Plantas Iberoamericanas y Del Caribe

Publicada por | Published by: Cooperación Latinoamericana y Caribeña en Plantas Medicinales y Aromáticas

Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas ISSN 0717 7917

Aloysia triphylla Volumen 9, Número 1, Enero de 2010 Revisiones | Reviews

− De Souza et al. (Brasil) Produtos Naturais com atividade inibitória da Translocase I, uma promissora classe de compostos contra tuberculose. Artículos | Articles

− Domínguez-Ortiz et al. (Mexico) Antioxidant and anti-inflammatory activity of Moussonia deppeana. − Ascari et al. (Brasil) Phytochemical and biological investigations of Caryocar brasiliense Camb. − Oliva et al. (Argentina) Antimicrobial activity of essential oils of Aloysia triphylla (L`Her.) Britton from different regions of Argentina. − Cortadi et al. (Argentina) Estudio farmacobotánico de hojas, cortezas y leños de Simaroubaceae sensu lato de Argentina. Parte I. Alvaradoa subovata Cronquist, Picramnia

parvifolia Engl., Picramnia sellowii Planch. y Castela coccinea Griseb. − Rojas et al. (Argentina) Composición química y efecto antibacteriano del aceite esencial de Aloysia triphylla (L’Hér.) Britton contra patógenos genito-urinarios. − Kader et al. (Bangladesh-Reino Unido) Zederone from the rhizomes of Zingiber zerumbet and its anti-staphylococcal activity. − Letelier et al. (Chile) A protocol for evaluating the safety of herbal preparations in a rat model: the case of a supercritical fluid extract of Saw Palmetto.

Comunicaciones | Communications − Pérez Colmenares et al. (Venezuela) Volatile components from the leaves of Solanum hypomalacophyllum Bitter.

Indexada por | Indexed by: SCOPUS, Science Citation Index Expanded (SCISEARCH), Journal Citation Reports/Science Edition, Biological Abstracts y BIOThomson Reuters Master Journal List , Chemical Abstracts (CAS), NAPRALERT, CAB International (CAB Abstracts), GlobalHEALTH, Index Copernicus, IMBIOMED, LATINDEX, QUALIS, REDALYC, Biblioteca Virtual da Saude (BVS).

© 2010 The Authors

© 2010 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (1), i

BLACPMA ISSN 0717 7917

Comité Editorial | Editorial Board

BLACPMA es una publicación de la Cooperación Latinoamericana y Caribeña de Plantas Medicinales y Aromáticas

This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. () which permits to copy, distribute and transmit the

work, provided the original work is properly cited. You may not use this work for commercial purposes. You may not alter, transform, or build upon this work. Any of these conditions can be waived if you get

permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights.

Este es un articulo de Acceso Libre bajo los términos de una licencia Atribución Creativa Común-No Comercial-No trabajos derivados 3.0 Internacional. Usted es libre de copiar, distribuir y comunicar

públicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los créditos de la obra de la manera especificada por el autor o el licenciador (pero no de una manera que sugiera que tiene su

apoyo o apoyan el uso que hace de su obra). No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede alterar, transformar o generar una obra derivada a partir de esta obra.

Al reutilizar o distribuir la obra, tiene que dejar bien claro los términos de la licencia de esta obra. Alguna de estas condiciones puede no aplicarse si se obtiene el permiso del titular de los derechos de autor. Nada

en esta licencia menoscaba o restringe los derechos morales del autor.

EDITOR JEFE | EDITOR IN CHIEF

José L. Martínez (Santiago, Chile)

EDITORES CIENTIFICOS | SCIENTIFIC

EDITORS

José María Prieto (London, UK)

Peter Taylor (Caracas, Venezuela)

EDITOR EJECUTIVO | MANAGING EDITOR

Damaris Silveira (Brasilia, Brasil)

EDITORES | EDITORS

Carla Delporte (Santiago, Chile)

Gabino Garrido (Antofagasta, Chile)

Martha Gattuso (Rosário, Argentina)

Jeannette Gavillán (San Juan, Pto Rico)

Leonora Mendoza (Santiago, Chile)

Horacio Olivo (Iowa, USA)

Edgar Pastene (Concepción, Chile)

Verónica Rivas (Monterrey, México)

Gabriela Ricciardi (Chaco, Argentina)

Luis A. Simeoni (Brasília, Brasil)

Beatriz Varela (Buenos Aires, Argentina)

EDITOR HONORARIO | HONORARY EDITOR

Jorge Rodríguez Chanfreau (La Habana, Cuba)

CONSEJO EDITORIAL | EDITORIAL

ADVISORY BOARD

Julio Alarcón (Chillán, Chile)

Talal Aburjai (Amman, Jordan)

Arnaldo Bandoni (Buenos Aires, Argentina)

Elizabeth Barrera (Santiago, Chile)

Armando Cáceres (Guatemala, Guatemala)

Salvador Cañigueral (Barcelona, España)

Bruce Cassels (Santiago, Chile)

Geoffrey Cordell (Illinois, USA)

Rosa Degen (Asunción, Paraguay)

Marco Dehesa (Quito, Ecuador)

Olga Lock (Lima, Perú)

Rodolfo Juliani (New Jersey, USA)

Patricia Landázuri (Armenia, Colombia)

Norman Farnsworth (Illinois, USA)

Elisabeth Williamson (London, UK)

Michael Heinrich (London, UK)

Peter Houghton (London, UK)

Luis Kanzaki (Brasilia, Brasil)

Ana Ladio (Bariloche, Argentina)

Francisco Morón (La Habana, Cuba)

Patrick Moyna (Montevideo, Uruguay)

Pulok Mukkerjee (Jadavpur, India)

Luca Rastrelli (Salerno, Italia)

Vicente Martínez (Guatemala, Guatemala)

John A. O. Ojewole (Natal, Sudafrica)

Edison Osorio (Medellín, Colombia)

Mahendra Rai (Maharashtra, India)

Elsa Rengifo (Iquitos, Perú)

José Luis Ríos (Valencia, España)

Lionel Robineau (Pointe à Pitre, Guadalupe)

Gloria Saavedra (Cochabamba, Bolivia)

Marcelo Wagner (Buenos Aires, Argentina)

Talal Zari (Arabia Saudita)

© 2010 The Authors

© 2010 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (1), ii-iii


Nota Editorial | Editorial Notes


This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. () which permits to copy, distribute and transmit the

work, provided the original work is properly cited. You may not use this work for commercial purposes. You may not alter, transform, or build upon this work. Any of these conditions can be waived if you get

permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights.

Este es un articulo de Acceso Libre bajo los términos de una licencia Atribución Creativa Común-No Comercial-No trabajos derivados 3.0 Internacional. Usted es libre de copiar, distribuir y comunicar

públicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los créditos de la obra de la manera especificada por el autor o el licenciador (pero no de una manera que sugiera que tiene su

apoyo o apoyan el uso que hace de su obra). No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede alterar, transformar o generar una obra derivada a partir de esta obra.

Al reutilizar o distribuir la obra, tiene que dejar bien claro los términos de la licencia de esta obra. Alguna de estas condiciones puede no aplicarse si se obtiene el permiso del titular de los derechos de autor. Nada

en esta licencia menoscaba o restringe los derechos morales del autor.

Una nueva década José L MARTINEZ1, Damaris SILVEIRA2, José M PRIETO3, Gabino GARRIDO4 & Peter TAYLOR5

1Universidad Santo Tomás, Talca, Chile; 2Universidad de Brasilia, Brasil; 3Universidad de Londres, Inglaterra; 4Universidad de

Antofagasta, Chile; 5IVIC, Caracas, Venezuela

Estamos comenzando una nueva década del s.

XXI y un nuevo año, el noveno, del Boletín

Latinoamericano y del Caribe de Plantas Medicinales

y Aromáticas (BLACPMA) con muchas novedades.

Durante el año anterior nos sometimos a evaluación

en SCIELO y recibimos muchas recomendaciones

que nos han permitido mejorar. Sin embargo una

entre ellas nos ha causado especial dolor de corazón y

cabeza: SCIELO nos llamo a re-configurar el Comité

Editorial ya que en su opinión estaba sobrecargado y

no permitía el crecimiento sostenible de la revista.

BLACPMA ha sido siempre un foro de profesionales

unidos en la pasión de divulgar ciencia, y siempre

hemos considerado plasmar a todos ellos en nuestro

comité editorial lo cual era consustancial a esta

filosofía. Sin embargo la profesionalización de este

boletín parece incompatible con ello y hemos debido

aceptar cambios profundos. Empezando por los roles

principales, asumiendo Peter Taylor (Venezuela) y

Damaris Silveira un rol mas importante dentro de la

jerarquía, aliviando un poco a Gabino Garrido

(Chile).y a José María Prieto (Inglaterra) que habían

llevado un gran peso hasta ahora. En los roles de

Editores hemos decidido convocar a aquellos colegas

que se mostraron mas proactivos durante el 2009:

desde Argentina Martha Gattuso (Rosario), Gabriela

Ricciardi (Resistencia) y Beatriz Varela (Buenos

Aires), además de Verónica Rivas (México) y

Horacio Olivo (USA) y Edgar Pastene (Chile).

Así que ¿qué paso con todos nuestros grandes

amigos que no podemos mantener nominalmente en

el comité editorial? Simplemente siguen vinculados

igual que antes al BLACPMA a través de la naciente

CLACPMA (ver boletín de XXX 2009). CLACPMA

ya se está mostrando como un catalizador del trabajo

en red de sus integrantes y como resultados de la

misma han sido dos proyectos presentados antes que

desafortunadamente no resultaron agraciados, pero

no por ello nos daremos por vencidos. Próximamente

su Presidente José María Prieto tratará de quitarse

horas de sueño y editar un boletín de noticias

CLACPMA para dinamizar esta asociación y darla a

conocer allende Latinoamérica.

Peter Taylor (Venezuela) ha tomado para si la

ingrata tarea de reconsiderar todo el flujo editorial

elaborando un cronograma, un manual de funciones y

un texto con las instrucciones a autores y revisores

que se dará a conocer en próximos números. Con esto

se pretendemos agilizar aun más los procesos

evaluativos ya que la entrada en ISI de BLACPMA

ha supuesto una carga extraordinaria para el equipo

editorial y educar a autores y revisores en cuanto a

los mínimos de calidad exigibles por nuestra revista.

Por otro lado, nuevamente hemos recibido el

interés de SILAE para establecer una Asociación

entre nuestra publicación y su Sociedad. Desde ya

estamos invitados a su reunión en Cagliary (Italia)

para Septiembre de 2010 pero esperamos poder

reunirnos en forma previa en el primer semestre de

este año.

Una de nuestras principales colaboradoras la Dra.

Gabriela Ricciardi y amiga de muchos años, este mes

de febrero de 2010 contrae matrimonio, como Comité

Ejecutivo de BLACPMA deseamos para ella un

mundo lleno de felicidades en esta nueva vida que

inicia.

Todas estas buenas nuevas se han empañado.

Hace poco más de un mes, el pueblo Haitiano ha

sufrido una de sus peores tragedias y como Boletín

Latinoamericano y del Caribe no nos hemos querido

mantener al margen. No tenemos las herramientas

para poder estar más cerca pero de todos modos

Prieto et al. Guia de Autores

www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol. 9 (1) 2010 | iii

queremos enviar a través de estas líneas un noble

saludo de apoyo y solidaridad. El Editor Jefe ha

invitado al Sr. Benito Baranda, Presidente de la

Organización de Voluntariado “Fundación América

Solidaria” para que en un número próximo nos ilustre

un poco sobre la situación de ese país caribeño y

quizás nos invité de alguna forma a solidarizar ya sea

a través de la Fundación que él dirige o de otras

instancias. Lamentablemente para este número su

tiempo ha sido escaso debido a sus innumerables

labores para apoyar a Haití.

Queremos terminar agradeciendo a aquellos que

de una u otra manera hacen que este Boletín ocupe el

sitial que tiene en este momento todos los autores y

revisores que durante el 2009 han contribuido a esta

revista y especialmente por su importante apoyo a

Arnaldo Bandoni y Mariela Marinoff (Argentina),

Guillermo Padrón (México), Carlos Céspedes (Chile)

y Carolina Baquero (Colombia).

Les dejamos ya con este su Boletín, y como

siempre les rogamos nos hagan llegar sus sugerencias

y contribuciones que siempre serán bienvenidas y

acreditadas.

© 2010 The Authors © 2010 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (1), 1 - 12

BLACPMA ISSN 0717 7917 Revisión | Review


This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. (http://creativecommons.org/licenses/by-nc-nd/3.0/ ) which permits to copy, distribute and transmit the work, provided the original work is properly cited. You may not use this work for commercial purposes. You may not alter, transform, or build upon this work. Any of these conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights. Este es un articulo de Acceso Libre bajo los términos de una licencia “Atribución Creativa Común-No Comercial-No trabajos derivados 3.0 Internacional” (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.es) Usted es libre de copiar, distribuir y comunicar públicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los créditos de la obra de la manera especificada por el autor o el licenciador (pero no de una manera que sugiera que tiene su apoyo o apoyan el uso que hace de su obra). No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede alterar, transformar o generar una obra derivada a partir de esta obra. Al reutilizar o distribuir la obra, tiene que dejar bien claro los términos de la licencia de esta obra. Alguna de estas condiciones puede no aplicarse si se obtiene el permiso del titular de los derechos de autor. Nada en esta licencia menoscaba o restringe los derechos morales del autor.

Produtos Naturais com atividade inibitória da Translocase I, uma promissora classe de compostos contra tuberculose

[Natural Products with Translocase I inhibitory activity, as new lead compounds against tuberculosis]

Marcus Vinícius Nora DE SOUZA* , Victor FACCHINETTI, Danielle CARDINOT, Claudia Regina Brandão GOMES

Fundação Oswaldo Cruz, Instituto de Tecnologia em Fármacos-Far Manguinhos, R. Sizenando Nabuco 100, Manguinhos, 21041-250, Rio de Janeiro, RJ, Brazil.

Abstract

Nowadays, Tuberculosis (TB), a contagious infectious disease caused by Mycobacterium tuberculosis, is becoming again a worldwide health problem. The major causes that increase TB cases in the twenty one century are the rapid spread of multi-drug resistant strains, and TB association with the human immunodeficiency virus (HIV) infection, which started in the mid-1980s. Considering that, the development of new drugs is urgently needed or a human tragedy could happen. In this context, Translocase I, an enzyme involved in the biosynthesis of peptidoglycan, can be an important target for the development of new drugs against this disease. Considering that, the aim of the present review is to highlight a series of new promising anti-TB agents, which have been reported as Translocase I inhibitors namely Liposidomicines, Caprazamicines, Capuramicines, Pacidamicines, Mureidomicines e Napsamicines, Muraimicines, and Tunicamicines all of these structural templates isolated from Streptomyces species.

Keywords: tuberculosis; translocase I; streptomyces

Resumo

Atualmente, a tuberculose (TB), doença infecto-contagiosa cujo agente etiológico é o Mycobacterium tuberculosis é um grave problema de saúde mundial. Os principais fatores responsáveis pelo ressurgimento dessa doença no século vinte um foram o rápido desenvolvimento de cepas multiresistentes e a associação do Mycobacterium tuberculosis com o vírus da imunodeficiência humana (HIV) no início da década de 1980. Nesse contexto, torna-se necessário o desenvolvimento de novos fármacos ou uma tragédia humana pode ocorrer. Considerando esses fatores, a translocase I, enzima envolvida na biossíntese de peptideoglicanas, pode ser um importante alvo no desenvolvimento de novos fármacos no combate a essa doença. Assim sendo, o objetivo dessa revisão é destacar uma série de novas substâncias promissoras para o tratamento da TB, isoladas de diferentes espécies de Streptomyces, que vem sendo relatadas como inibidores da translocase I como Liposidomicinas, Caprazamicinas, Capuramicinas, Pacidamicinas, Mureidomicinas e Napsamicinas, Muraimicinas, and Tunicamicinas.

Palavras Chave: tuberculose; translocase I; Streptomyces

List of Abbreviations

AIDS (Acquired Immune Deficiency Syndrome); BCG (Bacilo de Calmette-Guérin); CDC (Center for Disease Control and Prevention); CPZs (caprazamicinas); LPMs (liposidomicinas); LPS (lipopolissacarídeo); MDR (Multidrug resistant); MIC (Minimum inhibitory concentration); MRYs (muraimicinas); OMS (Organização Mundial de Saúde); Human Immunodeficiency Virus (HIV); Tuberculosis (TB); TCM (tunicamicinas); UPAs (uridil peptídeos); XDR (Extensively Drug Resistant)

Recibido | Received: July, 10, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: September 8, 2009. Publicado en Línea | Published Online 15 December 2010 Declaración de intereses | Declaration of interests: authors have no competing interests. Financiación | Funding: This work has not received funds. This article must be cited as: Marcus Vinícius Nora de Souza*, Victor Facchinetti, Danielle Cardinot, Claudia Regina Brandão Gomes. 2009. Produtos Naturais com atividade inibitória da Translocase I, uma promissora classe de compostos contra tuberculose. Bol Latinoam Caribe Plant Med Aromat 9(1):1 – 12. {EPub 15 DEcember 2009 }.

