Isoquinoline alkaloids from the roots of Zanthoxylum rigidum as multi-target inhibitors of cholinesterase, monoamine oxidase A and Aß1-42 aggregation.

Multifactorial neurodegenerative disorders such as Alzheimer's disease (AD) are considered a growing public health problem due the rising incidence and low effectiveness of current treatments [6]. Since pharmacotherapy based on a single target has been insufficient for drug development in complex diseases, the emerging multi-target approach is a promising strategy for the search of new anti-AD drug candidates. Herein described natural isoquinoline alkaloids were investigated for multi-target activity on key mechanisms associated with the AD's pathogenesis, i.e. cholinergic depletion, beta amyloid (Aß) aggregation and oxidative stress. Alkaloid isolation from root extract of Zanthoxylum rigidum was carried out using multi-step chromatography and TLC-bioautography against acetylcholinesterase (AChE) giving eight purified isoquinoline alkaloids. Isolated compounds were tested for inhibitory activity against cholinesterase (AChE and BChE), monoamine oxidase (MAO-A and B) and Aß aggregation. Our study revealed two benzophenanthridine alkaloids, nitidine (5) and avicine (7), as the most potent multi-target candidates. Both showed dual cholinesterase inhibition, being more active against AChE over BChE, with IC50 values in sub-micromolar range in AChE. Kinetic analysis with cholinesterase showed, that both compounds are reversible-mixed inhibitors, where avicine (7) presented highest potency with Ki values of 0.063 µM (EeAChE), 0.511 µM (HrAChE) and 0.123 µM (EqBChE). In addition, these alkaloids presented moderate Aß1-42 anti-aggregation activity and MAO-A inhibition with IC50 values between 0.5 and 2 µM. Our findings suggest that avicine (7) is a promising natural compound and multifunctional candidate representing a suitable starting point for the development of new therapeutic agents for Alzheimer's disease.

Metabolomic profiling of Zanthoxylum species: Identification of anti-cholinesterase alkaloids candidates.

The isolation of bioactive compounds from natural sources is a key step in drug discovery and development, however, this procedure is usually expensive and difficult due to the complexity and the limited amounts of the metabolites in the extracts. Thus, rational or targeting isolations are becoming more popular to reduce the bottlenecks in bioactive natural products research. In this study, we used a LC-MS-based metabolomic approach and biochemometric statistical tools (PCA and OPLS-DA) to identify potential anti-cholinesterase alkaloids predictors in Zanthoxylum genus (Rutaceae). For this purpose, 41 alkaloid extracts from nine Colombian Zanthoxylum species were screened by UHPLC-UV-HRMS and inhibitory activity against Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE). Based on the screening results, a multivariate statistical analysis (MVA) and selection of anti-cholinesterase candidates were performed using the S-plot from the OPLS-DA model. The supervised analysis (OPLS-DA) paring the anti-cholinesterase screening and LC-HRMS data showed at least 11 ChE inhibition markers which could have contributed in the differentiation of active and inactive extracts. The predictors were tentatively identified by comparing chromatographic retention times (Rt) and accurate mass and MS2 fragmentation patterns. In general, the inhibition markers correspond to four types of isoquinoline alkaloids: tetrahydroprotoberberines, protoberberines, dihydrobenzophenanthridines and benzophenanthridines. The most active extracts from Z. schreberi and Z. monophylum showed the highest presence of berberine and chelerythrine, previously reported as cholinesterase inhibitors. Thus, to validate the results of the OPLS-DA model, three alkaloids from the bark of Z. schreberi (identified as berberine, chelerythrine and columbamine) were bio-directed isolated, and all of them showed strong inhibition against both enzymes. These findings support our statistical models and contribute to the rational search of anticholinesterase alkaloids. Therefore, LC-MS-based metabolomic approach combined with chemometric statistical analysis are shown as useful tools for the isolation of targeted bioactive natural products, contributing to improve the research and development stages of lead compounds.

New butyrolactone and other metabolites from the bark of Endlicheria arenosa against of the phytopathogen Colletotrichum tamarilloi.

