitors (AChE), in view that the acethylcholine is the main neurotransmitter affected. Nevertheless, the therapeutic agents used present collateral effects at gastrointestinal level, so it motivates the search for new agents. From the autochthonous species Huperzia saururus we have isolated 10 alkaloids until the present, some of which were active on memory retention, investigation developed in vitro and in vivo. After these promising results, these studies will be continued, as well as the search for the pharmacophore. For this reason, chemical modifications on functional groups will be done and the obtained products will be evaluated on their activity on memory as well. In line with this, the different families of compounds present in H. saururus will be studied in view that the popular use claims its aphrodisiac effect. The search for a perfect aphrodisiac that increase the sexual desire, the pleasure and performance was a constant since the old times. In all the cases it was looked a better sexual performance or fight against the erectile dysfunction (DE). For investigate the biological activity of H. saururus, the extracts and the isolated compounds will be evaluated on the NO generation, which will be estimated by means of nitrite accumulation, stable metabolite of NO, in supernatants of endothelial cell cultures (HUVEC). The compounds that appear effectives will be evaluated by in vivo assays such as sexual male behavior, and mount behavior. As for alkaloids, toxicity of active compounds will be studied to follow the pre-clinic methodology research of potential medicines.

Natural aphrodisiacs: studies of commercially-available herbal recipes, and phytochemical investigation of "Erythroxylum vacciniifolium" Mart. (Erythroxylaceae) from Brazil

De tout temps, hommes et femmes ont cherché par tous les moyens à développer, préserver ou recouvrer leurs propres capacités sexuelles mais également à stimuler le désir du partenaire. L?utilisation d?aphrodisiaques naturels a été l?un des recours les plus répandus. De nos jours, la commercialisation de nouvelles "love drugs" de synthèse, e.g. Viagra®, Cialis®, Levitra®, a remis au goût du jour les aphrodisiaques classiques et à relancer la recherche sur des molécules nouvelles. La pratique croissante de l?automédication, le matraquage publicitaire sur les aphrodisiaques naturels, la prolifération sur le marché de compléments alimentaires non contrôlés et l?absence de véritable législation accroissent les risques qui pèsent sur la santé publique. Dans le but d?évaluer les risques potentiels sur le consommateur de produits aphrodisiaques commercialisés, le développement et la validation d?une méthode rapide d?analyse qualitative et quantitative de la yohimbine dans ces préparations du marché sont exposés dans la première partie de ce travail. La yohimbine est un antagoniste ?2-adrénocepteur du système nerveux central et périphérique, elle est employée depuis plus d?un siècle dans le traitement des dysfonctionnements érectiles. Cette méthode analytique utilise la chromatographie liquide couplée à l?ultraviolet et à la spectrométrie de masse (LC-UV-MS) et au total, vingt préparations aphrodisiaques ont été étudiées. La dose journalière de yohimbine mesurée s?est révélée très variable selon les produits puisqu?elle varie de 1.32 à 23.16 mg. La seconde partie de ce travail concerne l?étude phytochimique et pharmacologique d?Erythroxylum vacciniifolium Mart. (Erythroxylaceae), une plante, appelée localement catuaba, utilisée dans la médecine traditionnelle brésilienne comme tonique et aphrodisiaque. Dans un premier temps, l?extrait alcaloïdique a été analysé par chromatographie liquide haute performance (HPLC) couplée soit à un détecteur UV à barrette d?iode (LC-UV-DAD), soit à un spectromètre de masse (LC-MS), ou soit à un spectromètre de résonance magnétique nucléaire (LC-RMN). L?interprétation de ces données spectrales enregistrées en ligne a permis d?obtenir des informations structurales et d?identifier partiellement près de 24 alcaloïdes appartenant à la classe des tropanes et potentiellement originaux. Par des méthodes classiques de chromatographie liquide sur l?extrait alcaloïdique de la plante, dix sept tropanes nouveaux ont ensuite été isolés dont les catuabines et leurs dérivés, et les vaccinines. Tous ces composés sont des tropane-diols ou triols estérifiés par au moins un groupe acide 1-méthyl-1H-pyrrole-2-carboxylique. Un de ces composés a été identifié comme un tropane N-oxyde. Toutes les structures ont été déterminées par spectrométrie de masse haute résolution et spectroscopie RMN multi-dimensionnelle. Parmi les nombreux tests biologiques réalisés sur ces tropanes, seuls les tests de cytotoxicité se sont révélés faiblement positifs pour certains de ces composés.<br/><br/>Throughout the ages, men and women have incessantly pursued every means to increase, preserve or recapture their sexual capacity, or to stimulate the sexual desire of selected individuals. One of the most recurrent methods has been the use of natural aphrodisiacs. Nowadays, the commercialization of new synthetic "love drugs", e.g. Viagra®, Cialis® and Levitra®, has fascinated the public interest and has led to a reassessment of classical aphrodisiacs and to the search for new ones. The practice of self-medication by an increasing number of patients, the incessant aggressive advertising of these herbal aphrodisiacs, the invasion of the medicinal market with uncontrolled dietary supplements and the absence of real directives amplifies the potential health hazards to the community. In order to evaluate the possible risks of commercialized aphrodisiac products on consumer health, the development and validation of a rapid qualitative and quantitative method for the analysis of yohimbine in these products, is reported in the first part of the present work. Yohimbine, a pharmacologically well-characterized ?2-adrenoceptor antagonist with activity in the central and peripheral nervous system, has been used for over a century in the treatment of erectile dysfunction. The analytical method is based on liquid chromatography coupled with ultraviolet and mass spectrometry (LC-UV-MS) and in total, 20 commercially-available aphrodisiac preparations were analyzed. The amount of yohimbine measured and expressed as the maximal dose per day suggested on product labels ranged from 1.32 to 23.16 mg. The second part of this work involved the phytochemical and pharmacological investigation of Erythroxylum vacciniifolium Mart. (Erythroxylaceae), a plant used in Brazilian traditional medicine as an aphrodisiac and tonic, and locally known as catuaba. With the aim of obtaining preliminary structure information on-line, the alkaloid extract was analyzed by high performance liquid chromatography (HPLC) coupled to diode array UV detection (LC-UVDAD), to mass spectrometry (LC-MS) and to nuclear magnetic resonance spectroscopy (LCNMR). Interpretation of on-line spectroscopic data led to structure elucidation and partial identification of 24 potentially original alkaloids bearing the same tropane skeleton. Seventeen new tropane alkaloids were then isolated from the alkaloid extract of the plant, including catuabines D to I, their derivatives and vaccinines A and B. All compounds were elucidated as tropane-diol or -triol alkaloids esterified by at least one 1-methyl-1H-pyrrole-2-carboxylic acid. One of the isolated compounds was identified as a tropane alkaloid N-oxide. Their structures were determined by high resolution mass spectrometry and multi-dimensional NMR spectroscopy. Among the numerous bioassays undertaken, only the cytotoxicity tests exhibited a weak positive activity of certain compounds.