*Contactos | Contacts: [email protected]

mailto:[email protected]

De Souza et al. Translocase I, um importante alvo terapêutico no combate à tuberculose

www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol. 9 (1) 2010 | 2

INTRODUÇÃO A tuberculose (TB) é uma doença crônica

endêmica na maioria dos países em desenvolvimento, transmitida pelo ar e existente há milhares de anos, causada pelo agente etiológico Mycobacterium tuberculosis, que, por ser uma bactéria aeróbia, desenvolve-se principalmente nos pulmões, mas também pode atacar outras áreas do corpo humano.

Atualmente, estima-se que um terço da população mundial é portadora assintomática do Bacilo de Koch, dos quais 5% a 10% irão manifestar a doença que tem como principais sintomas tosse crônica persistente, suor noturno, dor no tórax e perda de peso devido à falta de apetite. ( De Souza and Vasconcelos, 2005a)

A prevenção da TB é feita por meio da vacinação de recém-nascidos em seus primeiros 30 dias de vida com a vacina BCG (Bacilo de Calmette-Guérin), e o tratamento da doença é realizado, preferencialmente, através da combinação de quatro fármacos: Isoniazida, Rifampicina, Pirazinamida e Etambutol, utilizados durante um período de 6 meses que pode ser estendido para 9 meses em casos especiais. Apesar de ser um tratamento eficaz e barato, a taxa de abandono é extremamente elevada em alguns países por diversos motivos como a longa duração do tratamento, falta de informação e de acompanhamento médico e a grande quantidade de efeitos colaterais associados ao uso desses fármacos. (De Souza, 2006a, 2006b)

A questão da TB torna-se ainda mais delicada quando inserida no contexto da pandemia de HIV/AIDS, já que a co-infecção por M. tuberculosis e HIV tem se mostrado uma combinação letal. Dados da Organização Mundial de Saúde (OMS) indicam a ocorrência de mais de 300 casos anuais de TB a cada 100.000 habitantes em áreas da África subsaariana, local em que há maior incidência do vírus HIV. Nessas áreas, mais de dois terços das pessoas infectadas por TB estão co-infectadas por esse vírus. No Brasil, dados da OMS mostram que no período entre 2000 e 2006 foram notificados quase 700.000 casos de TB, sendo o Rio de Janeiro o estado com maior número de casos registrados por ano, e pouco mais de 60.000 óbitos causados por essa doença. Aproximadamente 20% dessas mortes estão associadas a pacientes co-infectados pelo vírus HIV. (WHO, 2009)

MATERIAIS E MÉTODOS

O presente review foi organizado basicamente em três secções: A primeira delas apresenta a tuberculose e o seu ressurgimento, devido ao aparecimento de super-bactérias resistentes aos fármacos utilizados. A segunda seção destaca a importância dos produtos naturais no tratamento da tuberculose, bem como uma importante enzima conhecida como translocase presente na parede cellular do Mycobacterium tuberculosis, agente etiológico da TB. Finalmente, a última seção destaca produtos naturais e sua relação estrutura-atividade capazes de inibir essa enzima.

MULTIDRUG RESISTANT (MDR) E EXTENSIVELY DRUG RESISTANT (XDR) TUBERCULOSE

O abandono do tratamento tem provocado o aparecimento de cepas resistentes aos fármacos de 1ª escolha. Multidrug resistant (MDR) TB é definido, segundo a OMS (Organização Mundial da Saúde), como doenças causadas pelo M. tuberculosis resistente a Isoniazida e Rifampicina que são os fármacos mais eficazes no tratamento da tuberculose. Essas cepas resistentes foram identificadas no final dos anos 80 e início dos anos 90, representando uma ameaça ao controle da doença.

De acordo com a OMS, as infecções por cepas MDR-TB devem ser tratadas com pelo menos quatro medicamentos de segunda escolha nunca usados anteriormente pelos pacientes, incluindo Capreomicina, Amicacina ou Canamicina injetáveis e um derivado fluorquinolônico como a Ciprofloxacina e a Ofloxacina. O uso dos fármacos de segunda escolha apresenta como desvantagens a maior quantidade de efeitos colaterais, alto custo e maior tempo de tratamento, que pode chegar a até 24 meses de duração.

No tratamento de casos de infecções por cepas MDR-TB, também é comum o uso de Etionamida e Ácido p-aminossalicílico e dos fármacos de primeira escolha Pirazinamida e Etambutol. (De Souza, 2006c)

Uma nova ameaça ao controle da TB é a identificação recente de cepas do tipo Extensively Drug Resistant (XDR) TB, que é definida, de acordo com a OMS, como cepas do tipo MDR-TB resistentes a fluorquinolonas e a pelo menos um dos três medicamentos injetáveis de segunda escolha anteriormente citados. Como essas bactérias apresentam resistência aos fármacos de primeira e de


segunda escolha mais eficazes, a doença se torna virtualmente incurável através da utilização dos medicamentos atualmente existentes no mercado para tratamento da TB. (CDC, 2009)

Uma pesquisa conduzida pela OMS em conjunto com o CDC (Center for Disease Control and Prevention) entre 2000 e 2004 identificou cepas do tipo XDR-TB em todo o mundo, principalmente nos países da antiga União Soviética e da Ásia. Outra pesquisa realizada pela OMS, dessa vez na África do Sul, mostrou resultados alarmantes. De 544 pacientes estudados, 221 estavam infectados com cepas do tipo MDR-TB e desses, 53 foram enquadrados na definição de XDR-TB, sendo 44 HIV-positivos. Dos 53 pacientes XDR-TB, 52 morreram em até 25 dias, incluindo os beneficiados pelo uso de antirretrovirais, fato esse que desperta a preocupação e demonstra a urgência de novos esforços com relação à identificação de novos alvos terapêuticos e ao desenvolvimento de novos fármacos no combate à TB. (WHO, 2009)

IMPORTÂNCIA DOS PRODUTOS NATURAIS NO TRATAMENTO DA TUBERCULOSE

De maneira similar ao que aconteceu com outras doenças, tais como câncer, a malária, e certas doenças inflamatórias, o sucesso dos produtos naturais merece destaque também na descoberta do tratamento e cura da tuberculose. Vale ressaltar que a descoberta da estreptomicina (Figura 1), isolada a partir de culturas de Streptomyces griséus, pela equipe liderada pelo bioquímico norte-americano, Selman Waksman, em 1943, foi uma das mais relevantes da história da medicina moderna e da humanidade. Esse medicamento foi responsável pela cura e pelo controle da tuberculose, doença que na época de seu surgimento causava a morte de um número incontável de indivíduos. (De Souza, 2009) Figura 1. Estrutura da Estreptomicina.

Após a descoberta da estreptomicina, outros aminoglicosídeos foram descobertos e utilizados até os dias de hoje no tratamento e controle da tuberculose, podendo-se destacar os aminoglicosídeos canamicida, obtida a partir da cultura de fungos Streptomyces capreolus, amicacina, derivado semisintético obtido partir da canamicina A e capreomicina 1A, obtida a partir da cultura de fungos Streptomyces Kanamyceticus. Além dos aminoglicosídeos, pode-se mencionar também a D-cicloserina, obtida a partir da fermentação de Streptomyces sps.

Com o crescente surgimento de super bactérias resistentes a praticamente todos os fármacos utilizados no tratamento da tuberculose, diversos grupos de pesquisa tem demonstrado mais uma vez, o papel fundamental da mãe natureza na descoberta de novos fármacos no combate à tuberculose. Nesse contexto, pode-se destacar o crescente número de publicações científicas na área, identificando diversos produtos naturais isolados de diversas fontes com promissoras perspectivas no combate a tuberculose multiresitente. Na figura abaixo se encontra alguns exemplos de produtos naturais obtidos de plantas com atividade tuberculostática. (De Souza, 2005b) (De Souza, 2006d). Figura 2: Produtos naturais com atividade tuberculostática.

O

OO O

OH

(+) - Calanolide A

7

8

12

A

B

C

MIC = 3.13 μ g mL-1

O

H3C OCOCH3

CH3

CH3

H

O

H3CHN

O H

H OH

H

H CH2OH

MIC = 3.13 μ g mL-1

O

OOH

HO

O

OH

MIC = 6.3 μ

H

OAcO OAc

HO

MIC = 12.5 μ g mL-1

N

NH

N

CHO

NH

Solsodomina A

MIC = 10 μ g mL-1

g mL-1

(XU et al., 2004)(CHUMKAEW et. al, 2003)

(KANOKMEDHAKUL et al., 2005)

(KANOKMEDHAKUL et al., 2005)

(SAYED et al., 1998)

MYCOBACTERIUM E TRANSLOCASE I

Em geral, o citoplasma bacteriano é separado do meio extracelular por uma membrana citoplasmática formada por uma bicamada lipídica com boa fluidez, que age como uma barreira seletivamente permeável,



e por uma parede celular constituída por peptideoglicanas que confere a resistência necessária para suportar a alta pressão osmótica interna (Nikaido and Saier JR, 1994). A biossíntese da parede celular vem sendo explorada como alvo farmacológico para a pesquisa de novos antibióticos desde a descoberta da penicilina por Fleming em 1929.

Bactérias Gram-positivas como o Staphylococcus aureus possuem parede celular formada por uma espessa camada de peptideoglicanas que confere pouca resistência à difusão de pequenas moléculas. Já as bactérias Gram-negativas, como a Escherichia coli, contêm em sua parede celular, além da camada de peptideoglicanas, uma outra membrana bilipídica externa. A superfície externa dessa membrana é recoberta por um lipídeo não usual, o lipopolissacarídeo (LPS), que possui estrutura de pouca fluidez. Foi demonstrado que até mesmo moléculas lipofílicas apresentam dificuldades para atravessar a parte hidrofóbica desse lipídeo, o qual se torna uma barreira eficiente contra a rápida difusão de antibióticos lipofílicos. Em contraste, a parede celular das micobactérias é constituída por três subestruturas covalentemente interligadas: as peptideoglicanas, arabinogalactanas e os ácidos micólicos, sendo os últimos formados por longas cadeias de ácidos graxos contendo diferentes grupos funcionais, como ligações duplas, cetona, éster, epóxido, metóxi e ciclopropano. Esses compostos são de extrema importância para a sobrevivência das micobactérias, pois dificultam a penetração de drogas hidrofóbicas, evitam a desidratação e permitem que a bactéria se desenvolva no sistema imune do hospedeiro (Figura 3). (Bugg and Walsh, 1992), (Heijenoort, 2001), (Kimura and Bugg, 2003) (De Souza, 2008) Figura 3. Representação da parede celular de bactérias.

A biossíntese de peptideoglicanas é essencial para

a sobrevivência da bactéria (seres que possuem células procarióticas) e se torna um alvo interessante no desenvolvimento de antibióticos, já que não existe correspondência em células eucarióticas (presente

nos animais e na maioria das plantas). A biossíntese do peptideoglicano consiste é constituida de três estágios: síntese do lipídeo I e do lipídeo II, seguido da polimerização do lipídeo II por transpeptidação e transglicosilação. Como exemplo de fármacos em uso clínico que inibem a polimerização do lipídeo II na superfície da bactéria, pode-se mencionar as β-lactamas e as vancomicinas. Esses fármacos possuem um mecanismo de ação diferente dos utilizados no tratamento para tuberculose, tornando-se possível combater as infecções causadas por micobactérias multirresistentes. (Boyle and Donachie, 1998), (Hirano et al, 2008a)

Estudos têm sido realizados visando à descoberta de substâncias capazes de inibir a síntese do lipídeo I. Nessa etapa, a enzima fosfo-MurNAc-pentapeptideo translocase, também chamada de translocase I (MraY), catalisa a reação entre o UDP-MurNAc-pentapeptideo e o undecaprenil-fosfato formando UMP e o Lipídeo I, que é o primeiro intermediário da síntese de peptideoglicanas. Essa catálise requer a presença de Mg+2 como cofator para a ativação da enzima (Figura 4).(Struve et al, 1966), (Heydanek et al, 1969) Figura 4: Biossíntese do Lipídeo I

Várias substâncias têm sido relatadas como

inibidoras da enzima translocase I, dentre elas as liposidomicinas, as caprazamicinas, as capuramicinas e as pacidamicinas, que serão abordadas a seguir.

INIBIDORES DA TRANSLOCASE I

Liposidomicina As liposidomicinas (LPMs) são produtos naturais

da classe dos antibióticos 6’-N-alquil-5’-β-O-aminoribosilgliciluridina. Em 1998, foram reportadas liposidomicinas do tipo I, II, III e IV, isoladas da cepa mutante de Streptomyces griseosporeus SN-1061M. As LPMs originais do tipo I possuem as porções sulfato e ácido 3-metilglutárico. Os compostos do tipo II não contêm a porção ácido 3-metilglutárico, os do tipo III não contêm a porção sulfato e os do tipo 4 não possuem nenhuma das duas porções (Figura 5). (Kimura et al, 2003)



A capacidade das liposidomicinas (LPMs) em inibir seletivamente a síntese de peptideoglicanas foi descoberta em 1985. (Isono et al, 1985). Testes in vitro indicam que as LPMs do tipo I e III são os melhores inibidores da enzima translocase I, quando comparadas as LPMs do tipo II e IV, indicando que a porção ácido 3-metilglutárico exerce papel fundamental na inibição da translocase I. Entretanto, as LPMs do tipo I, assim como a LPMs do tipo II, apresentam baixa atividade antimicrobiana in vivo devido à presença da porção sulfato, hidrofílica, na posição 2” da 5-aminopentose, fato que confere às LPMs baixa permeabilidade através da membrana celular. As LPMs do tipo III e IV são mais lipofílicas devido à ausência da porção sulfato, consequentemente, possuem maior atividade antimicobacteriana in vivo.

A presença do ácido graxo nessa classe de substâncias também é extremamente importante para a atividade biológica, provavelmente porque confere um aumento da lipofilicidade. (Kimura et al, 1998) Figura5: Liposidomicinas dos tipos I, II, III e IV.

O

HO OH

N

HNO ON

N

O

O

R

O

OCH3O

HOO

CH3

H3CHO2C

Liposidomicina I

Liposidomicina III

2'''

3'''6'

5'

Liposidomicina II

Liposidomicina IV

Liposidomicinas R

I-A; II-A; III-A; IV-A H3C

(H3C)2HCI-B; III-B

H3CI-C; II-C; III-C; IV-C

H3CI-G; III-G

H3CI-H; III-H

H3CI-K; III-K

I-L; III-L (H3C)2HC

I-M; III-M

I-N; III-N

I-Z; III-Z

III-X

III-Y

H3C

H3C

H3C

H3C

H3C

O

O

H2N

HO

HO3SO

O

HO OH

N

HNO O

5'

O

O

H2N

HO

HO3SO

N

N

O

O

R

O

CH3

H3CHO2C

2'''

3'''6'

N

N

O

O

R

O

OCH3O

HOO

CH3

H3CHO2C

6'

3'''

2''' O

HO OH

N

HNO O

5'

O

O

H2N

HO

HO

N

N

O

O

R

O

CH3

H3CHO2C

2'''

3'''6'

O

HO OH

N

HNO O

5'

O

O

H2N

HO

HO

Dini e colaboradores em 2000 e 2001 sintetizaram várias substâncias baseadas em simplificações

estruturais das LPMs, que foram testadas como inibidoras da translocase I. Algumas dessas moléculas estão representadas na figura 6. Figura 6. Simplificações estruturais das liposidomicinas

Substância R R1 MraY IC (μΜ)

1 H NH2 50

2 (R) - CH2OH NH2 425

3 (S) - CH2OH NH2 5

4 H CH(NH)NH 30

5 H H2NC(NH)NH 25

6 H MeNH 45

7 H EtNH 150

8 H n-PrNH 140

O

HO OH

N NH

O

OO

R

O

HO OH

R1 O

R3' R2'

N NH

O

OOO

R3" R2"

H2N5"

Substância R2' R3' R2'' R3''

9 H OH OH OH

10 OH H OH OH

11 OH OH H OH

12 OH OH OH H

13 H H OH OH

MraY IC (μΜ)

80

10

115

>1000

120

A substância 1 apresentou moderada atividade

inibitória (IC50: 50µM). A introdução do grupamento CH2OH na posição 5”, indicou que o isômero R (2) é inativo, enquanto o isômero S (3) possui maior atividade inibitória do que a substância 1 (IC50: 5µM). (Dini et al, 2000)

Um grupamento básico, tal como o grupamento amino, aminas secundárias, amidina e guanidina são importantes para a atividade inibitória da MraY. Sendo que, no caso das aminas secundárias, o tamanho da cadeia lateral influência diretamente na atividade inibitória. A metilamina (6) é ativa (IC50: 45µM), enquanto que aminas com grupamento alquílico (7 e 8) maior são inativas. (Dini et al, 2001a)

A comparação da atividade inibitória dos compostos 9-13 indicou que presença da hidroxila na posição 3” é essencial para a atividade. Entretanto, o composto 3’-dexoxi é 5 vezes mais ativo do que o correspondente derivado 3’-hidroxilado, sugerindo que apenas a hidroxila na posição 3” é essencial para a inibição da translocase I. (Dini et al, 2001b)

Caprazamicinas As caprazamicinas (CPZs) (Figura 7) são

produtos naturais que, assim como as LPMs, pertencem a classe dos antibióticos 6’-N-alquil-5’-β-O-aminoribosilgliciluridina. As CPZs são isoladas a partir de culturas de Streptomyces sp. e mostram uma excelente atividade contra bactérias Gram-positivas. (Igarashi et al, 2005)



Figura 7. Estrutura das Caprazamicinas.