In this work, screening of Lauraceae species for their antifungal activity against Collectotrichum tamarilloi was carried out and the ethanol extract derived from the bark of Endlicheria arenosa was found to be the best candidate. From the ethanolic extract of the bark of E. arenosa, the hexane and chloroform fractions were found to be active, from these five fatty acids were identified and two lactones were isolated. The most active fatty acid was the dodecanoic acid with a minimal inhibitory concentration (MIC) of 78.0 µM. The butyrolactone 3R,4R-licunolide A, it has not previously reported, and licunolide B show both the lowest MIC (55.3 µM). This is the first report of compounds of natural origin as growth inhibitors of C. tamarilloi.

Anticonvulsant profile of 2-ethylthio-7-methyl-4-(4-methylphenyl)pyrazolo[1, 5-a][1, 3, 5]triazine

This work evaluates the central nervous effects in ICR strain mice of 2-ethylthio-7-methyl-4-(4-methylphenyl)pyrazolo[1,5-a][1,3,5]triazine (MH4b1), a compound obtained by an efficient one-step reaction of S,S-diethyl 4-methylbenzoylimidodithiocarbonate with 5-amino-3-methyl-1H-pyrazole, in order to assess its neuro-pharmacological profile. The tests applied were: maximal electroshock seizure (MES), pentylenetetrazole (PTZ) seizures, forced swimming, plus maze, marble burying, sleeping time, rota-rod and catalepsy. In addition, MH4b1 binding to the benzodiazepine site of the GABA-A receptor and MH4b1 inhibition of monoamine oxidase (MAO) subtypes A and B were evaluated. MH4b1 showed anticonvulsant effects in a dose dependent manner (30-300 mg/kg, p.o.) against MES and inhibition of MAO-B (IC50: 24.5 µM) without activity at the benzodiazepine site. These data suggest that MH4b1 has anticonvulsant properties related to MAO-B inhibition.

Este trabalho avalia o efeito do 2-etiltio-7-metil-4-(4-metilfenil)pirazol[1,5-a][1,3,5]triazina (MH4b1) no sistema nervoso central de camundongos ICR. O MH4b1 foi obtido por a reação de 4-metilbenzoilimidoditiocarbonato de S,S-dietil e 5-amino-3-metil-1H-pirazol em uma única etapa. O perfil neurofarmacológico foi realizado por testes de convulsão induzida por eletrochoque (MES) e pentilenotetrazol (PTZ) e por testes de nado forçado, labirinto em cruz, esconder as esferas, sono barbitúrico, rota-rod e catalepsia. Também foi avaliada a união do MH4b1 ao o local de ligação de benzodiazepínicos do receptor GABA-A e a capacidade inibitória do MH4b1 sobre a monoaminoxidase (MAO) A e B. O MH4b1 mostrou efeito anticonvulsivante dependente da dose (30-300 mg) no teste do MES e apresentou atividade inibitória da MAO-B (CI50: 24.5 µM) sem interagir com o local de ligação de benzodiazepínicos do receptor. Os resultados sugerem que o MH4b1 tem atividade anticonvulsivante relacionada com a inibição da MAO-B.

Croton schiedeanus Schltd prevents experimental hypertension in rats induced by nitric oxide deficit

Croton schiedeanus Schltd (N.V.: "almizclillo") is a plant used in traditional medicine as an antihypertensive in Colombia. It contains flavonoid, diterpenoid and fenilbutanoid metabolites that have vasodilatation effects linked to the NO/cGMP pathway. This work aimed to assess the capacity of a 96% EtOH extract to prevent the hypertension induced by nitric oxide (NO) deficiency in rats. The NO synthase inhibitor L-NAME (10 mg/kg/d, i.p) was administered during five weeks to three groups of rats (6-7 animals): C. Schiedeanus (200 mg/kg/d, p.o), enalapril (reference, 10 mg/kg/d, p.o) and vehicle (control: olive oil 1 ml/kg/d, p.o). In addition, the blank group received only vehicle. The arterial blood pressure (BP) and heart rate (HR) were measured daily for six weeks. After sacrificing the animals, the aortic rings were isolated, contraction was triggered with phenylephrine (PE 10-6 M) and relaxant responses were achieved with cumulative concentrations of acetylcholine (ACh, 10-10 - 10-4 M). L-NAME increased the systolic arterial pressure in the control group, attaining mean values of 131 mm Hg at week 5, whereas the C. schiedeanus, enalapril and blank groups maintained blood pressure under 100 mm Hg. The capacity of PE to contract aortic rings was greater in the C. schiedeanus, enalapril and blank groups than in the control group (2157, 2005, 1910 and 1646 mg, respectively). The pEC50 values for ACh were as follows: C. Schiedeanus (6.89) >enalapril (6.39) > blank (5.68) > control (5.09). These results give support to C. Schiedeanus as a natural antihypertensive source.