Influência de diferentes meios de cultura sobre o crescimento de Pfaffia glomerata (Spreng.) Pedersen (Amaranthaceae) para conservação in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Validated high-performance liquid chromatographic method for the standardisation of Ptychopetalum olacoides Benth., Olacaceae, commercial extracts

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Controle de qualidade dos extratos polares de Turnera diffusa Willd. ex Schult., Turneraceae

Este trabalho descreve o desenvolvimento de metodologia para o controle de qualidade dos extratos etanólicos e infusão de Turnera diffusa Willd. ex Schult., Turneraceae, usando como marcadores os compostos fenólicos. Essa espécie é usada popularmente como afrodisíaca e antiulcerogênica. Foram usadas técnicas cromatográficas: CLAE acoplada com detector de ultravioleta e cromatografia em camada delgada. Foram detectados flavonóides como os principais componentes da planta.

Sazonalidade na produção de raízes e teor de β-ecdisona em acessos de fáfia

As raízes de espécies de Pfaffia são utilizadas há muito na medicina popular no Brasil, especialmente como tônicas, afrodisíacas e antidiabéticas. Nas margens e ilhas do Rio Paraná, vegeta naturalmente a espécie Pfaffia glomerata (Spreng.) Pedersen, que corre risco de extinção pela coleta intensiva de suas raízes. Para preservá-la, é preciso desenvolver sistemas que permitam seu uso sustentável. Nesse contexto, foi avaliada a sazonalidade da produção de biomassa e o teor de β-ecdisona em raízes de diferentes acessos de P. glomerata. A coleta dos acessos foi realizada ao longo dos rios Paraná (A1 e A3), Ivaí (A2) e Paranapanema (A4). Mudas produzidas a partir desses acessos foram plantadas experimentalmente em área de ocorrência natural da espécie. O delineamento utilizado foi blocos casualizados, com parcelas subdivididas (acessos nas parcelas; épocas de colheita nas subparcelas), quatro repetições e subparcelas com seis plantas na área útil. Foram realizadas quatro colheitas: oito, dez, doze e quatorze meses após o plantio. As maiores produtividades em massa seca (MS) de raízes foram alcançadas na terceira colheita, com 38,41 g planta-1. Já o acesso A4 foi significativamente mais produtivo que os demais, tanto para massa fresca (94,67 g planta-1), quanto para MS de raízes (26,39 g planta-1). A relação MF/MS das raízes, com média de 26,9% (3,7:1), foi pouco influenciada pela origem dos acessos, embora tenha se alterado significativamente ao longo das colheitas (de 4,1:1, na primeira colheita, a 3,3:1, na terceira colheita, que correspondeu ao valor máximo). O teor de β-ecdisona foi estatisticamente similar nas três primeiras colheitas, variando de 0,26 a 0,38%, com redução drástica na última colheita (0,16%). Os acessos A2 e A3 foram aqueles com teor de β-ecdisona mais altos, respectivamente 0,36 e 0,30%. Considerando os resultados obtidos, sugere-se que a terceira colheita, que corresponde a doze meses após o transplante, como a mais adequada para colheita das raízes.