Caprazamicinas

Caprazamicinas R

H3C

H3C

G

(H3C)2HC

O

HO OH

N NH

O

O

O

O

HO

HO

NH2

N

N O

O

HO2CMe

Me

O

R

O

OMeO

OO

MeO

Me

OMeOMe

1'5'6'

2'''

3'''1''

5''

5

A

B

C

D

F

E

(H3C)2HC

H3C

(H3C)2HC

H3CCH3

As CPZs apresentam atividade in vitro contra Mycobacterium tuberculosis, tanto em cepas sensíveis como em cepas multirresistente e não demonstraram toxicidade significativa em ratos.

Devido às excelentes propriedades biológicas, as CPZs são substâncias promissoras para a síntese de novos agentes anti-TB, com um novo mecanismo de ação. Nesse contexto, quando comparadas às LPMs, as CPZs possuem importantes semelhanças estruturais e biológicas sugerindo que essas classes de compostos possuem o mesmo mecanismo das LPMs. (Ichikawa, 2008)

Hirano e colaboradores sintetizaram diversos análogos da caprazamicina (Figura 8), incluindo o caprazol e o palmitoilcaprazol, que foram testados contra o Mycobacterium sp. Figura 8. Análogos das Caprazamicinas.

O

HO OH

N NH

O

O

O

O

HO

HO

NH2

N

N O

O

HO2CMe

H

OO

O

HO OH

N NH

O

O

O

O

HO

HO

NH2

N

N O

O

HO2CMe

Me

14(MIC = 25 µg/mL)

Palmitoilcaprazol(MIC = 6,25 µg/mL)

O

HO OH

N NH

O

O

O

O

HO

HO

NH2

N

N O

HO

HO2CMe

Me

O

HO OH

N NH

O

O

O

O

HO

HO

NH2

H2N

N O

O

HO2CMe

O

O

HO OH

N NH

O

O

OHN

N O

O

HO2CMe

Me

O

Me

O

O

HO

HO

NH2

N

N O

O

HO2CMe

Me

O

Caprazol15

16 17

(MIC = 2,50 µg/mL)(inativa)

(inativa) (inativa)

O palmitoilcaprazol, quando testado contra Mycobacterium smegmatis ATCC607, apresentou MIC (Minimum inhibitory concentration – concentração inibitória mínima) de 6,25 µg/mL, similar ao das CPZs, e quatro vezes maior do que o do composto 14 (25 µg/mL), mostrando que a ausência do grupamento metila na porção da diazepanona influencia negativamente a atividade antimicobacteriana. (Hirano et al, 2008a). Outros análogos foram testados contra Mycobacterium tuberculosis H37Rv, sendo que o composto 15 apresentou atividade ligeiramente reduzida quando comparado ao palmitoilcaprazol (MIC = 2,50 e 6,25 µg/mL, respectivamente), sugerindo que a abertura do anel diazepanona diminui a atividade inibitória sem, contudo, tornar a substância inativa. A fragmentação da molécula nas porções aminoribose ou uridina tornou as moléculas 16 e 17 inativas e, portanto, são cruciais para a atividade antibacteriana. (Hirano et al, 2008b) O caprazol, análogo das CPZs sem a porção alquílica no anel diazepanona, também não apresentou atividade antimicobacteriana. Essas alterações realizadas na parte lipofílica da cadeia lateral são de fundamental importância para a permeabilidade na célula bacteriana. (Hirano et al, 2008a)

Capuramicina A capuramicina (figura 9) foi originalmente

isolada a partir de culturas de Streptomyces griseous 466-S3 e possui a capacidade de inibir a enzima translocase I, no entanto seu espectro de ação é baixo.



Figura 9. Análogos da Capuramicina e sua attividade antimicrobiana (Hotoda et al, 2003a)

O

RO OH

N NH

O

O

CONH2

OOHN

O

OHOH

HN

O

Capuramicina (R = H)

A-500359A (R = Me)

O

HO OH

N NH

O

O

CONH2

OOR1

O

OHOH

R

Análogos da Capuramicina 18-24

Compostos R1 1a 2b 3c 4d 5e

Capuramicina - 10 12,5 8 8 8

A-500359A - 10 6,25 8 4 16

A-500359E MeO- 27 >100 - - -

18 PhNH- 6,5 6,25 16 4 8

19 3-Me-PhNH- 7,6 12,5 4 1 8

20 3-F-PhNH- 10 6,25 2 2 8

21 4-F-PhNH- 37 6,25 4 2 2

22 3,4-di-F-PhNH-

9 6,25 2 0,5 1

23 4-Cl-PhNH- 18 6,25 4 2 16

24 4-Br-PhNH- 20 6,25 8 0,5 8

Rifampicina - - - 0,125 0,125 0,125

Isoniazida - - -


a Translocase I, IC50 (ng/mL) bM. smegmatis SANK75075, MIC (µg/mL) c M. avium NIHJ1605, MIC (µg/mL) dM. intracellulareATCC1954 E-3, MIC (µg/mL) eM. kansasii ATCC12478, MIC (µg/mL)

Em geral, a capuramicina apresenta maior atividade frente à micobactérias e pouca atividade frente a bactérias Gram-positivas. É interessante notar que as micobactérias apresentam resistência contra muitos derivados β-lactâmicos, medicamentos que pertencem à mesma classe das capuramicinas, devido à presença da enzima β-lactamase. A atividade dessas substâncias poderia ser então explicada pela estabilidade do seu anel β-lactâmico frente a essas enzimas. O mecanismo de permeabilidade das capuramicinas através da membrana das micobactérias ainda não está completamente elucidado, sendo atualmente, muitos análogos de capuramicinas vêm sendo isolados, sintetizados e testados como inibidores da enzima translocase I. Como exemplo, pode-se citar análogos da capuramicina que não contém a porção azepan-2-ona (Figura 7) obtidos por Hotoda e Colaboradores, a partir da reação entre diversas aminas e o produto natural A500359E, também isolado de cepas S. griseous.

As substâncias obtidas foram testadas frente ao M. smegmatis e a enzima Translocase I, ficando evidente a importância do grupamento NH da amida para a atividade dessas substâncias. Os melhores resultados foram observados com os compostos 18-24 que foram, então, testados frente às micobactérias de maior relevância clínica: Mycobacterium avium NIHJ1605, Mycobacterium intracellulare ATCC1954 E-3 e Mycobacterium kansasii ATCC12478. Suas atividades foram comparadas com a da rifampicina e isoniazida sendo que o análogo 18 apresentou MIC semelhante ao da capuramicina, variando entre 4 e 16 μg/mL. (Hotoda et al, 2003a)

Hotoda e Colaboradores também obtiveram derivados acilados a partir das moléculas de capuramicina e de seu derivado metilado A500359A (Figura 10)

Observou-se que o aumento no tamanho da cadeia lateral provoca diminuição na atividade das moléculas frente à enzima Translocase I, porém, cadeias de tamanho ideal como a duodecanoíla e a decanoíla presentes, respectivamente, nos derivados da capuramicina (25, MIC variando entre 0,063 e 3,13 μg/mL) e do A-500359A (26, MIC variando entre 0,063 e 6,25 μg/mL), apresentaram excelente atividade frente às micobactérias, provavelmente devido à lipofilicidade conferida a esses compostos que permite uma melhor penetração através da membrana celular desses microorganismos. Os valores de MIC observados para estes compostos foram iguais ou melhores do que os observados para a isoniazida (MIC entre 0,125 e 0,25 μg/mL) e a rifampicina (MIC entre 1 e 2 μg/mL). (Hotoda et al, 2003b) Figura 10. Análogos acilados da Capuramicina e sua attividade antimicrobiana (Hotoda et al, 2003b)

O

MeO O

N NH

O

O

CONH2

OOHN

O

OH

HN

O

OH

R

O

(CH2)nH25 e 26

Compostos R n 1a 2b 3c 4d 5e

Capuramicina H - 10 12,5 8 8 8

A-500359A Me - 10 6,25 8 4 16

25 H 11 n.d. 3,13 <0,063 0,125 0,125

26 Me 9 50 6,25 <0,063 <0,063 <0,063

Rifampicina - - n.d 0,125 0,125 0,125 0,25

Isoniazida - - n.d - 1 8 2 a Translocase I, IC50 (ng/mL); bM.smegmatis SANK75075, MIC (µg/mL);c M. Avium NIHJ1605, MIC (µg/mL); dM.


intracellulareATCC1954 E-3, MIC (µg/mL); eM. Kansasii ATCC12478, MIC (µg/mL)

Os compostos (RS-124922) e (RS-118641)

(Figura 11), sintetizados pela empresa japonesa Sankyo, foram avaliados frente ao Mycobacterium tuberculosis, apresentando melhor atividade contra M. tuberculosis (Cepa H37Rv) (MIC = 8 μg/mL e MIC = 1 μg/mL, respectivamente), do que o composto A500359A (MIC = 16 μg/mL). A atividade dessas substâncias frente à cepas MDR-TB não sofreu variações estatisticamente significativas, quando comparadas às observadas nas cepas H37Rv. Não foram realizados testes in vivo para o composto (RS-124922) devido a sua baixa solubilidade no modelo de tratamento utilizado. Na avaliação in vivo dos outros, observou-se uma considerável redução no número de organismos viáveis nos pulmões em comparação com o grupo de controle. Os estudos desenvolvidos mostram que os análogos de capuramicinas possuem grande potencial antimicobacteriano e são bons candidatos a posterior avaliação no tratamento de infecções causadas por M. tuberculosis. (Koga et al, 2004) Figura 11. Análogos da capuramicina sintetizados pela empresa japonesa Sankyo e sua attividade antimicrobiana (Koga et al, 2004).

O

MeO OO

CONH2

OOHN

O

OHOH

HN

O

Me

O

N NH

O

O

MeO OH

N NH

O

O

CONH2

OOHN

O

OHOH

F

F

A500359-A

O

MeO OH

N NH

O

O

CONH2

OOHN

O

OHOH

HN

O

Me

(RS-118641)

(RS-124922)

Substâncias Cepa H37Rv MIC ( μg/mL)

Cepa MDR-TB MIC ( μg/mL)

A500359-A 16 16 (RS-124922) 8 4 (RS-118641) 1 0,5 Rifampicina < 0,03 > 32 Isoniazida 0,06 4


Pacidamicinas, mureidomicinas e napsamicinas As pacidamicinas são antibióticos da classe uridil

peptídeos (UPAs), assim como as mureidomicinas e napsamicinas (Figura 12), capazes de inibir a enzima translocase I (MraY), isoladas de cultura de Streptomices sp. (Isono et al, 1992), (Boojamra et al,

2003), (Chatterjee et al, 1994), (Chen et al, 1989), (Fernandes et al, 1989) Figura 12. Estrutura das napsamicinas, mureidomicinas e pacidamicinas.

Napsamicina R R1

A H uracil

B CH3 uracilC H diidrouracil

D CH3 diidrouracil

O

OH

NHO

HN

ONH

O

NH

CO2H

SCH3

OHCH3

NCH3

O

NH

HO

R R1 O

OH

NHO

HN

ONH

O

NH

CO2H

SCH3

OHCH3

CH3

O

R1

HN

R

OH

O

OH

NHO

HN

ONH

O

NH

R1

CO2H

CH3CH3

NCH3

OHN

R

OH

N NH

O

O

Mureidomicina R R1

A H uracil

B H diidrouracilC glicina uracil

D glicina diidrouracil

Pacidamicina R R1

1 alanil 3-indol

2 alanil fenil

3 alanil 3-hidroxi-fenil

4 H 3-indol

5 H fenil

6 glicil 3-indol

7 glicil fenil

Compostos da classe das UPAs apresentam

excelente atividade contra Pseudomonas aeruginosa devido as suas propriedades toxicológicas e farmacológicas favoráveis. Cepas bacterianas resistentes, a ofloxacin e β-lactamas, permanecem sensíveis à substâncias dessa classe de antibióticos. Entretanto, os UPAs possuem espectro de ação muito estreito, sendo ativo especificamente contra cepas de Pseudomonas, e muito pouco ativo contra bactérias Gram-negativas e Gram-positivas, o que pode ser explicado pelo fato desses compostos possuírem acesso restrito à célula bacteriana, devido a sua polaridade e ao seu grande potencial para fazer ligações de hidrogênio com a água. (Isono et al, 1992), (Boojamra et al, 2001), (Boojamra et al, 2003)

Boojamra e colaboradores, sintetizaram várias diidropacidamicinas D, a partir da hidrogenação do produto natural (pacidamicina 4), ou de síntese total, introduzindo diferentes aminoácidos na cadeia acíclica (Figura 13). (Boojamra et al, 2003) As substâncias sintetizadas foram testadas contra quatro cepas de Mycobacterium tuberculosis, das quais duas, a W e a P, são resistentes a todo o tipo de tratamento anti-tuberculose, observando-se que as moléculas em que NHCH(R3)COOH é um resíduo de um aminoácido aromático, e as porções H2NCH(R1)C(O) e (CO)CH(R2)NH possuem resíduos hidrofóbicos


(substâncias 27 e 32-35) apresentaram melhor atividade antimicobacteriana (MIC variando entre 4 e 10 µg/mL), inclusive quando comparadas a pacidamicina 4 (MIC maior que 30 µg/mL), sendo ativo inclusive frente às cepas multirresistentes. Figura 13. Derivados da diidropacidamicina 4 e sua attividade antimicrobiana (Boojamra et al, 2003)

SubstânciasR1

H2NO R2

NHO

R3

NH CO2H

Pacidamicina 4 meta-tirosina alanina triptofano

27 alanina 4-flúor-fenilalanina tirosina

28 glicina leucina triptofano

29 alanina metionina tirosina

30 alanina fenilalanina (2-naftil)alanina

31 leucina fenilalanina triptofano

32 alanina (4-bifenil)alanina triptofano

33 alanina fenilalanina triptofano

34 alanina 4-flúor-fenilalanina triptofano

35 alanina 4-trifluormetilfenilalanina triptofano

ONHO

HN

ONH

O

NH

R3

CO2H

R2

NCH3

OH2N

N NH

O

OOH

R1

CH3

Substâncias 1a 2b 3c 4d

Pacidamicina 4 >30 >30 >30 >30

27 10 8 8 10

28 >30 30 10 10

29 >30 >30 >30 >30

30 30 30 30 10

31 30 30 30 10

32 8 4 4 10

33 8 8 4 8

34 8 8 4 8

35 8 4 4 8


aCepa H37Rv, MIC (µg/mL) bCepa TN913, MIC (µg/mL) cCepa TN565 (W), MIC (µg/mL) dCepa TN1618(P), MIC (µg/mL)

Muraimicinas As muraimicinas (MRYs) são substâncias

estruturalmente semelhantes aos antibióticos uridil peptídeo (UPAs), como as mureimicinas, napsamicinas, pacidamicinas e liposidomicina. Dezenove muraimicinas foram isoladas a partir de

culturas de Streptomices sp. (Figura 14). (McDonald et al, 2002) Dentre estas, as muraimicinas A e B, que possuem um grupamento éster com uma cadeia alquilica longa, geralmente possuem melhor atividade antibacteriana do que as muraimicinas que não apresentam o grupamento éster, demonstrando a necessidade da porção lipofílica para a penetração na parede celular das bactérias.

McDonald e colaboradores relataram que algumas muraimicinas (muraimicinas A1, A5, B6, C2 e C3) inibem a síntese do lipídeo II e a biossíntese da camada peptideoglicana em concentrações de 0,027 µg/mL, indicando que a atividade antibacteriana das MRYs é comparável a da LPM C e a da mureidomicina A, que inibem a translocase de E. colii, in vitro, com valores de IC50 de 0,05 e 0,03 µg /mL, respectivamente.(Ichikawa and Matsuda, 2007) Figura 14. Estrutura das muraimicinas.

Muraimicina R1 R2

A1 O2C(CH2)12N(OH)C(NH)NH2 OCH3

A2 O2C(CH2)10N(OH)C(NH)NH2 OCH3

A3 O2C(CH2)12NHC(NH)NH2 OCH3

A4 O2C(CH2)12N(OH)C(NH)NH2 OH

B1 O2C(CH2)6CH(CH3)CH2CH3 OCH3

B2 O2C(CH2)6CH(CH3)2 OCH3

B3 O2C(CH2)4CH(CH3)CH2CH3 OCH3


B5 O2C(CH2)5CH(CH3)2 OH


B7 O2C(CH2)4CH(CH3)2 OH

O

HO OH

N NH

O

OO

CO2HHN

H

H

HN

O

R1

NH

OHN

HN

NH

H

HO

O

HNHO2C

O

HO R2

H2N

O

HO OH

N NH

O

O

CO2HHN

HHN

O

R1

NH

OHN

HN

NH

H

HO

O

HNHO2C

HOH

Muraimicina R1 R2

C1 OH OCH3

C2 OH OH

C3 OH H

D1 H OCH3

D2 H OH

D3 H H

Muraimicina R1

A5 O2C(CH2)12N(OH)C(NH)NH2

C4 OH

Lin e colaboradores sintetizaram derivados da muraimicina C1, através de reações seletivas nos grupamentos amino primário e secundário (Figura 15).