Croton schiedeanus Schltd (NV: "almizclillo") é utilizado na medicina tradicional da Colômbia para o tratamento da hipertensão arterial. Outras pesquisas demonstraram que a planta tem metabólitos como os flavonoides, os diterpenoides e os fenilbutanoides, os quais têm comprovados efeitos vasodilatadores vinculados com a via NO/GMPc. O objetivo deste estudo foi avaliar a capacidade do extrato de Croton schiedeanus Schltd em EtOH a 96% na prevenção da hipertensão induzida pela deficiência de óxido nítrico (NO), em ratos. O inibidor da NO sintetase L-NAME (10 mg/kg/d, ip) foi administrado durante cinco semanas em três grupos de ratos (6-7 animais): C. schiedeanus (200 mg/kg/d, v.o.), enalapril (referência, 10 mg/kg/d, v.o.) e o veículo (controle: azeite de oliva 1 mL/kg/d, v.o.). O grupo branco recebeu somente o veículo. A pressão sanguínea (BP) e a frequência cardíaca (FC) foram medidas diariamente em um período de seis semanas. Após o sacrifício, os anéis aórticos foram isolados e contraídos, utilizando fenilefrina (PE 10-6 M) e as respostas para a relaxação foram obtidas com doses acumulativas de acetilcolina (ACh, 10-10-10-4 M). Os resultados demonstraram que o L-NAME provocou incremento significativo da pressão nos ratos do grupo controle, obtendo-se valores médios de 131 mm Hg na quinta semana. No entanto, os grupos C. schiedeanus, enalapril e branco mantiveram a pressão arterial aos níveis médios iniciais 100 mm Hg. A capacidade da PE para fazer a contração dos anéis da aorta foi maior nos grupos C. schiedeanus, enalapril e branco do que no grupo controle (2157, 2005, 1910 and 1646 mg, respectivamente). Os valores de pCE50 de ACh foram os seguintes: C. schiedeanus (6,89) > enalapril (6,39) > branco (5,68) > controle (5,09). Pode-se afirmar que estes resultados dão suporte à C. schiedeanus como fonte natural anti-hipertensiva.

Efecto citotóxico de algunos compuestos naturales aislados de plantas Laureaceae y derivados sintéticos / Cytotoxic effect of some natural compounds isolated from Lauraceae plants and synthetic derivatives

Introducción. El efecto contra la proliferación celular de once neolignanos, dos lignanos y un diterpeno, aislados de tres plantas de la familia Lauraceae, y cuatro benzofuranos y dos biciclooctanos sintéticos, fue evaluado in vitro sobre cinco líneas celulares derivadas de tumores sólidos de alta incidencia en Colombia.Objetivo. Evaluar el efecto citotóxico de veinte compuestos sobre las líneas tumorales HeLa, A-549, Hep-2, PC-3 y MCF-7. Materiales y métodos. Los 14 compuestos de origen natural fueron aislados de tres plantas nativas colombianas (Pleurothyrium cinereum, Ocotea macrophylla y Nectandra amazonum) por técnicas cromatográficas y se establecieron sus estructuras por métodos espectroscópicos, y los seis derivados sintéticos fueron preparados mediante reacción de oxiarilación y metilación con diazometano. El efecto contra la proliferación y la recuperación celular se hicieron mediante tratamiento in vitro de las líneas tumorales con los compuestos , evaluando la viabilidad celular por tinción con resazurina.Resultados. Entre los compuestos evaluados, solamente ocofilal A, cinerina D, ácido kaurenoico, dos benzofuranos y la (-)-cinerina A sintética presentaron actividad contra la proliferación celular en diferentes niveles. Los biciclooctanos, así como el ácido kaurenoico, fueron activos contra todas las líneas celulares, mientras que los benzofuranoides mostraron actividad selectiva contra HeLa. Además, la (-)-cinerina A exhibió un efecto letal total contra todas las líneas celulares, mientras que el ácido kaurenóico presentó efecto letal total contra PC-3, Hep-2 y A549.Conclusión. Los compuestos evaluados que exhibieron actividad contra la proliferación celular mostraron resultados interesantes, lo cual sugiere su potencial uso como cabezas de serie o moléculas plantilla en el desarrollo de agentes anticancerígenos.