Application of low-pressure gas chromatography-ion-trap mass spectrometry to the analysis of the essential oil of Turnera diffusa (Ward.) Urb.

Turnera diffusa Willd. var. afrodisiaca (Ward) Urb. (syn. T. aphrodisiaca) belongs to the family of Turneraceae and is an aromatic plant growing wild in the subtropical regions of America and Africa. It is widely used in the traditional medicine as e.g. anti-cough, diuretic, and aphrodisiac agent. This work presents a 3 min chromatographic analysis using low-pressure (LP) gas chromatography (GC)-ion-trap (IT) mass spectrometry (MS). The combination of a deactivated 0.6 m x 0.10 mm i.d., restrictor with a wide-bore CP-Wax 52 capillary column (10 m x 0.53 mm i.d., 1 mum) reduces the analysis time by a factor of 3-7 in comparison to the use of a conventional narrow bore column. Chromatographic conditions have been optimized to achieve the fastest separation with the highest signal/noise ratio in MS detection. These results allow fast and reliable quality control of the essential oil to be achieved. (C) 2003 Elsevier B.V. All rights reserved.

Development of an analytical method for the quantification of pfaffic acid in Brazilian ginseng (Hebanthe eriantha)

Hebanthe eriantha (Poir.) Pedersen (Amaranthaceae), which is known as Brazilian ginseng is widely used in folk medicine as an aphrodisiac and antidiabetic tonic. The anti-tumor activity, attributed to the pfaffic acid present in roots of H. eriantha, is responsible for the great interest in the commercialization of this species. In Brazil, the species H. eriantha is mainly used in commercial preparations, although other plants of the genus Pfaffia and Hebanthe have been marketed as Pfaffia paniculata or Brazilian ginseng. The pfaffic acid present in the roots is mainly conjugated with sugars (pfaffosides) and can be used as an active marker of H. eriantha, which helps to differentiate this species from others marketed as Brazilian ginseng. The main objective of this study was to develop and validate a liquid chromatographic method to quantify pfaffic acid in the roots of H. eriantha. The extraction and hydrolysis conditions were optimized using an univariate and experimental design, respectively, and the quantification of pfaffic acid by high performance liquid chromatography with diode-array detection (HPLC-DAD) was validated. This method was used to evaluate the pfaffic acid content in 30 different genotypes of the species from a germplasm collection. The content of pfaffic acid ranged from 0.97 to 4.29% (w/w) on a dry weight basis. © 2013 Elsevier B.V.

Avaliação da atividade anti-inflamatória intestinal de Pfaffia glomerata (Spreng.) Pedersen

Pós-graduação em Ciências Biológicas (Farmacologia) - IBB

Propagação assexuada e desenvolvimento inicial de mudas propagadas sexuadamente de Ficus adhatodifolia Schott ex Spreng. (Moraceae)

Pós-graduação em Agronomia (Horticultura) - FCA

Study of the ventral prostate of Wistar rats treated with Heteropterys tomentosa (A. Juss.)

Heteropterys tomentosa is a Brazilian plant traditionally used as an aphrodisiac and stimulant. Previous studies suggested possible androgenic and antioxidant effects after long term administration of H. tomentosa infusion. The aim of the present study was to evaluate the effects of this plant infusion on the rat ventral prostate: an androgen responsive organ. Wistar rats were treated, by gavage, with H. tomentosa roots infusion (treated group, n=6) or water (control group, n=6) for 56 days. Morphological, morphometrical and stereological analyses were employed to study the ventral prostate tissue, as well as androgen receptor and apoptotic cell staining. The ultrastructure of the prostatic epithelium was also analyzed. No alteration was observed in the stereological and morphometrical analyses. The pattern of androgen receptor expression and the apoptotic index were identical in the control and treated group. Ultrastructural analysis showed no alterations caused by H. tomentosa. These results suggested that treatment with H. tomentosa infusion, although considered a strong aphrodisiac, did not cause any major damage nor benefit to the prostate tissue.