Figura 15. Derivados da muraimicina C1.

O

HO OH

N NH

O

OO

N

H

HN

O

OH

NH

OHN

HN

NH

H

HO

O

HNHO2C

O

HO OMe

H2N

NO R

O

36: R = (CH2)4CH3 (MIC = 6,25 µg/mL)

37: R = CH2Ph (MIC = 6,25 µg/mL) Os derivados onde ocorreu substituição tanto na

amina primária como na secundária foram inativos como inibidores da MraY, em concentrações < 100 µg/mL. Entretanto, os compostos onde houve substituição apenas na amina secundária apresentaram boa atividade inibitória, que demonstrou estar correlacionada com a lipofilicidade dos substituintes introduzidos. Dentre estes, destacou-se as hidantóinas 36 e 37, que inibiram a MraY na mesma concentração que a muraimicina C1 (6,25 µg/mL). (Lin et al, 2002)

Tunicamicinas A classe de produtos naturais conhecidas como

tunicamicinas (TCM) (Figura 16) foi isolada da fermentação de Streptomyces lysosuperficius sendo capazes de inibir a replicação de vírus, bactérias e fungos baseada na glicosidação de proteínas.

Tunicamicina R

I (CH2)7CH(CH3)2

II (CH2)8CH(CH3)2

III (CH2)10CH3

IV (CH2)11CH3

V (CH2)9CH(CH3)2

VI (CH2)11CH(CH3)2

VII (CH2)10CH(CH3)2

VIII (CH2)12CH3

O

OH

N NH

O

OHO

OHO

O

HO OHNH

O

R

O

OH

HOHO

H3C(OC)HN

Figura 16. Estrutura das Tunicamicinas.

Além de sua ampla atividade, as Tunicamicinas

servem como ferramentas na elucidação de mecanismos bacterianos. (Inuka et al, 1993), (Ichikawa, 2008)

DISCUSSÃO

Tendo em vista o grande número de efeitos colaterais associados aos fármacos mais utilizados no tratamento da TB, a duração de seu tratamento e os adventos MDR e XDR-TB, faz-se necessário o desenvolvimento de novos medicamentos ou uma grande tragédia poderá ocorrer, voltando-se ao tempo em que não se existia a cura para essa doença. Nesse contexto, a translocase I (Mra Y), uma das enzimas envolvidas na fase inicial da biossíntese de peptideoglicanas, tem se mostrado um alvo promissor na busca de novos fármacos, sendo representada por diversos produtos naturais e seus derivados sintéticos, representando uma nova classe no combate às infecções bacterianas modernas. Nesse contexto, podemos destacar as liposidomicinas (LPMs) e as caprazamicinas (CPZs), produtos naturais da classe dos antibióticos 6’-N-alquil-5’-β-O-aminoribosilgliciluridina, que apresentam excelentes atividades frente a enzima translocase. Dentre os compostos avaliados dessas classes, merece destaque, a capuramicina, substância pertencente a família das CPZs, que apresentou excelentes perspectivas sendo o estudo de sua estrutura atividade importante para identificação de novos derivados mais potentes, simplificados, com melhores propriedade farmacocinéticas, bem como capazes de ajudar na melhor compreenção desse mecanismo de ação.

REFERÊNCIAS Boojamra CG, Lemoine RC, Lee JC, Léger R, Stein KA,

Vernier NG, Magon A, Lomovskaya O, Martin PK, Chamberland S, Lee MD, Hecker S J, Lee VJ. 2001. Stereochemical elucidation and total synthesis of dihydropacidamycins D, a semisynthetic pacidamicyn. J. Am. Chem. Soc. 123(5): 870-874.

Boojamra CG, Lemoine RC, Blais J, Vernier NG, Stein K A, Magon A, Chamberland S, Hecker S J, Lee V J. 2003 Synthetic dihydropacidamycin antibiotics: A modified spectrum of activity for the pacidamycin class. Bioorg. Med. Chem. Lett. 13: 3305-3309.

Boyle DS, Donachie WD. 1998. mraY is an essential gene for cell growth in Echeriachia Coli. J. Bacteriol. 180(23) 6429-6432.

Bugg TDH, Walsh, CT. 1992. Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: Enzymology, antibiotics and antibiotic resistance. Nat. Prod. Rep. 9: 199-215.

Centers For Disease Control And Prevention. Extensively Drug-Resistant Tuberculosis (XDR TB). http://www.cdc.gov/tb/xdrtb/ (consulted June 29, 2009)


http://www.cdc.gov/tb/xdrtb/



Chatterjee S, Nadkarni SR, Vijayakumar EKS, Patel MV, GANGULI B N. 1994. J. Antibiot. 47(5): 595-598.

Chen R H, Buko AM, Whittern DN, Mcalpine JB. 1989. Pacidamycins, a novel series of antibiotics with anti-pseudomonas aeruginosa activity. II. Isolation and structural elucidation. J. Antibiot. 42(4): 512-520.

Chumkaew P, Karalai C, Ponglimanont C, Chantrapromma K. 2003. Antimycobacterial Activity of Phorbol Esters from the Fruits of Sapium indicum J. Nat. Prod. 66: 540-543.

De Souza MVN, Vasconcelos TRA. 2005a. Fármacos no combate à tuberculose: passado, presente e futuro. Quím. Nova. 28(4): 678-682.

De Souza MV. 2005b. Plants and fungal products with activity against tuberculosis. ScientificWorldJournal. 5:609-28.

De Souza MVN. 2006a. Tuberculose em pacientes HIV-positivos: um grave problema de saúde pública mundial. Rev. Bras. Farm. 87: 42-44.

De Souza MVN. 2006b. Current status and future prospects for new therapies for pulmonary tuberculosis. Curr. Opin. Pulm. Med. 12(3): 167-171.

De Souza MVN. 2006c. Promising drugs against tuberculosis. Recent Pat. Antiinfec. Drug Discov. 1(1): 33-45.

De Souza MVN. 2006d. Marine natural products against tuberculosis. ScientificWorldJournal. 6:847-861.

De Souza MVN, Ferreira ML, Pinheiro AC, Saraiva MF, Almeida MV, Valle MS. 2008. Synthesis and biological aspects of mycolic acids: an important target against mycobacterium tuberculosis. ScientificWorldJournal. 8: 720-751.

De Souza MVN. 2009. Promising candidates in clinical trials against multidrug-resistant tuberculosis (MDR-TB) based on natural products. Fitoterapia. Edizione Farmaceutica. 80: 453-460.

Dini C, Collette P, Drochon N, Guillot JC, Lemoine G, Mauvais P, Aszodi J. 2000. Synthesis of the nucleoside moiety of liposidomycins: elucidation of the pharmacophore of this family of MraY inhibitors. Bioorg. Med. Chem. Lett. 10: 1839-1843.

Dini C, Drochon N, Feteanu S, Guillot JC, Peixoto C, Aszodi J. 2001a. Synthesis of analogues of the O-β-D-ribofuranosyl nucleoside moiety of liposidomicyns. Part 1: contribution of the amino group and the uracil moiety upon the inhibition of MraY. Bioorg. Med. Chem. Lett. 11: 529-531.

Dini C, Drochon N, Guillot JC, Mauvais P, Walter P, Aszodi J. 2001b. Synthesis of analogues of the O-β-D-ribofuranosyl nucleoside moiety of liposidomicyns. Part 2: role of the hydroxyl groups upon the inhibition of MraY. Bioorg. Med. Chem. Lett. 11: 533-536.

Fernandes P B, Swanson R N, Hardy D J, Hanson C W, Coen L, Rasmussen R R, Chen R H. 1989. Pacidamycins, a novel series of antibiotics with anti-

Pseudomonas aeruginosa activity. III. Microbiologic profile. J. antibiotic. XLII (4). 521-526.

Heijenoort, JV .2001. Recent advances in the formation of bacterial peptidoglycan monomer unit. Nat. Prod. Rep. 18:503-519.

Heydanek MG, Struve WG, Neuhaus FC. 1969. Initial state in peptidoglycan synthesis. III. Kinetics and uncoupling of phospho-N-acetylmuramylpentapeptide translocase (uridine 5'-phosphate). Biochem. 63(8): 1214-1221.

Hirano S, Ichikawa S, Matsuda A. 2008a. Synthesis of caprazamycin analogues and their structure activity relationship for antibacterial activity. J. Org. Chem. 73(2): 569-577.

Hirano S, Ichikawa S, Matsuda A. 2008b. Structure-activity relationship of truncated analogs of caprazamycins as potential anti-tuberculosis agents. Bioorg. Med. Chem. 16: 5123-5133.

Hotoda H, Furukawa M, Daigo M, Murayama K, Kaneko M, Muramatsu Y, Ishii M M, Miyakoshi S, Takatsu T, Inukai M, Kakuta M, Abe T, Harasaki T, Fukuoka T, Utsui Y, Ohya S. 2003a. Synthesis and antimycobacterial activity of capuramycin analogues. Part 1: substitutions of the azepan-2-one moiety of capuramycin. Bioorg. Med. Chem. Lett. 13: 2829-2832.

Hotoda H, Daigo M, Furukawa M, Murayama K, Hasegawa C A, Kaneko M, Muramatsu Y, Ishii M M, Miyakoshi S, Takatsu T, Inukai M, Kakuta M, Abe T, Fukuoka T, Utsui Y, Ohya S. 2003b. Synthesis and antimycobacterial activity of capuamycin analogues. Part 2: acylated derivatives of capuramycin-related compounds. Bioorg. Med. Chem. Lett. 13: 2833-2836.

Ichikawa S. 2008. Nucleoside chemistry based on nucleoside natural products synthesis. Chem Pharm. Bull. 56(8): 1059-1072.

Ichikawa S, Matsuda A. 2007. Nucleoside natural products and related analogs with potential therapeutic properties as antibacterial and antiviral agents. Expert Opin. Ther. Pat. 17(5): 487-498.

Igarashi M, Takahashi Y, Shitara T, Nakamura H, Naganawa H, Miyake T, Akamatsu Y. 2005. Caprazamycins, novel lipo-nucleoside antibiotics, from Streptomices sp. J. Antibiot. 58(5): 327-337.

Inukai M, Isono F, Takatsuki A. 1993. Selective inhibition of the bacterial translocasereaction in peptidoglycan synthesis by mureidomycins. Antimicrob. Agents Chemother.37(5): 980-983.

Isono K, Uramoto M, Kusakabe H, Kimura K I, Igasi K, Nelson C C, McCloskey JA. 1985. Liposidomycins: novel nucleoside antibiotics which inhibit bacterial peptidoglycan synthesis. J. Antibiotics. 38(11): 1617-1621.

Isono F, Kodoma K, Inukai M. 1992. Susceptibily of Pseudomonas species to the novel antibiotics


mureidomycins. Antimicob. Agents Chemother. 36(5): 1024-1027.

Kanokmedhakul S, Kanokmedhakul K, Kanarsa T, Buayairaksa. 2005. New Bioactive Clerodane Diterpenoids from the Bark of Casearia grewiifolia. J. Nat. Prod. 68: 183-188.

Kanokmedhakul, S, Kanokmedhakul K, Nambuddee K, Kongsaeree P. 2004. New Bioactive Prenylflavonoids and Dibenzocycloheptene Derivative from Roots of Dendrolobium lanceolatum. J. Nat. Prod. 67: 968-972.

Kimura KI, Iked Y, Kagami S, Yoshihama M, Suzuki K, Osada H, Isono K. 1998. Selective inhibition of the bacterial peptidoglycan biosynthesis by the new types of liposidomycins. J. Antibiotics. 51(12): 1099-1104.

Kimura K, Bugg TDH. 2003. Recent advances in antimicrobial nucleoside antibiotics targeting cell wall biosynthesis. Nat. Prod. Rep. 20: 252-273.

Koga T, Fukuoka T, Doi N, Harasaki T, Inoue H, Hotoda H, Kakuta M, Muramatsu Y, Yamamura N, Hoshi M, Hirota T. 2004. Activity of capuramycin analogues against Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare in vitro and in vivo. J. Antimicrob. Chemother. 54: 755-760.

Lin Y, Li Z, Francisco GD, Mcdonald LA, Davis RA, Singn G, Yang Y, Mansour T S. 2002. Muraymicyns, novel peptidoglycan biosynthesis inhibitors:

semisynthesis and SAR of their derivatives. 12(17): 2341-2344.

McDonald LA, Barbieri LR, Carter GT, Lenoy E, Lotvin J, Petersen PJ, Siegel MM, Singh G, Williamson R T. 2002. Structures of muraymicyns. novel peptidoglycan biosynthesis inhibitors. J. Am. Chem. Soc. 124 (35): 10260-10261.

Nikaido H, Saier JR MH. 1994. Transport proteins in bacteria: common themes in their design. Science. 258(5084): 936-942.

Sayed KAE, Hamann MT, EL-Rahman HA A, Zaghloul AM. 1998. New Pyrrole Alkaloids from Solanum sodomaeum. J. Nat. Prod. 61: 848-850.

Struve WG, Sinha RK, Neuhaus FC. 1966. On the initial stage in peptidoglycan synthesis. Phospho-N-acetylmuramylpentapeptide tranlocase (uridine monophosphate). Biochem. 5(1): 82-93.

World Health Organization. Tuberculosis (TB). http://www.who.int/tb/en/ (consulted June 16, 2009)

Xu ZQ, Barrow WW, Suling WJ, Westbrook L, Barrow E, Lin Y M, Flavin M .T. 2004. Anti-HIV natural product (+)-calanolide A is active against both drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis. Bioorg. Med. Chem. 12: 1199-1207.


http://www.who.int/tb/en/

© 2009 The Authors

© 2009 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (1), 13 - 19


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Antioxidant and anti-inflammatory activity of Moussonia deppeana

[Actividad antioxidante y anti-inflamatoria de Moussonia deppeana]

Miguel A. DOMÍNGUEZ-ORTIZ,1*

Omar MUÑOZ-MUÑIZ,2*

Rosa Virginia GARCÍA-RODRÍGUEZ,2

Maribel VÁZQUEZ-HERNÁNDEZ,2 Janeth GALLEGOS-ESTUDILLO,

2 Jesús Samuel CRUZ-SÁNCHEZ.

1

1 Instituto de Ciencias Básicas, Universidad Veracruzana; 2 Unidad de Servicios de Apoyo en Resolución Analítica, Universidad

Veracruzana, Luis Castelazo Ayala S/N, Col. Industrial Ánimas, CP. 91190 Xalapa, Ver. México.

Abstract

Moussonia deppeana (Schldl. & Cham) Hanst is a species of Mexican Medicinal Flora used in Veracruz state, to treat sufferings related to stomach

pain, renal diseases, cough, tumors and inflammation. Obtained results showed that EtOAc extract was the most active in free radical scavenging test DPPH (CI50 18.3±3.4 µg/mL) with 41% of reducing power respect to ascorbic acid and total content of polyphenols was smaller (328.9±7.6 mg GAE/g) than the

found in the ethanol extract (388.6±6.2 mg GAE/g). Anti-inflammatory activity was evaluated by topical application of the extracts (doses 2 mg/ear) giving a

greater inhibition in hexane and EtOAc extracts (39 and 28%, respectively). The model of paw edema was evaluated in EtOAc extract, observing a similar inhibition to indomethacin (43% with 100 mg of dose) at the first hour. These results support the biological effect attributed in their traditional use.

Keywords: Anti-inflammatory, Antioxidant, Moussiana deppeana, Ethnopharmacology, Medicinal Plants.

Resumen

Moussonia deppeana (Schldl. & Cham) Hanst, es una especie de la Flora Medicinal Mexicana usada en el estado de Veracruz, para tratar padecimientos relacionados con dolor estomacal, enfermedades renales, tos, tumores e inflamación. Los resultados obtenidos mostraron que el extracto de EtOAc fue el más

activo en la prueba de DPPH (CI50 18.3±3.4 µg/mL), con un poder reductor de 41% respecto al ácido ascórbico y el contenido total de polifenoles fue menor

(328.9±7.6 mg GAE/g) al encontrado en el extracto etanólico (388.6±6.2 mg GAE/g). La actividad anti-inflamatoria evaluada mediante aplicación tópica de los extractos (dosis de 2 mg/oreja) dio mayor inhibición con el extracto hexánico, seguida del EtOAc (39 y 28%, respectivamente). El modelo del edema

plantar fue evaluado únicamente en el extracto de EtOAc observándose una inhibición similar a indometacina (43% a dosis de 100 mg de extracto) en la primera hora. Los resultados apoyan el efecto biológico atribuido en su uso tradicional.