Introduction. The antiproliferative effect of eleven neolignans, two lignans and one diterpene isolated from three Lauraceae plants, four benzofurans and two bicyclooctanes synthetic derivatives was evaluated in vitro on a set of five human cancer cells from solid tumors with a high incidence in Colombia.Objective. To evaluate the cytotoxic effect of twenty compounds on the tumor cell lines HeLa, A-549, Hep-2, PC-3, and MCF-7.Materials and methods. Fourteen natural compounds were isolated by chromatographic techniques from three native colombian plants (Pleurothyrium cinereum, Ocotea macrophylla and Nectandra amazonum), whose structures were established by spectroscopic methods; six synthetic derivatives were prepared by oxyarylation and diazomethane methylation. Antiproliferative effect and cell recovery were performed by means of in vitro treatment of tumor cell lines with test compounds, evaluating cell viability by resazurin staining.Results. Among test compounds, only neolignans ocophyllal A, cinerin D, kaurenoic acid, two benzofuran-derivatives, and synthetic (-)-cinerin A were found to have antiproliferative effect at different levels. Bicyclooctanoids as well as kaurenoic acid exhibited activity against all human cancer cells while benzofuranoids showed selective activity against HeLa. Furthermore, compounds (-)-cinerin A and kaurenoic acid exhibited total lethal effect against all-five cell lines and PC-3, Hep-2, and A549 cell lines, respectively.Conclusion. Test compounds exhibiting antiproliferative activity showed interesting results, which would promote their use as lead compounds on further studies for anticancer agents development.

Leishmanicidal and cytotoxic activities of extracts and naturally-occurring compounds from two Lauraceae species.

The in vitro leishmanicidal effects of ethanolic extracts and fifteen naturally-occurring compounds (five lignans, eight neolignans, a diterpene and a dihydrochalcone), obtained from Pleurothyrium cinereum and Ocotea macrophylla, were evaluated on promastigotes of Leishmania panamensis and L. braziliensis. In addition, in order to determine the selective action on Leishmania species as a safety principle, in vitro cytotoxicity on J774 cells was also evaluated for test compounds and extracts. One extract and seven compounds showed activity against Leishmania parasites at different levels. Dihydroflavokawin B (8) was found to be the most potent antileishmanial compound on both parasites, whilst (+)-otobaphenol (14), was found to be the most selective compound on L. panamensis.

Cytotoxic effect of some natural compounds isolated from Lauraceae plants and synthetic derivatives.

INTRODUCTION: The antiproliferative effect of eleven neolignans, two lignans and one diterpene isolated from three Lauraceae plants, four benzofurans and two bicyclooctanes synthetic derivatives was evaluated in vitro on a set of five human cancer cells from solid tumors with a high incidence in Colombia. OBJECTIVE: To evaluate the cytotoxic effect of twenty compounds on the tumor cell lines HeLa, A-549, Hep-2, PC-3, and MCF-7. MATERIALS AND METHODS. Fourteen natural compounds were isolated by chromatographic techniques from three native Colombian plants (Pleurothyrium cinereum, Ocotea macrophylla and Nectandra amazonum), whose structures were established by spectroscopic methods; six synthetic derivatives were prepared by oxyarylation and diazomethane methylation. Antiproliferative effect and cell recovery were performed by means of in vitro treatment of tumor cell lines with test compounds, evaluating cell viability by resazurin staining. RESULTS: Among test compounds, only neolignans ocophyllal A, cinerin D, kaurenoic acid, two benzofuran-derivatives, and synthetic (-)-cinerin A were found to have antiproliferative effect at different levels. Bicyclooctanoids as well as kaurenoic acid exhibited activity against all human cancer cells while benzofuranoids showed selective activity against HeLa. Furthermore, compounds (-)-cinerin A and kaurenoic acid exhibited total lethal effect against all-five cell lines and PC-3, Hep-2, and A549 cell lines, respectively. CONCLUSION: Test compounds exhibiting antiproliferative activity showed interesting results, which would promote their use as lead compounds on further studies for anticancer agents development.