La palmera datilera y la palmera canaria en la medicina tradicional de España

En la actualidad son muy pocos los usos vigentes. Aunque los dátiles son la materia utilizada con mayor frecuencia, también se han empleado la savia, el polen y el cogollo tierno o palmito. Los dátiles de Phoenix dactylifera se utilizaron como analgésico y para tratar la anemia y trastornos digestivos, o para fortalecer las encías, en el tratamiento de la disfunción eréctil y como afrodisiacos, para facilitar el parto y calmar los dolores postparto, y tratar el prolapso de la matriz o para el exceso de flujo menstrual. También se utilizaron como diuréticos, para la disuria y en trastornos de la vejiga. El uso que más claramente ha persistido es el tratamiento de diversos problemas respiratorios. En uso externo se utilizaron para tratar problemas de la piel, heridas, hemorragias y hemorroides. De la palmera de Canarias (Phoenix canariensis), especialmente en la isla de la Gomera, la savia cruda o guarapo, su concentrado o miel de palma y los resultantes de su fermentación (vino de palma) se consumen como alimento y también se utilizan como diurético, remedio de trastornos génitourinarios, digestivo, para infecciones de la cavidad bucal, expectorante, antitusígeno y para las irritaciones de garganta. En el Toledo de Al-Andalus las espatas de P. dactylifera se utilizaron, hace casi mil años, en el tratamiento de la debilidad, los dolores, nefritis, las enfermedades de la vejiga, trastornos hepáticos (también como preventivo), diarrea, trastornos digestivos, dolores en el abdomen y en el estómago, excesivo sangrado menstrual, úlceras en la piel y sarna, dolores articulares y trastornos cardiacos. La fitoterapia racional debería prestar atención a este recurso, considerar la evidencia científica disponible (farmacológica e incluso clínica) e incorporarlo a nuestro repertorio terapéutico.

Extinciones históricas: el caso de la enigmática planta del Silfio

This review refers to the Silphium (Apiaceae), one of the most enigmatic plants in the history of the Mediterranean. In Greco-Roman world, it was a panacea and especially, a powerful aphrodisiac which left many written historical references, in addition to their image mosaics and coins. Silphium extinction, due to over-exploitation is certainly a good example for the conservation of biodiversity and for the defense of sustainable use of natural resources.

Avaliação dos efeitos tóxicos in vitro e in vivo do extrato hidroetanólico dos frutos de Genipa americana L. (Rubiaceae) em camundongos Swiss

The therapeutic use of medicinal plants has contributed since antiquity in a beneficial way for health. However, many species lacks of scientific evidence which provide basis for their use in therapeutic practice. In this context is the Genipa americana L. species (Rubiaceae), popularly known as jenipapo and used to treat syfilis, ulcer and hemorrhagic disturbs. It's also used against bruising, as tonic and as aphrodisiac. Due this species lacks toxicological studies, the aim of this study was to evaluate the toxicity in vivo (acute and sub-chronic toxicity) and in vitro (cytotoxicity) of the hydroethanolic extract from G. americana fruits. The hydroethanolic extract of G. americana fruits was prepared by maceration. A preliminary phytochemical analysis was performed to assess the presence of secondary metabolites in the extract. The cytotoxicity study of the extract (0.1, 1.0, 10, 100 and 1000 mg / 100 ul) were performed against normal cells (3T3) and tumor (786-0, HepG2 and B16), analyzed by the MTT assay. To evaluate the acute (single dose of 2000 mg / Kg) and subchronic (100, 500 and 1000 mg / kg for 30 days) toxicity Swiss mice of both sexes were used. At the end of the experiment, blood samples and organs were collected for analysis. Data between groups were compared by t test or ANOVA with Dunnett's post-test with 5% significance level. The phytochemical study of the extracts mainly indicated the presence of iridoids. Results for cytotoxicity tests showed up to 70% inhibition of B16 cell line at a dose of 1000 mg / 100 ul, and up to 29% inhibition of 786-0 at a dose of 10 ug / 100 ul. The extract did not cause death in 3T3 and HepG2 cells. During the in vivo assays, there were no animal deaths. Analysis of blood samples revealed that the animals submitted to the evaluation of acute toxicity had changes in AST and ALT, and that the animals evaluated for subchronic toxicity showed changes in the relative wet weight of the kidney and plasma urea concentration. No differences were observed between groups on histopathological evaluation of the collected organs. Despite the changes found in the in vivo toxicity tests, using the criteria described by the OECD Guidelines, it is suggested that the hydroethanolic extract of the fruits of the G. americana is classified as low toxicity. The cytotoxicity of the extract suggests that they have potential against melanoma cell lines (B16).