Palabras Clave: Anti-inflamatorio, Antioxidante, Moussiana deppeana, Etnofarmacología, Plantas Medicinales.

Recibido | Received: May, 29, 2009.

Aceptado en Versión Corregida | Accepted in Corrected Version: October 15, 2009.

Publicado en Línea | Published Online 15 December 2009

Declaración de intereses | Declaration of interests: authors have no competing interests. Todos los autores contribuyeron de igual manera en el manuscrito.

Financiación | Funding: This work was financed by Universidad Veracruzana.

This article must be cited as: Miguel A. Domínguez-Ortiz, Omar Muñoz-Muñiz, Rosa Virginia García-Rodríguez, Maribel Vázquez-Hernández, Janeth Gallegos-Estudillo, Jesús

Samuel Cruz-Sánchez. 2010. Antioxidant and anti-inflammatory activity of Moussonia deppeana. Bol Latinoam Caribe Plant Med Aromat 9(1):13 – 19. {EPub 15 December

2009}.

*Contactos | Contacts: [email protected]; Phone: +52-228-841-8900 ext. 13554; Fax: +52-228-841-8917.


Domínguez-Ortíz et al. Antioxidant and Anti-inflammatory Activity of Moussonia deppeana

www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol.9 (1) 2010 | 14

INTRODUCTION

Moussonia deppeana (Schldl. & Cham)

Hanst, belongs to Gesneriaceae family (synonyms:

Kolheria deppeana, Gesneria deppeana, Moussonia

elongate), this species is broad distributed since

Mexico to Panama. In folklore medicinal is

commonly known as clachichinole, tlachichinole,

tochomitillo or valletina (Escalante, 1988). This plant

is frequently used by Mexican people in traditional

medicine because of their curative properties

(stomach inflammation, diarrhea, ulcer, kidney

disease, vaginal infection and some tumors). In this

sense, several extracts of these plants have been

studied as antiprotozoal (Calzada et al., 1998) and

some reports revealed the presence of β-sitosterol, β-

D-glucosyl-sitosterol, ursolic acid, oleanolic acid,

2β,3β-dihydroxy-olean-12-en-28-oic acid, 2α,3α-

dihydroxy-olean-12-en-28-oic acid (Noguera et al.,

1994); 2-methyl-anthraquinone, chromanone and

stigmasterol (Reyes-Blas, 1995).

Gesneriaceae family is very extensive and

includes tropical herbs and shrubs; many of them are

growth as ornamentals (Martínez, 1969; Alcántara

and Luna, 2001). In some members of this family

have been isolated anthocyanins, flavonoids and

flavones (Díaz, 1976; Gould and Lister, 2006). Of

particular interest was the isolation of some

glycosylated rutine derivatives by Robinson and

Tood (Robinson et al., 1934), which have a well-

defined anti-inflammatory activity.

By the other hand, is well known that oxygen

and nitrogen reactive species play important roles in

normal physiological processes, protection from

pathogens, cellular signaling pathways, and

regulation of vascular tone (Valko et al., 2007); also,

they are related to development of tissue damage in

various human diseases such as cancer, aging,

neurodegenerative disease, malaria and pathological

events in living organism (Gutteridge, 1994).

In this sense, antioxidant capacity of

medicinal plants and herbs has been linked to in vivo

protection from oxidative stress in numerous studies

(Prior et al., 2005) but rarely has been associated with

anti-inflammatory capacity (Jensen et al., 2008). For

this reason, the present study is aimed to the

evaluation of antioxidant and acute anti-inflammatory

effect of several extracts of M. deppeana growing in

Mexico by using 1,1-diphenyl-2-picrylhydrazyl

(DPPH) radical scavenging assay (Brand-Williams et

al., 1995; Miliauskas et al., 2004); reducing power

with FeCl3 test (Oyaizu, 1986); total polyphenols by

Folin-Ciocalteau assay (Spanos and Wrosltad, 1990)

as well as 12-O-tetradecanoylphorbol 13-acetate

(TPA) induced mouse ear edema model (Young and

De Young, 1989) and carrageenan induced mouse

paw edema model (Levy, 1969) for determination of

anti-inflammatory properties.

MATERIALS AND METHODS

All chemical used were analytical grade. 1, 1-

Diphenyl-2-picryl hydrazyl (DPPH), Folin-

Ciocalteu´s reagent, potassium ferricyanide

(K3Fe[CN]6), ferric chloride (FeCl3), gallic acid

monohydrate, ascorbic acid, carrageenan lambda, 12-

O-tetradecanoylphorbol 13-acetate and solvents were

obtained from Sigma-Aldrich (México).

Absorbance in colorimetric determinations

was measured in a UV-Vis Varian spectrophotometer

Cary100 model.

Vegetal material

M. deppeana was collected in Rancho Viejo-

Cinco Palos Municipality of Coatepec, Veracruz

State, Mexico in October 2007. The taxonomic

identification of plants was confirmed by Luis

Hermann Bojorquez Galván, a taxonomist. A

voucher specimen (CIB8987) has been deposited in

the Instituto de Investigaciones Biológicas Herbarium

of Universidad Veracruzana.

Preparation of crude plant extracts

About 800 g of aerial part of plant material

were cut into small pieces, dried at room temperature

and extracted by exhaustive maceration in darkness

with different solvents. Three extracts were obtained

successively and solvent was removed using rotary

evaporator (Hexane, 1.68 g; EtOAc, 2.01 g and

EtOH, 7.3 g). Crude extracts were kept in amber

colored glass vials at room temperature for further

use.

Phytochemical analysis

Phytochemical analysis of the plant extracts

was undertaken using standard qualitative methods

(color test and/or Thin Layer Chromatography, TLC).

Two milligrams of each extract were dissolved in

chloroform (5 mL) before application to TLC plates

(2×6 cm). The elution systems were benzene/acetone

(9:1) for hexane and EtOAc extracts meanwhile for

the ethanol extract was used a mixture butanol/water/



acetic acid (6:3:1). The revealing agents were:

Dragendorff solution (for alkaloids), AlCl3 1% in

ethanol (for flavonoids), ZnCl2 (for sapogenins),

KOH 10% in ethanol (for coumarins), perchloric acid

(for sterols) and NaOH (for quinones) (Domínguez,

1973; Kaufman et al., 2006).

Animals

All animals employed in the experiments

were CD1 male mice (20-25 g) obtained from

Facultad de Medicina of the Universidad

Veracruzana, Xalapa. The animals were acclimated

for one week in photoperiods adjusted to 12 hours of

light and 12 hours darkness daily and 50-55%

relative humidity with standard pellet diet (Rodent

chow) and drink ad libitum. This work was

performed according to the Guide for the Care and

Use of Laboratory Animals (National Academy of

Sciences, 1996) and the Official Mexican Norm

(NOM-062-ZOO-1999).

Determination of Antioxidant Capacity

Free radical scavenging by the use of DPPH

radical

The DPPH radical scavenging capacity of

each extract was determined according to Brand-

Williams method modified by Miliauskas, 2004.

DPPH radicals have and absorption maximum at 517

nm, which disappears with reduction by an

antioxidant compound. The DPPH radical solution in

methanol (9 x 10-5

M) was freshly prepared, and 2.9

mL of this solution was mixed with 100 µL of

methanolic solutions of plant extracts at several

concentrations (33, 16.5 and 8.25 µg/mL). The

samples were incubated for 30 min at 37 °C in a

water bath, and decrease in absorbance at 517 nm

was measured (AE). A blank sample containing 100

µL of methanol in the DPPH radical solution was

prepared daily, and its absorbance was measured

(AB). Radical scavenging activity was calculated

using the following formula:

100% xA

AAInhibition

B

EB

Determination of total phenolic content

The total phenolic concentration was

determined using the Folin-Ciocalteu´s reagent

according to the Spanos and Wrosltad, 1990. To 50

µL of each sample (1 mg/mL, three replicates), 2.5

mL 1/10 dilution of Folin-Ciocalteu´s reagent and 2

mL of Na2CO3 (7.5 %, w/v) were added and

incubated at 45 °C for 15 min. The absorbance of all

samples was measured at 765 nm using a UV-Vis

spectrophotometer. Results were expressed as gallic

acid equivalent (µg/mL) by using the following

equation, which was obtained from standard gallic

acid graph (range 20 to1000 µg/mL).

075.0)]/([001.0 mLgGAEAbsorbance

Determination of reducing power

The reducing power was determined

according to the method described by Oyaizu, 1986.

A 0.125 mL aliquot of extract (1 mg/mL) was mixed

with 1.25 mL of 200 mM sodium phosphate buffer

(pH 6.6) and 1.25 mL of 1% of potassium

ferricyanide. The mixture was then incubated at 50

°C for 20 min. After 1.25 mL of 10% trichloroacetic

acid (w/v) were added, the mixture was centrifugated

at 650 g for 10 min. A 2.5 mL aliquot of the upper

layer was mixed with 5 mL of destilled water and 1

mL of 0.1% ferric chloride, and the absorbance at

700 nm was measured. The obtained value was

compared with the ascorbic acid value as standard.

Anti-inflammatory evaluation

12-O-tetradecanoylphorbol 13-acetate (TPA)-

induced mouse ear edema.

Irritant dermatitis was induced on the right

ear by topical application of 2.5 g TPA in 25 L of

acetone according to the methodology reported by

Young and De Young, 1989. TPA was applied on

both the inner and outer surfaces of the ears. The

extracts (doses 2 mg/ear) and the indomethacin

(doses 2 mg/ear) were applied topically 30 minutes

after TPA to the right ear (E´t), the left ear received

vehicle (E´0). In the control group the right ear

received only TPA (Et) and the other ear acetone (E0).

In all groups the edema was allowed to develop for 6

hours; afterwards the animals were sacrificed by

dislocation cervical and plugs (diameter of 6 mm) of

the central portion were taken from both ears and

weighted. The inhibition of auricular edema was

calculated with the difference between weight ears of

the animal treatment with the extract or indomethacin

and the control group.



100)(

)´´()(%

0

00 xcontrolEE

treatedEEcontrolEEInhibition

t

tt

Carrageenan-induced mouse paw edema

20 µL/paw of a 1% carrageenan solution was

injected into the sub-plantar region of the left hind

paw of the mouse. The increment in the paw thick

was determined with a digital micrometer at 1, 3, 5

and 7 h after carrageenan administration. A reference

group was administered intraperitoneally with

indomethacin (doses 5 mg/kg). The extract was

administered at doses of 100 and 300 mg/kg by the

same via before carrageenan administration. The

control group received vehicle only. The percent

edema inhibition was calculated for each animal

group in comparison to group treated with vehicle

(10% Tween 80) according to the Olajide et al, 2000.

Statistical analysis of data

Data are presented as means ± S. D. of at

least triplicate experiments. For in vivo experiments,

data is reported as the means ± S.E.M. Significant

differences between groups were determined by

analysis of variance (ANOVA) complemented with

Dunnett´s test using the software Statistica version 7

from StatSoft, Inc. (2004), p<0.05 was considered

significant.

RESULTS AND DISCUSSION

Reactive oxygen species (ROS) may be

involved in the etiologies of several human diseases

as atherosclerosis, ischemic injury, cancer and

neurodegenerative diseases, as well as in processes

like inflammation and ageing (Edmonds, 2000). Also,

there is evidence that antioxidants may be useful in

preventing the deleterious consequences of oxidative

stress and there is increasing interest in the protective

biochemical functions of natural antioxidants

containing in medicinal plants. In this sense, our

attention has been focused in the antioxidant and

anti-inflammatory properties of M. deppeana, a

medicinal plant for the treatment of diabetes, stomach

inflammation, kidney diseases, cough, and tumoral

disease (Cano-Asseleih, 1997).

The phytochemical screening (Table 1)

revealed the presence of phenolic compounds

(flavonoids and coumarins) in EtOAc and EtOH

extracts. In the hexane and EtOAc extracts were

possible to observe a positive result for sterols and

quinones, which supports the presence of some

metabolites previously reported (Noguera et al., 1994

and Reyes-Blas, 1995).

Table 1. Phytochemical screening.

Test Extracts

Hexane EtOAc EtOH

Alkaloids - - -

Flavonoids - + +

Sapogenins - - +

Coumarins - + +

Quinones + + -

Sterols + + -

In many cases, the presence of flavonoids,

quinones and coumarins are associated with

antioxidant properties due to their role in several

human diseases where ROS could be involved. On

the other hand, these components could be presents

actively in inflammatory processes, in fact,

antioxidant/anti-inflammatory activity have been

related intrinsically to these chemical substances

(Takahashi and Shibamoto, 2008; Jensen et al.,

2008).

In order to evaluate the antioxidant efficiency

of plant extracts, the radical scavenging capacity

based in DPPH assay was determined and the results

are shown in Table 2. In this sense, the percentage of

inhibition of the DPPH radical varied from 41.1% for

the hexane extract to 92.4% for the EtOAc extract,

which represents a variation of approximately 2-fold

respect to the hexane extract. The EtOAc extract

showed the highest antioxidant activity index (AAI)

(Scherer and Teixeira-Godoy, 2009) following EtOH

extract and Hexane extract (1.9, 1.6 and 0.9,

respectively).

Table 2. Antioxidant capacity of the extracts of M. deppeana

based on DPPH test.

Extract DPPH

Inhibition

(%)

DPPH

IC50

(µg/mL)

AAI*

Hexane 41.1 ± 1.2 40.5 ± 1.3 0.9

EtOAc 92.4 ± 2.1 18.3 ± 3.4 1.9

EtOH 70.1 ± 1.8 22.0 ± 0.4 1.6

Phenylbutazone 47.8 ± 4.4 34.4 ± 3.2 1.0

Rutin 93.5 ± 1.1 4.8 ± 0.1 7.4

Ascorbic acid** 100 0.6 ± 0.1 61.2

Data expressed as mean ± SD (n=3). DPPH radical solution in

methanol (9 x 10-5 M, 35.48 µg/mL) using (33µg/mL) of plant

extract and ascorbic acid and rutin (5 µg/mL). * AAI = Final

concentration of DPPH (μg.ml-1) / IC50 (μg.ml-1). ** No

significance differences in IC50 were observed between this study

and results previously reported (Sharma and Bhat, 2009).



The DPPH free radical scavenger capacity of

the extracts was compared with well known

antioxidant (ascorbic acid and rutin) and anti-

inflammatory compound phenylbutazone. Despite the

fact that extracts had been a significant antioxidant

activity index (ca. 2, see Table 2), the obtained values

were lower than rutine and ascorbic acid (7.4, 61.2

respectively) but slightly higher than phenylbutazone

in the case of EtOAc extract (1.0 vs 1.9).

Taking into account the results in radical-

scavenging assay, it was expected that total phenol

content and reducing power had presented the same

behavior (Paśko et al., 2009); the EtOH extract had

the highest concentration of total phenol (388.6±6.2

µg GA/mL, Table 3) close to the EtOAc extract

(328.9±7.6 µgGA/mL, Table 3). In the reducing

power assay the EtOAc extract had shown the highest

value over the EtOH extract (41.3% vs 29.0%

respectively, Table 3). These observations indicated a

linkage between phenolics concentration, reducing

power and antioxidant activity; on the other hand, in

all assays hexane extract always showed the lowest

values in each test (Table 2 and 3).

Table 3. Total phenolic content and power reducing of the

extracts of M. deppeana.

Extract Total phenols Reducing power

(%)

Hexane 20.6 ± 1.5 5.3 ± 0.4

EtOAc 328.9 ± 7.6 41.3 ± 0.5

EtOH 388.6 ± 6.2 29.0 ± 1.1

Data expressed as mean ± SD (n=3). Total phenols expressed in

mgs. of Gallic acid equivalent per g of sample (mgGAE/g).

Reducing power is expressed with respect to ascorbic acid

(33µg/mL).

The propagation of free radical can bring

many adverse reactions leading to extensive tissue

damage. Lipids, proteins and DNA are very

susceptible to attack by free radical (Yu et al, 1992).

In this way, all antioxidant test evaluated in this work

showed that M. deppeana has a great amount of

antioxidant compounds that may offer resistance

against oxidative stress by scavenging the free

radicals and inhibiting lipid peroxidation.

Given the results in the antioxidant assays,

our attention now was focused in the evaluation of

the anti-inflammatory activity. In the ear edema

induced with topic application of TPA, the hexane

and EtOAc extracts produced the mayor inhibition of

the edema, 39 and 28%, respectively (Table 4). At 2

mg/ear dose, the inhibition edema was similar

between hexane extract and esculetin (39 and 38%

respectively, Table 4) but lower than indomethacin

(69%). In this test, ethanol extract did not show a

significant inhibition.

Table 4. Anti-inflammatory activity of M. deppeana on ear

edema induced with TPA.

Treatment Extract Dose

(mg/ear)

Edema

inhibition

(mg)

Inhibition

(%)

Control 14.4 ± 0.6 0

M. deppeana

Hexane

2

8.8 ± 0.6*

39

EtOAc

Ethanol

2

2

10.4 ± 0.7

13.8 ± 0.5

28

4

Indomethacin 2 4.5 ± 0.5* 69

β-sitosterol 1 9.9 ± 0.5* 31

Esculetin 1 8.9 ± 0.8* 38

Values are expressed as mean ± S. E. M. The inhibition (%) was

calculated respect to control group. * ANOVA-Dunnett´s, p <

0.05, n = 6.