Evaluación in vitro de la actividad de aceites esenciales de plantas colombianas sobre Leishmania braziliensis / In vitro evaluation of essentials oils activity from Colombians plants on Leishmania braziliensis

Las leishmaniosis son zoonosis que en el hospedero humano pueden afectar la piel, las mucosas o las vísceras, resultado del parasitismo de los macrófagos por un protozoario flagelado del género Leishmania, introducido al organismo por la picadura de un insecto flebótomo. En Colombia, la leishmaniosis cutánea es causada por parásitos del subgénero Viannia, al cual pertenecen las especies Leishmania (L) panamensis, L. braziliensis y L. guyanensis. El tratamiento clínico requiere la administración de medicamentos que, si bien son efectivos, generan efectos adversos en el individuo. Además, se ha detectado una resistencia del parásito, lo que ha favorecido la disminución de la eficacia de los tratamientos usados convencionalmente para controlar la enfermedad. En la búsqueda de nuevas y más seguras alternativas terapéuticas, los productos naturales son una fuente importante de agentes con potencial actividad antileishmanial. Éste es el caso de los aceites esenciales que por sus características físico-químicas, principalmente por su untuosidad, pueden constituirse en una alternativa tópica para el control de la leishmaniosis cutánea (lc). En este estudio, la actividad antileishmanial de trece aceites esenciales de diferentes familias de plantas colombianas (Lauraceae, Rutaceae, Verbenaceae, Lamiaceae, Zingiberaceae, Myristicaceae, Cardiopteridaceae y Pinaceae) se evaluó sobre los promastigotes de L. braziliensis. Si bien, es necesaria la valoración del efecto citotóxico que sobre los macrófagos, como células blanco de la infección por Leishmania, puedan generar estos aceites; resulta promisoria la actividad antileishmanial directa sobre las formas promastigotes, la cual puede ser comparable con la observada para pentamidina, fármaco que a diferencia de las sales antimoniales pentavalentes, no es una prodroga que requiera ser descompuesta en metabolitos activos una vez es incorporada al macrófago.

Leishmaniasis is zoonosis that in human host can affect skin, mucosa and viscera, when a flagelar protozoo is phagocyted by macrophages after bit of Phlebotomineo insect. In Colombia, cutaneous leishmaniasis is produced by subgenus Viannia parasites, genus that includes species like Leishmania panamensis (L), L. braziliensis y L. guyanensis. Clinical treatment requires the administration of effective drugs that induce severe adverse effects in the patient treated. Additionally, the parasite had generated a drug-resistance, which promotes a reduction in this kind of therapeutic schedule. In the searching of new and safer therapeutics alternatives to leishmaniasis control, natural products are a important source of active molecules with pharmacological activity. In this study we reported the antileishmanial activity of 13 essentials oils of different Colombians plants (Lauraceae, Rutaceae, Verbenaceae, Lamiaceae, Zingiberaceae, Myristicaceae, Cardiopteridaceae y Pinaceae) using L. braziliensis promastigotes. The effective concentration 50 (EC50) was 87,8 +/- 55,51 μg/mL, 265,5 +/- 7,5 μg/mL and 17,4 +/- 0,43 μg/mL for essentials oils of Persea caerulea fruit, Lippia alba leaves and Rosmarinus officinalis leaves, respectively, which permit suggest us, a parasitocide property. Although, it is necessary the cytotoxic effect study on macrophages as infection target cells, result promissory the antileishmanial effect direct to promastigote form, which can be comparable with the effect observed to pentamidine, drugs to a difference of antimonial drugs, is not a prodrug that require to be metabolized into macrophage. In other words, essentials oils would contain active principles lethal against free forms and maybe, against intracellular Leishmania spp forms that need further studies to be considered as therapeutic alternative to leishmaniasis cutaneous control.

COX, LOX and platelet aggregation inhibitory properties of Lauraceae neolignans.

The anti-inflammatory potential of 26 neolignans (14 of the bicyclooctane-type and 12 of the benzofuran-type), isolated from three Lauraceae species (Pleurothyrium cinereum, Ocotea macrophylla and Nectandra amazonum), was evaluated in vitro through inhibition of COX-1, COX-2, 5-LOX and agonist-induced aggregation of rabbit platelets. Benzofuran neolignans were found to be selective COX-2 inhibitors, whereas bicyclooctane neolignans inhibit selectively the PAF-action as well as COX-1 and 5-LOX. The neolignan 9-nor-7,8-dehydro-isolicarin B 15 and cinerin C 7 were found to be the most potent COX-2 inhibitor and PAF-antagonist, respectively. Nectamazin C 10 exhibited dual 5-LOX/COX-2 inhibition.