In this regard, inflammation induced by TPA

leads to protein kinase C activation, which is related

to activation of phospholipase A2 with releases

arachidonic acid from membrane cells that is

metabolized to prostaglandins and leukotrienes

(Recio et al., 2000). The inhibition observed in

hexane extract could be related to this mechanism.

According to the obtained results in

preliminary anti-inflammatory test (TPA), the hexane

and EtOAc extract have the major inhibition;

however, we chosen the EtOAc, due to the low

solubility presents in hexane extract, in order to

confirm the biological effect with the paw edema

induced with the carrageenan model in mouse applied

by systemic route.

The EtOAc extract of M. deppeana (100 and

300 mg/kg dose) applied 30 min before of

carrageenan did not reduce the edema formation

significantly respect to the control group. In contrast,

the indomethacin to 5 mg/kg dose reduced the edema

formation in all time registered.

Surprisingly, paw edema decrease 43% with

lower doses of the extract (100 mg/Kg) only at the

first hour; this effect was similar to indomethacin

(Table 5).

In the acute inflammation induced by

carrageenan, levels of substance P in inflamed paw

increase within 15 minutes after induction, these

levels remained elevated during the first 2 h of

inflammation (Gilligan et al., 1994). Other mediators

of inflammation as serotonin, histamine and

bradykinin play important roles during the early



phase of carrageenan edema (Di Rosa et al., 1971).

According that the observed effect in EtOAc extract

could be related to some of these inflammation

mediators.

Table 5. Anti-inflammatory activity of EtOAc extract of M.

deppeana on paw edema produced with carrageenan test.

Time

(h)

Paw edema (mm) and % inhibition

Control Indomethacin

5 mg/kg M. deppeana

100 mg/kg M. deppeana

300 mg/kg

1 0.7±0.1 0.4 ± 0.1*

(43%)

0.4± 0.1*

(43%)

0.6 ± 0.1

(14%)

3 1.1±0.1 0.7 ± 0.1*

(36%)

1.0 ± 0.1

(9%)

1.0 ± 0.1

(9%)

5 1.2±0.1 0.7 ± 0.1*

(42%)

1.0 ± 0.1

(17%)

1.1 ± 0.1

(8%)

7 1.0±0.1 0.7 ± 0.1*

(30%)

1.1 ± 0.2

(NE)

1.0 ± 0.1

(NE)

Values are expressed as mean ± S.E.M. The inhibition (%) was

calculated respect to control group. * ANOVA-Dunnett´s, p <

0.05, n = 8.

With these results, it was not possible to find

a correlation between antioxidant and anti-

inflammatory effects on the models used. In fact,

those observations indicate that responsible

compounds of both activities are not the same;

because in the anti-inflammatory topic test (TPA) the

hexanic extract was the best and the EtOAc extract

had the higher antioxidant activity. Further

investigations in the phytochemistry field and other

biological activities are being developed in our group

in order to get more information respect to action

mechanism and chemical composition.

CONCLUSION

Anti-inflammatory activity was observed in

the hexanic and EtOAc extracts of M. deppeana only

by topical application. The systemic administration of

low doses of EtOAc extract produced a similar

inhibition percent observed with indomethacine only

a short period of time. In the case of the antioxidant

activity, the EtOAc was the best extract evaluated

following by EtOH and Hexane extract; however, it

was not possible to find a correlation between the

anti-inflammatory and antioxidant activities with the

used assays. Finally, the biological activity of M.

deppeana observed in the polar extracts supports its

traditional use.

REFERENCES

Alcántara O, Luna I. 2001. Análisis florístico de dos áreas

con bosque mesófilo de montaña en el estado de

Hidalgo, México: Eloxochitlán y Tlahuelompa. Acta

Botánica Mexicana. 54: 51-87.

Brand-Williams W, Cuvelier ME, Berset C. 1995. Use of a

free radical method to evaluate antioxidant capacity.

LWT. 28(1): 25-30.

Calzada F, Meckes M, Cedillo-Rivera R, Tapia-Contreras

A, Mata R. 1998. Screening of Mexican Medicinal

Plants for Antiprotozoal Activity. Pharm. Biol. 36(5):

305-309.

Cano-Asseleih LM. 1997. Flora medicinal de Veracruz,

pp. 224. Ed. Universidad Veracruzana, Xalapa,

Veracruz-México.

Di Rosa M, Giroud JP, Willoughby DA. 1971. Studies of

the mediatiors of the acute inflammatory response

induced in rats in different sites by carrageenan and

turpentine. J. Pathol. 104:15-29.

Díaz JL. 1976. Índice y sinonimia de las plantas

medicinales de México. pp. 59, 259. Ed. Instituto

Mexicano para el Estudio de las Plantes Medicinales,

México.

Domínguez XA. 1973. Métodos de Investigación

Fitoquímica, pp. 81-85. Ed. Limusa, México.

Edmonds S. 2000. Do antioxidants have a role in the

therapy of human inflammatory diseases?, pp. 241-

252. In Winyard PG, Blake DR, Evans CH: Free

radicals and inflammation. Ed. Birkhäuser, Boston,

USA.

Escalante LI. 1988. Notas sobre el Tlachichinole. Tesis de

licenciatura, Universidad Nacional Autónoma de

México, México, D. F., pp. 9-11.

Gilligan JP, Lovato SJ, Erion MD, Jeng AY. 1994.

Modulation of carrageenan-induced hind paw edema

by substance P. Inflammation. 18: 285-292.

Gould KS and Lister C. 2006. Flavonoid functions in

Plants, pp. 397-442. In Andersen ØM and Markham

KR: Flavonoids: chemistry, biochemistry, and

applications. Ed. CRC Press, Boca Raton, FL.

Gutteridge JMC. 1994. Biological origin of free radicals

and mechanism of antioxidant protection. Chem. Biol.

Interact. 91: 133-140.

Jensen GS, Wu X, Patterson KM, Barnes J, Carter SG,

Scherwits L, Beaman R, Endres JR, Schauss AG.

2008. In vitro and in Vivo Antioxidant and Anti-

inflammatory Capacities of an Antioxidant-Rich Fruit

and Berry Juice Blend. Results of a Pilot and

Randomized, Double-Blinded, Placebo-Controlled,

Crossover Study. J. Agric. Food Chem. 56(18): 8326-

8333.

Kaufman PB, Hoyt JE, Bergman S, Madsen BJ, Perry M,

Warber S, Peschel K. 2006. Case Studies, pp. 263-

318. In: Cseke LJ, Kirakosyan A, Kaufman PB,

Warber SL, Duke JA, Brielmann HL: Natural

Products from Plants. Ed. CRC Press, Boca Raton,

FL, USA.

Levy L. 1969. Carrageenan paw oedema in the mouse.

Life Sciences. 8: 601-606.



Martínez M. 1969. Plantas medicinales de México, Ed.

Botas, México, pp. 321.

Miliauskas G, Venskutonis PR, Van Beek TA. 2004.

Screening of radical scavenging activity of some

medicinal and aromatic plant extracts. Food Chem.

85: 231-237.

National Academy of Sciences. 1996. Guide for the care

and use of laboratory animals. Ed. National Academy

Press, Washington D. C. USA.

Noguera B, Camacho MR, Mata R. 1994. Constituents of

Kholeria deppeana. Fitoterapia. LXV(2): 182.

Norma Oficial Mexicana. 2001. NOM-062-ZOO-1999.

Especificaciones técnicas para la producción, cuidado

y uso de los animales de laboratorio. Diario Oficial de

la Federación. 6 de diciembre de 1999.

Olajide OA, Awe SO, Makinde JM, Ekhelar AI, Olusola

A, Morebise O, Okpako DT. 2000. Studies on the

anti-inflammatory, antipyretic and analgesic

properties of Alstonia boonei stem bark. J.

Ethnopharmacol. 71: 179-186.

Oyaizu M. 1986. Studies on products of browning

reaction: antioxidative activity of products of

browning reaction prepared from glucosamine. J.

Nutr. 44: 307-315.

Paśko P, Bartoń H, Zagrodzki P, Gorinsein S, Fołta M,

Zachwieja Z. 2009. Antocyanins, total polyphenols

and antioxidant activity in amaranth and quinoa seeds

and sprouts during their growth. Food Chem. 115:

994-998.

Prior RL, Wu X, Schaich K. 2005. Standarized methods

for determination of antioxidant capacity and

phenolics in foods and dietary supplements. J. Agric.

Food Chem. 53: 4290-4302.

Recio MC, Ginet RM, Uribun L, Máñez S, Cerdán M, De

la Fuente JR, Rios JL. 2000. In vivo activity of

pseudoguaianolide sesquiterpene lactone in acute and

chronic inflammation. Life Sciences, 66: 2509-2518.

Reyes-Blas H. 1995. Estudio Fitoquímico de la raiz de

Kolheria deppeana. Tesis de Licenciatura,

Universidad Veracruzana, México, pp 1-71.

Robinson GM, Robinson R, Todd AR. 1934. J. Chem. Soc.

809.

Scherer R, Teixeira-Godoy H. 2009. Antioxidant activity

index (AAI) by the 2,2-diphenyl-1-picrylhydrazyl

method. Food Chem. 112: 654-658.

Spanos GA.; Wrosltad, R. E. 1990. Influence of processing

and storage on the phenolic composition of Thompson

seedless grape juice. J. Agric. Food Chem. 38: 1565-

1571.

Takahashi N and Shibamoto T. 2008. Chemical

compositions and antioxidant/anti-inflammatory

activities of steam distillated from freeze-dried onion

(Allium cepa L.) sprout. J. Agric. Food Chem. 56(22):

10462-10467.

Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M,

Telser J. 2007. Free radicals and antioxidants in

normal physiological functions and human disease.

Int. J. Biochem. Cell. B. 39: 44-84.

Young JM, De Young LM. 1989. Coetaneous models of

inflammation from the evaluation of topical and

systemic pharmacological agents, pp. 215-231. In

Chang, J. Y.; Lewis, A. J.; Alan, R.: Pharmacological

methods in the control of inflammation. Ed. Liss Inc.

New York, USA.

Yu BP, Suescun EA, Yang SY. 1992. Effect of age-related

lipid peroxidation on membrane fluidity and

phospholipids A2: modulation by dietary restriction.

Mech. Ageing Dev. 65: 17.


BLACPMA ISSN 0717 7917 Articulo Original | Original Article



Phytochemical and biological investigations of Caryocar brasiliense Camb [Estudios fitoquímicos y biológicos sobre Caryocar brasiliense Camb]

Jociani ASCARI , Jacqueline Aparecida TAKAHASHI, Maria Amélia Diamantino BOAVENTURA*

Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, CEP 31270-970. Belo Horizonte – MG, Brasil.

Abstract

Caryocar brasiliense epicarp and external mesocarpe were chemically and biologically evaluated. From the phytochemical study, ethyl gallate, 5-hydroxyfurfural, gallic acid, methyl shikimate, and mixtures of β-D-fructopyranose and β-D-fructofuranose, α-and β-D-glucose, lupeol and oleic acid and β-sitosterol, stigmasterol and oleic acid were isolated and spectroscopically identified by NMR (1D and 2D). Tests on the antioxidant, allelopathic and antimicrobial activities were carried out for the crude extract, fractions and pure compounds. Extract and pure compounds showed good activities in all bioassays.

Keywords: Caryocar brasiliense Camb.; phenolic compounds; triterpenes; antimicrobial; antioxidant; allelopathic activity

Resumo

El epicarpo y el mesocarpo externo de Caryocar brasiliense Camb. fueron evaluados química y biologicamente. Del estudio fitoquímico, fueron aislados e identificados por RMN (1D y 2D) galato de etilo, 5-hidroximetilfurfural, ácido gálico, chiquimato de metilo y mezclas de β-D-fructopiranosa y β-D-fructofuranosa, α- y β-D-glucosa, lupeol y ácido oléico, β-sitosterol, estigmasterol y ácido oléico. Fueron realizados ensayos de evaluación del efecto anti-oxidante, anti-microbiano y alelopático para el extracto etanólico crudo, fraciones y compuestos puros, los cuales presentaron buena actividad.

Palavras Chave: Caryocar brasiliense Camb.; compuestos fenólicos; triterpenos; actividad anti-oxidante; actividad antimicrobiana; actividad alelopática.

Recibido | Received: August, 9, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: October 26, 2009. Publicado en Línea | Published Online December 15, 2009. Declaración de intereses | Declaration of interests: authors have no competing interests. Financiación | Funding: CNPq, JAT and MADB for grants. FAPEMIG, for financial support. This article must be cited as: . Jociani Ascari , Jacqueline Aparecida Takahashi, Maria Amélia, Diamantino Boaventura. 2010. Phytochemical and biological investigations of Caryocar brasiliense Camb. Bol Latinoam Caribe Plant Med Aromat 9(1):20 – 28. {EPub 15 Dec 2009 }.



Ascari et al. Phytochemical and biological investigations of Caryocar brasiliense Camb


INTRODUCTION

Caryocaraceae is a small botanic family widely distributed in Central and South America. It is constituted by two genera, Caryocar and Anthodicus, which include 25 species. Caryocar genus presents the higher number of species (16), being the most economically important since their very nutritive fruits are used as source of edible oils and in the preparation of juices and liqueurs (Prance, 1990).

Caryocar brasiliense Camb., known in Brazil as pequizeiro, deserves a distinguished position due to the commercial, nutritional and gastronomic importance of its fruit, named pequi. Pequi is a spherical green fruit, presenting 1-4 segments. Its structure is composed by an epicarp (very thin peel), an external pulpy mesocarp, internal mesocarp (light-yellow, pulpy, rich in oil, vitamins and proteins), that involve a layer of thin and rigid endocarp spines (approximately 2-5 mm large) and white nut (seed). Together, internal mesocarp, needle endocarp and seed constitute one segment (Damiani, 2006). The fruit is used in Central Brazil culinary; fruits and leaves of C. brasiliense are used in the folk medicine to treat cold, edema, bronchitis, cough, burns and is also used as a scaring agent (Vieira and Martins, 2000; Magalhães et al., 1988).

From C. brasiliense leaves β-amyrin, oleanoic and ellagic acids and a mixture of β-sitosterol and stigmasterol were isolated. Edible pulp showed to be rich in vitamins A, C, riboflavin, thiamin and carotenoids (Azevedo and Rodriguez, 2004). The oil extracted from C. brasiliense fruits presented several biological properties such as anti-fungal (Passos et al., 2002), against Trypanosoma cruzi (Herzog-Soares et al., 2002) and Biomphalaria glabrata (Bezerra et al., 2002) activities and also showed to be an effective antioxidant agent (Paula-Junior et al., 2006).

The external mesocarp is the part that presents the biggest dimension in the pequi fruit but it is thrown away since this is a non edible part of the fruit. The disposal of the external mesocarp generates a huge amount of solid residues that could be used, adding value to the plant. In this work, the phytochemical study of this part of the fruit, together with the epicarp, is presented, since no systematic phytochemical study addressing these parts were found in the literature. Twelve compounds were isolated, pure or as constituents of mixtures. Pequi fruit ethanol extract, fractions and some of the isolated compounds were tested for their antioxidant activity using the radical DPPH (1,1-diphenyl-2-picrylhydrazyl)

spectrophotometric assay, as well as for their allelopatic activity, evaluating their effect on the growth of radicule and shoot of Lactuca sativa (lettuce).


General Experimental Procedures Melting points were determined with a Kofler

hot plate apparatus and are uncorrected. Infrared (IR) spectra were recorded with a Spectrum One with ATR-IR, from Perkin Elmer. Nuclear Magnetic Resonance (NMR) spectra were recorded in CD3OD, D2O, CDCl3 and C5D5N, at room temperature, on Bruker Avance DX-200 and DRX 400 MHz spectrometers. Absorbance, in DPPH assay, was measured in a Hitashi 2010 spectrophotometer. Silica gel Merck (Darmstadt, Germany) 100-200 and 200-425 mesh were used for column chromatography and silica gel Merck 60G was used for thin layer chromatography. Polyamide was purchased from Macherey Nagel (Düren, Germany). Solvents PA and HPLC grade were purchased from Vetec (Brazil) and Sigma Chemicals Co (St. Louis, USA), respectively. BHT (2,6-di-tert-butyl-4-methylphenol) and DPPH (1,1-diphenyl-2-picrylhydrazyl), were also purchased from Sigma.

Plant Material Fruits of Caryocar brasiliense were collected

in January 2008, in Curvelo region, Minas Gerais, Brazil, and identified by Dr. João Renato Stehmann. A voucher specimen (No. 120826) was deposited at the Herbarium of the Natural History Museum of UFMG, Belo Horizonte, Minas Gerais, Brazil.

Extraction and Isolation The fruit was open and the internal mesocarp

was discharged. The external mesocarp, together with the epicarp was grinded in ethanol, using a liquefier, and after 14 days at room temperature, the mixture was filtered through a cotton plug followed by Whatman filter paper. The extract was concentrated with a rotatory evaporator and 256.0 g of ethanol extract were obtained. A portion (130.0 g) of it was chromatographed over polyamide column (106.0 g), eluted with water, methanol and ethyl acetate pure and mixtures of decreasing polarities. Twenty-eight fractions of 100 mL each were collected, and reunited, by silica gel TLC, in five groups of fractions (G-1 to G-5). G-1 (72.6 g) was dissolved in water and extracted with diethyl ether; subsequent evaporation of



the solvents afforded 6.0 g of ethereal fraction (G-1E) and 66.6 g of insoluble residue (T1).

G-1E (6.0 g) was submitted to a silica gel column chromatography, with CH2Cl2, CH3OH and H2O, as eluent, either pure or in mixtures of increasing polarity: 48 fractions of 50 mL each were collect, and pooled in 11 groups of fractions. Group of fractions 2 (635.8 mg) was rechromatographed on silica gel column with hexane, CH2Cl2 and ethyl acetate as eluent. A white solid precipitated from several fractions and, by washing with CH2Cl2 and filtration, 1.4 g of a pure compound were obtained. This compound, pure by TLC, was identified as ethyl gallate (1). The solvents from the filtration were evaporated and the residue (150.6 mg) was rechromatographed on silica gel column: group of fractions 31-36 (41.0 mg) was found to be pure by TLC and was identified as 5-hydroxy-methylfurfural (2)

Group of fractions 6 (618.1 mg), from G-1E, was submitted to silica gel column chromatography (eluents: hexane, dichloromethane and ethyl acetate, either pure or in mixtures of increasing polarity). From group of fractions 2 (fractions 20-24), gallic acid (3) was isolated (201.0 mg). From group of fractions 8, from G-1E (594.6 mg), after silica gel column chromatography, 8.0 mg of pure methyl shikimate (4) were isolated.

Part of G-2 (9.5 g), from polyamide column, was chromatographed over silica gel column, with dichloromethane, ethyl acetate and methanol, as eluent. Eight group of fractions were obtained and group of fractions 2 (fractions 16-19) furnished additional 1.03 g of ethyl gallate (1), by precipitation. Group of fractions 4 (fractions 25-28, 2.1 g), from G-2, after rechromatography over silica gel column, gave 146.0 mg of a mixture of β-D-fructopyranose (5) and β-D-fructofuranose (6). Group of fractions 5 (fractions 29-30, 2.3 g), from G-2, was also submitted to chromatography on silica gel and lead to the isolation of a white solid (8.5 mg), that was identified as a mixture of α-D-glucose (7) and β-D-glucose (8). Part of G-3 (8.0 g), from polyamide column, after submitted to silica gel column chromatography, using the same eluent described, gave 9 groups of fractions. Group of fractions 1 (0.3 g) was rechromatographed on silica gel column (eluent hexane/acetone, either pure or in mixtures of increasing polarity), and 23 fractions were obtained. From fractions 1-7 (0.27 g), 8.0 mg of lupeol (9) and oleic acid (10) mixture and 20.0 mg of β-sitosterol (11), stigmasterol (12) and

oleic acid (10) mixture were isolated. From fractions 7-18, 1.15 g of ethyl gallate (1) were obtained by filtration.

Spectrometric data Compound 1 (Ethyl gallate): 3.58 g, 14.0 % yield. white solid, m.p.: 170.2-172.1 oC. IR (KBr, cm-1): 3446, 3290, 2973, 2933, 1704, 1615, 1533, 1469, 1309 and 1250. 1H NMR (400 MHz, C5D5N) δH (H, m, J in Hz): 7.9 (H-2), 7.9 (H-6), 4.3 (H-8, q, 7.2); 1.2 (H-9, t, 7.2). 13C NMR (100 MHz, C5D5N) δC: 121.8 (C-1); 110.6 (C-2); 148.0 (C-3); 141.3 (C-4); 148.0 (C-5); 110.6 (C-6); 167.5 (C-7); 60.8 (C-8); 14.8 (C-9). Compound 2 (5-Hydroxymethylfurfural): 41.0 mg, 0.02 % yield, viscous oil. IR (KBr, cm-1): 3369, 1658, 1582, 1519 and 1018. 1H NMR (200 MHz, CDCl3) δH (H, m, J in Hz): 7.2 (H-3, d, 3.4); 6.5 (H-4, d, 3.4); 9.6 (H-6, s); 4.7 (H-7, s); 2.7 (s, OH). 13C NMR (50 MHz, CDCl3) δC: 152.3 (C-2); 123.0 (C-3); 110.0 (C-4); 160.8 (C-5); 177.8 (C-6); 57.6 (C-7). Compound 3 (Gallic acid). 201.0 mg, 0.08 % yield. white solid, m.p.: 245-248 oC. IR (KBr, cm-1): 3500, 3450-2600, 1650, 1600, 1540 and 1350. 1H NMR (400 MHz, CD3OD) δH (H, m): 7.1 (H-2, H-6, s); 9.0 (OH, s). 13C NMR (100 MHz, CD3OD) δC: 122.1 (C-1); 110.5 (C-2 and C-6); 146.5 (C-3 and C-5); 139.7 (C-4); 170.6 (C-7); Compound 4: Methyl shikimate (8.0 mg, 0.003 % yield): white solid, m.p.: 108-109 oC. IR (KBr, cm-1): 3303, 1715, 1657 and 1233. 1H NMR (400 MHz, CD3OD) δH (H, m, J in Hz) : 6.8 (H-2, m); 4.4 (H-3, br s); 3.7 (H-4, dd, 6.8 and 3.6); 4.0 (H-5, m); 2.2 and 2.7 (H-6, m); 4.8 (H-8, s); 4.5 (OH, br s). 13C NMR (100 MHz, CD3OD) δC: 130.0 (C-1); 139.3 (C-2); 67.4 (C-3); 72.7 (C-4); 68.6 (C-5); 31.6 (C-6); 168.9 (C-7); 52.5 (C-8). Mixture 1 [β-D-fructopyranose (5) and β-D-fructofuranose (6)]: 146 mg, 0.06 % yield. β-D-Frutopyranose (5). 1H NMR (400 MHz, CD3OD) δH (H, m): 3.5 and 3.7 (H-1, m); 3.8 (H-3, br s); 3.9 (H-4, br s); 4.0 (H-5, d); 3.7 and 4.0 (H-6, m). 13C NMR (100 MHz, CD3OD) δC: 64.7 (C-1); 99.3 (C-2); 69.6 (C-3); 72.0 (C-4); 71.4 (C-5); 64.7 (C-6). β-D-fructofuranose (6). 1H NMR (400 MHz, CD3OD) δH (H, m): 3.6 and 3.7 (H-1, m); 4.0-4.1 (H-3, H-4 and H-5, m); 3.5 and 3.8 (H-6, m). 13C NMR (100 MHz, CD3OD) δC: 64.4 (C-1); 103.3 (C-2); 77.7 (C-3); 77.0 (C-4); 83.5 (C-5); 64.4 (C-6). Mixture 2 [α-D-glucose (7) and β-D-glucose (8)]: 8.5 mg, 0.0033 % yield. α-D-Glucose (7). 1H NMR (400



MHz, D2O) δH (H, m, J in Hz): 5.2 (H-1, d, 3.6); 3,4 (H-2, m); 3.7 (H-3, t, 9.6); 3.4 (H-4, t, 9.6); 3.7–3.8 (H-5, m); 3.7 (H-6, dd, 5.6 and 12.4). 13C NMR (100 MHz, D2O) δC: 92.1 (C-1); 71.5 (C-2); 72.8 (C-3); 69.6 (C-4); 71.3 (C-5); 60.6 (C-6). β-D-Glucose (8). 1H NMR (400 MHz, D2O) δH (H, m, J in Hz): 4.6 (H-1, d, 8.2); 3.2 (H-2, t, 8.8); 3.4-3.5 (H-3, H-4, H-5, m); 3.7–3.8 (H-6, m). 13C NMR (100 MHz, D2O) δC: 95.9 (C-1); 74.1 (C-2); 75.7 (C-3); 69.6 (C-4); 75.9 (C-5); 60.7 (C-6). Mixture 3 [lupeol (9) and oleic acid (10)]: 8.0 mg, 0.003 % yield: Lupeol (9). 13C NMR (100 MHz, CDCl3) δC: 38.4 (C-1); 27.5 (C-2); 79.4 (C-3); 39.0 (C-4); 55.6 (C-5); 18.7 (C-6); 34.6 (C-7); 41.2 (C-8); 50.8 (C-9); 37.5 (C-10); 21.3 (C-11); 25.0 (C-12); 38.4 (C-13); 43.2 (C-14); 27.5 (C-15); 35.9 (C-16); 43.3 (C-17); 48.3 (C-18, C-19); 151.3 (C-20); 29.4 (C-21); 40.3 (C-22); 28.3 (C-23); 15.7 (C-24); 16.4 (C-25); 16.3 (C-26); 14.9 (C-27); 18.2 (C-28); 109.6 (C-29); 19.6 (C-30). Oleic acid (10) 13C NMR (100 MHz, CDCl3) δ: 178.7 (C-1); 34.1 (C-2); 25.0 (C-3); 29.6 (C-4); 29.4 (C-5); 29.5 (C-6); 29.6 (C-7); 27.8 (C-8); 130.3 (C-9); 130.1 (C-10); 27.7 (C-11); 29.7 (C-12); 29.9 (C-13); 29.8 (C-14 and C-15); 32.2 (C-16); 23.0 (C-17); 14.4 (C-18). Mixture 4 [β-sitosterol (11), stigmasterol (12) and oleic acid (10)]: 20.0 mg, 0.0075 % yield. β-Sitosterol (11) and Stigmasterol (12). 13C NMR (100 MHz, CDCl3) δC: 37.3 (C-1); 31.7 (C-2); 71.9 (C-3); 42.3 (C-4); 140.8 (C-5); 121.8 (C-6); 32.0 (C-7); 31.9 (C-8); 50.2 (C-9); 36.2 (C-10); 21.1 (C-11); 39.7 (C-12); 42.3 (C-13); 56.9 (C-14); 24.4 (C-15); 28.9 (C-16); 56.0 (C-17); 12.1 (C-18); 19.4 (C-19); 36.2 (C-20 for β-sitosterol); 40.5 (C-20 for stigmasterol); 19.0 (C-21 for β-sitosterol); 21.2 (C-21 for stigmasterol); 34.0 (C-22 for β-sitosterol); 138.3 (C-22 for stigmasterol); 26.1 (C-23 for β-sitosterol); 129.8 (C-23 for stigmasterol); 45.9 (C-24 for β-sitosterol);51.3 (C-24 for stigmasterol); 29.2 (C-25 for β-sitosterol); 31.9 (C-25 for stigmasterol); 19.0 (C-26); 19.8 (C-27 for β-sitosterol); 19.0 (C-27 for stigmasterol); 22.7 (C-28 for β-sitosterol); 25.4 (C-28 for stigmasterol); 12.0 (C-29 for β-sitosterol); 12.3 (C-29 for stigmasterol).

DPPH Radical Scavenging Assay Radical scavenging activities of extracts and

flavonoids (Table 1) were determined according to the method described by Burda and Oleszek (2001). BHT (2,6-di-tert-butyl-4-methylphenol) was used as

reference compound. Samples and BHT (750.0 μL) were prepared in triplicate for each concentration used (1.0, 10.0 and 100.0 μg/mL) and, to each flask, the volume was adjusted to 2.0 mL by adding 1.5 mL of a 0.002 % p/v solution of DPPH in methanol. The solutions were shaken vigorously and kept in the dark for 30 min. The control was prepared as above without any extract or substance. Absorbance (measured on a Hitashi 2010 spectrophotometer) was measured at 517 nm and methanol was used for the baseline correction.

Radical scavenging activity was expressed as the inhibition percentage and was calculated: {(Abscontrol - Abssample)/Abscontrol} x 100

where Abscontrol = absorbance of DPPH radical in methanol and Abssample = absorbance of the extracts and pure substances in methanol + DPPH. Scavenging activities were expressed in μg/mL. IC50 values (μg/mL) was expressed the concentration of sample necessary to scavenge 50% of DPPH free radicals.

Allelopathic Bioassay Lactuca sativa (cv Grand Rapids) seeds were

purchased from Isla Pak, RS, Brazil. All undersized and damaged seeds were discarded. According to metodology described by Vieira et al. (2005), germination and growth were conducted in 100 mm Petri dishes containing 9.0 cm sheet of Whatman no. 1 filter paper as suport. Then, 25 lettuce seeds were placed per dish with 10 mL of a test (10-4, 10-6 and 10-8 M) or a control solution (without substance). All solutions were prepared with deionized water and their pH values [buffered with 10 mM 2-(N-morpholino) ethanesulfonic acid, MES] were adjusted to 6.0 - 6.5 with NaOH solution. Concentrations lower than 10-4 M were obtained by dilution series. All tests were triplicated. Dishes were covered with Parafilm to reduce evaporation and incubated in the dark at 25 oC, in a controlled-environment growth chamber, for 5 days. After this time, the number of germinated seeds were counted (a seed was considered to be germinated when the radicle was at least 0.2 mm long), the lengths of radicle and shoots were measured (using a paquimeter). During the measurement process, the dishes were kept at 4 oC to avoid subsequent growth. The osmotic pressure values were measured on a microsmometer and ranged between 30 and 38 mOsmolar.


26

25

27

O

OHH

O7

5

43

26

Ethyl galate (1) 5-Hydroxymethylfurfural (2) Galic acid (3)

COOCH2CH3

OH

OH

HO

12

4

6

7 8 9COOH

OH

OH

HO

12

4

6

7

O CH2OH

OHOHOH

OH6

43

12

O

OH

OHOH

CH2OH

CH2OH

2

34

5

6

1

Methyl shikimate (4) β-D-Frutopyranose (5) β-D-Frutofuranose (6)

1

O

910

Oleic acid (10) β-Sitosterol (11) Stigmasterol (12)

H OH

OH

OH

HOHO

H

HO

H

H

OHO

H

HOHO

H

HO

H

H

OH

HHO

3

1

29

30

19

28

17

27

14

26

810

25

2324

20

α-D-Glucose (7) β-D-Glucose (8) Lupeol (9)

1

3

56

18

1920

21

29

26

27

2522

23

1

35

6

18

1920

21 22

23

29

H

H H

H

H HHO

HO

12

3

4

56

7 8CO2CH3HO

HO

HO



Table 1 – Antioxidant activity of ethanol extract and fractions from epicarp and external mesocarp of C. brasiliense Camb. (pequi), determined by DPPH method, in three different concentrations.

Inhibition (%) Extracts /fractions

(1 µg/mL) (10 µg/mL) (100 µg/mL)

IC50 (µg/mL)

EE 0.0 63.7 94.4 28.3±6.6

G-1 5.4 51.3 96.7 9.3±6.3 G -1E 16.6 95.5 96.9 2,4±0.8 T1 3.6 16.9 91.7 22.2±2.5 BHT 40.4 45.6 84.2 16.4 ±3.6

Table 2 – Antimicrobial activity, in vitro, of G-1E fraction, obtained from epicarp and external mesocarp of C. brasiliense Camb. (pequi) towards several bacterial strain and the yeast C. albicans

Inhibition zones (mm)a Microrganisms G –1E Chloranfenicol Miconazol S. aureus 13.0±2.8 20±2.8 nt S. typhymurium 11.0±1.41 20±0.0 nt E. coli 12.0±2.8 23±1.4 nt C. freundi 11.0±0.0 22±0.7 nt B. cereus 11.0±2.8 24±1.4 nt L. monocytogenes 18.0±2.8 30±1.4 nt P. aeruginosa 10.0±1.4 14±2.1 nt C. albicans 0 nt 25±2.1

nt = not tested; aValues are mean ± SD of triplicate determinations.

Figure 2. Effect of ethanol extract (EE) and G-1 group from epicarp and external mesocarp of C. brasiliense Camb. (pequi) on radical and shoot length of L. sativa, in three different concentrations. Values are presented as percentage differences from the control, zero representing an observed value identical to the control (solution without substance), a positive value representing stimulation and a negative value representing inhibition.

-30

-20

-10

0

10

20

30

40

Radical

Leng

th (%

con

trol)

1.0 mg/ mL 0.2 mg/ mL 0.04 mg/ mL

EEG-1

EE G-1Shoot



Figure 3. Effect of ethereal fraction (G1-E), ethereal insoluble fraction (T1) and G-3, from epicarp and external mesocarp of C. brasiliense Camb. (pequi) on radical and shoot length of L. sativa, in three different concentrations. Values are presented as percentage differences from the control (solution without substance), zero representing an observed value identical to the control, a positive value representing stimulation and a negative value representing inhibition.

-75

-60-45-30-15

015

30

1.0 mg/ mL 0.2 mg/ mL 0.04 mg/ mL 0.008 mg/ mL 0.0016 mg/ mL

Rad

ical

G1-E T1

-30

-15

0

15

30Leng

th (%

con

trol)

Sho

ot

G1-E T1G1-E T1 G-3

G1-E T1 G-3

Figure 4. Effect of ethyl galate (1), hydroxymethylfurfural (2) and galic acid (3), isolated from epicarp and external mesocarp of C. brasiliense Camb. (pequi) on radical and shoot length of L. sativa, in three different concentrations. Values are presented as percentage differences from the control (solution without substance), zero representing an observed value identical to the control, a positive value representing stimulation and a negative value representing inhibition.

-40

-30

-20

-10

0

10

20

10-3 M 10-5 M 10-7 M

Leng

th (%

con

trol)

Shoot 3 21

3 21

Radical

Data Analysis The effect on germination and growth are

given as percent differences from control, and consist of the differences (in cm) between mean values of seeds with tested compounds and mean values for control (seeds grown without addition of tested compounds)/ mean values for control x 100. Thus, zero represents the control, positive values represent stimulation of the studied parameter and negative values represent inhibition. The data were evaluated

by using Student’s t-test and the differences between the experiment and control were significant at a value of P ≤0.05. The inhibitory and stimulatory activities, compared to those of the control, are shown in Figures 2, 3 and 4.

Antimicrobial Bioassay

Samples were tested in duplicate by disc diffusion method in agar with minor modifications (Lana et al., 2003). Microorganisms used were Staphylococcus aureus ATCC 29212, Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25922, Citrobacter freundi ATCC 8090, Bacillus cereus ATCC 11778, Listeria monocytogenes ATCC 15313, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 18804. For minimum inhibitory concentration test, carried out in duplicate, samples were serially diluted starting from concentration of 512 to 5.0 μg/ml for each test microorganism. Tubes were incubated for 18 hours at 35°c. The results are shown in Table 2

RESULTS AND DISCUSSION A total of twelve compounds were isolated

from the pequi fruit epicarp and external mesocarp parts. Their structures were determined based on the analysis of spectroscopic data, especially NMR, and literature data comparison. The pure compounds were identified as ethyl gallate (1) (Ceruks et al., 2007), 5-hydroxymethylfurfural (2) (Kuo et al., 2002), gallic acid (3) (Souza Filho et al., 2006) and methyl shikimate (4) (Liu et al., 2004; Adrio et al., 1997). Additionally, mixtures containing β-D-fructopyranose (5) and β-D-fructofuranose (6) (Sobolev et al., 2003; Breitmaier and Voelter, 1987); α- and β-D-glucose (7 and 8) (Collins and Ferrier, 1995); lupeol (9) (Mahato and Kundun, 1994) and oleic acid (10) (Oliveira et al., 2006); β-sitosterol (11), stigmasterol (12) (Goulart et al., 1993) and oleic acid (10) were obtained. The structures of these isolated compounds were assigned on the basis of spectroscopic data, including two-dimensional NMR methods and by comparison of their spectral data with values described in the literature. Structures of compounds 1-12 can be found in Figure 1.

Ethyl gallate (1) was isolated on a very high yield (14% from the crude ethanol extract, EE), whereas compounds 2, 3 and 4 were isolated on a very low yield from EE.




Ethyl gallate and gallic acid were previously isolated from leaves of Caryocar microcarpum (Kawanishi and Raffaud, 1986). Oleic acid, β-sitosterol and stigmasterol have already been isolated from C. microcarpum and C. villosum (Kawanishi and Raffaud, 1986; Marx et al., 1997).

Crude ethanol extract (EE), fractions G-1 obtained from polyamide column, G-1E (fraction soluble in ethyl ether, obtained from G-1) and T1 (ethyl ether insoluble residue, obtained from G-1) were evaluated for their antioxidant activities (Burda and Oleszek, 2001). Table 1 shows the average values for DPPH radical scavenging activity in each tested concentration, and IC50 values. G1-E presented the higher IC50, that can be associated to the presence of gallic acid and ethyl gallate in higher concentrations than in EE and T1. At 100 μg/mL, all extracts tested were more active than BHT, the reference compound.

The obtained results from allelopathic evaluation, according to methodology described by Vieira et al. (2005), of for crude ethanol extract, fractions and compounds 1, 2 and 3 are shown in Figures 2, 3 and 4. Both EE and G-1 showed inhibitory activity on radical growth and stimulatory activity on shoot growth of L. sativa. G-1 presented biggest inhibitory effect at 1.0 mg/mL, and also biggest stimulatory effect at 0.2 mg/mL (Figure 2). Fractions G-1E, T1 e G-3 (Figure 3) were tested at concentrations below 0.04 mg/mL, aimed to observe more stimulation on shoot and radical growth (Macías et al., 2000). However, only G1-E showed a slight stimulatory effect on radical growth at 1.6 x 10-

3 mg/mL and the inhibitory effects were bigger than those presented for EE and G-1, at analogous concentrations. On shoot, the best growth stimulatory effect was observed for G-3, at 8.0 x 10-3 mg/mL. The effect of pure compounds 1, 2 and 3 on radicle and shoot growth of L. sativa was mainly inhibitory (Figure 4). Gallic acid (3) presented the biggest inhibitory effect on radicle, at 10-3 M, and also the biggest stimulatory effect on shoot, at 10-7 M.

Crude ethanol extract (EB) and fractions G-1 and G-1E were tested for their antimicrobial activity (Lana et al., 2003), presenting inhibition zones of 7 mm. Only fraction G-1E presented expressive activity towards the tested microorganisms, according to Table 2. C. albicans was not affected by this fraction in the tested concentration. The minimum inhibitory concentration (MIC) found for

all tested microorganisms, except for S. typhimurium and C. albicans was 512 μg/mL.

CONCLUSIONS

This work pointed out for the possibility to use the external mesocarp of pequi fruit as a rich source of ethyl gallate, since this compound was isolated in an expressive yield (14 % from crude ethanol extract). The biological potential of this crude extract and isolated compounds were also noticeable (high IC50 in the antioxidant evaluation of extracts, and both inhibitory and stimulatory effect on radical and shoot growth of L. sativa, respectively from gallic acid), since plant material used is a residue produced in large scale in Brazil.

AKNOWLEDGEMENTS To CNPq, for JAT e MADB grants. To

FAPEMIG, for financial help.

REFERENCES Adrio J, Carretero JC, Ruano JLG, Cabrejas LMM.1997.

Enantioseletive synthesis of (+)-shikimic acid and (+)-5-epi-shikimic acid by assymetric Diels-Alder reaction of (+)-α-sulfinylacrylates. Tetrahedron Assym. 8:1623-1631.

Azevedo MCH, Rodriguez ADB. 2004. Confirmation of the identity of the carotenoids of tropical fruits by HPLC-DAD and HPLC-MS. J. Food Compos. Anal. 17:385-396.

Bezerra JCB, Silva IA, Ferreira HD, Ferri PH, Santos SC. 2002. Molluscidal activity against Biomphalaria glabrata of brazilian cerrado medicinal plants. Fitoterapia 73:428-430.

Breitmaier E, Voelter W. 1987. Carbon 13 NMR spectroscopy high- resolution methods and applications in organic chemistry and biochemistry. 3th ed., Weinheim: New York, 515 p..

Burda S, Oleszek PM. 2001. Antioxidant and antiradical activities of flavonoids. J. Agric. Food Chem. 49:2774-2779.

Ceruks M, Romoff P, Fávero AO, Lago, JHG. 2007. Constituíntes fenólicos polares de Schinus terebinthifolius Raddi (Anacardiaceae). Quim. Nova 30:597-599.

Collins P, Ferrier, R. 1995. Monosaccharides, their chemistry and their roles in natural products; 1st. ed, John Wiley & Sons: Chichester, 1995, 564 p.

Damiani, C. Qualidade e perfil volátil de pequi (Caryocar brasiliense Camb.) minimamente processado, armazenado sob diferentes temperaturas. 2006. MSc Thesis. Universidade Federal de Lavras. Brasil. 127p.


Marx F, Andrade EHA, Maia JG. 1997. Chemical composition of the fruit pulp of Caryocar villosum. Zeitsch. Lebensm. Unters.Forsch. 204:442-444.

Goulart MOI, Sant’Ana AEG, Lima RA, Cavalcante SH. 1993. Fitoconstituintes químicos isolados de Jatropha elliptica. Atribuição dos deslocamentos químicos dos átomos de carbono e hidrogênio dos triterpenos de jatrofolonas A e B. Quim. Nova 16:95-100.

Oliveira DM, Silva GDF, Duarte LP, Vieira Filho SA. 2006. Chemical constituents isolated from roots of Maytenus acanthophylla Reissek (Celastraceae). Biochem. Syst. Ecol. 34:661-665.

Herzog-Soares JD, Alves RK, Isac E, Bezerra JCB, Gomes MH, Santos SC, Ferri PH. 2002. Atividade tripanocida in vivo de Stryphnodendron adstringens (barbatimão verdadeiro) e Caryocar brasiliense (pequi). Rev Bras. Farmacogn. 12:1-2.

Passos XS, Santos SC, Ferri PH, Fernandes OF, Paula TF, Garcia AC, Silva MR. 2002. Antifungal activity of Caryocar brasiliense (Caryocaraceae) against Cryptococus neoformans. Rev. Soc. Bras. Med. Trop. 35:623-627.

Kawanishi K, Raffaud RF. 1986. Caryocar microcarpum: an ant repellent and fish poison of the nortweat amazon. J. Nat. Prod. 49:1167-1168. Paula-Junior W, Rocha FH, Donatti L, Fadel-Picheth

CMT, Weffrt-Santos AM. 2006. Leishmanicidal, antibacterial, and antioxidant activities of Caryocar brasilienses Cambess leaves hydrothanolic extract. Rev. Bras. Farmacogn. 16:625-630.

Kuo Y, Lee P, Wein Y. 2002. Four new compounds of seeds of Cassia fistula. J. Nat. Prod. 65:1165-1167.

Lana EJL, Carazza F, Takahashi JA. 2003. Antibacterial evaluation pf some new 2-aryl-3,5-dimethoxy-1,4-benzoquinone derivatives. J. Agric. Food Chem. 54:2053-2056.

Prance GT. 1990. The genus Caryocar L. (Caryocaraceae): an underexploited tropical resource. Adv. Econ. Bot. 8:177-188. Liu A, Liu ZZ, Zou ZM, Chen SZ, Xu LZ, Yang SL.

2004. Synthesis of (+)-zeylenone from shikimic acid. Tetrahedron 60:3689-3694.

Sobolev AP, Segre A, Lamanna R. 2003. Proton high-field NMR study of tomato juice. Magn. Reson. Chem. 41:237-245. Macías FA, Castellano D, Molinillo JMG. 2000. Search

for standard phytotoxic bioassay for allelochemicals. Selection for standard target species. J. Agric. Food Chem. 48:2512-2521.

Souza Filho APS, Santos RA, Santos LS, Guilhon GMP, Santos AS, Arruda MSP, Muller AH, Arruda AC. 2006. Potencial alelopático de Myrcia guianensis. Planta Daninha 24:649-652. Magalhães HG, Monteiro Neto H, Lagrota MH, Wigg MD,

Guimarães LAS, Loja MASO, Araújo RR. 1988. Estudo estrutural do pequizeiro Caryocar brasiliense Camb. Caryocaraceae, sob o aspecto farmacoquímico e botânico. Rev. Bras. Farm. 69:31-41.

Vieira RF, Martins MVM. 2000. Recursos genéticos de plantas medicinais de cerrado: uma compilação de dados. Braz. J Med. Plant. 3:13-36.

Vieira HS, Takahashi JA, Pimenta LPS, Boaventura MADZ. 2005. Effects of kaurane diterpene derivatives on germination and growth of Lactuca sativa seedlings. Z. Naturfo. C 60:72-78.

Mahato SB, Kundun AP. 1994. 13C NMR spectra of pentacyclic triterpenoids–a compilation and some salient features. Phytochemistry 37:1517-1575.



BLACPMA ISSN 0717 7917 Articulo Original | Original Article



Antimicrobial activity of essential oils of Aloysia triphylla (L`Her.) Britton from different regions of Argentina

[Actividad antimicrobiana de aceites esenciales de Aloysia triphylla (L`Her.) Britton procedentes de diversas regiones de Argentina]

María de las M. OLIVA1, Emilia BELTRAMINO1, Nicolás GALLUCCI1, Carina CASERO1, Julio ZYGADLO2, Mirta DEMO1

1 Universidad Nacional de Río Cuarto. Dpto de Microb e Inmunol. Ruta 36. Km 601. Rio Cuarto. Córdoba. Argentina. 2 Instituto Multidisciplinario de Biología Vegetal (IMBIV). Cat. Qca. Org. UNC. Córdoba. Argentina.

Abstract Essential oils are known to exert antimicrobial activity. Differences in the chemical composition of them influence this activity. This work intends

to study the variability in the chemical composition and the antimicrobial activity of essential oils obtained from plants of A. triphylla collected from different regions of Argentina. Essential oils were obtained by hydrodistillation and analyzed with GC-MS. The antimicrobial studies were carried out by the paper disc diffusion method. The essential oils shared common components but presented differences in the quantity and quality of the rest of them. The essential oil from La Paz showed the highest citral/limonene relation and the best antimicrobial activity. Yeasts resulted to be the most sensitive microorganisms, followed by the Gram positive bacteria. Statistical analysis showed significative differences in the antimicrobial activity. The differences in the biological activity of each essential oil could be attributed to the quantity and quality of the terpenic composition.

Keywords: Aromatic plants; Aloysia triphylla; Antibacterial; antifungic; terpenes.

Resumen

Los aceites esenciales poseen conocida actividad antimicrobiana. Esta actividad puede estar influenciada por la composición química de los aceites. El objetivo del presente trabajo fue estudiar la variabilidad en la composición química y la actividad antimicrobiana del aceite esencial obtenido a partir de plantas de A. triphylla recolectada de diferentes regiones de Argentina. Los aceites esenciales fueron obtenidos por hidrodestilación y analizados por GC-MS. Los estudios antimicrobianos se llevaron a cabo por la técnica de difusión en disco. Los aceites esenciales presentaron componentes mayoritarios comunes y presentaron diferencias en la cantidad y calidad del resto de los componentes. La mayor relación citral/limoneno y la mejor actividad antimicrobiana fue obtenida con el aceite esencial de La Paz. Las levaduras resultaron ser los microorganismos más sensibles, seguidos por las bacterias Gram positivo. El análisis estadístico mostró diferencias significativas en la actividad antimicrobiana de las distintas muestras. Las diferencias en la actividad biológica de cada aceite esencial podría ser atribuido a la cantidad y calidad de los terpenos lo constituyen.

Palabras Clave: Plantas Aromaticas; Aloysia triphylla; Antibacterianos; antifungicos; terpenos. Recibido | Received: September 9, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: December 10, 2009. Publicado en Línea | Published Online: December 15, 2009. Declaración de intereses | Declaration of interests: Authors have no competing interests. Financiación | Funding: We are grateful to SECyT of Universidad Nacional de Río Cuarto for financial support.

This article must be cited as: María de las M. Oliva, Emilia Beltramino, Nicolás Gallucci, Carina Casero, Julio Zygadlo, Mirta Demo. 2010. Antimicrobial activity of essential oils of Aloysia triphylla (L`Her.) Britton from different regions of Argentina. Bol Latinoam Caribe Plant Med Aromat 9(1):29 – 37. {EPub December 15, 2009}.

*Contactos | Contacts:. E-mail: [email protected]. Tel: +54-0358-4676434, Fax: +54-0358-4676231.


Oliva et al. Antimicrobial activity of essential oils of A. triphylla from different regions of Argentina


INTRODUCTION

For centuries, indigenous plants have been used in herbal medicine for curing various diseases. The acceptance of traditional medicine as an alternative form for health care and the development of microbial resistance to the available antibiotics have led scientific groups to investigate the antimicrobial activity of medicinal plants (Ozturk and Ercisli, 2007). In addition, a major problem in the food industry is that the microbial activity is a primary mode of deterioration of many foods. Currently there is a growing interest to use natural antibacterial compounds for the preservation of foods, as these possess a characteristic flavor and sometimes show antioxidant activity as well as antimicrobial activity (Schelz et al., 2006; Teixeira-Duarte et al., 2007).

Plants which are rich in a wide variety of secondary metabolites belonging to chemical classes (tannins, terpenoids, alkaloids, polyphenols) are generally superior in their biological activities suggesting that this strength is dependent on the diversity and quantity of such constituents (Geyid et al, 2005). Most of the antimicrobial activity in essential oils (EOs) appears to derive from oxygenated terpenoids as alcoholic and phenolic terpenes, while other constituents are believed to contribute little to the antimicrobial effect (Burt. 2004; Koroch et al. 2007.; Zygadlo and Juliani. 2000). Therefore, the determination of the compounds responsible for any biological activity would facilitate the selection of the plants for future investigation

Aloysia triphylla (L`Her.) Britton, (Aloysia citriodora Palau,) popularly known as “cedrón”, is a member of the Verbenaceae Family. It is perennial and grows widely in North and South America and also in northeast, northwest and central regions of Argentine. It is cultivated from Mexico till the South region of the continent. It is a bush with white flowers and fruits, with an intense scent lemon-like, sweet, lightly floral, and herbaceous (Barboza y col. 2001; Gil et al. 2007). This specie is used in folk medicine to treat many digestive disorders, as antiinflamatory, analgesic, antipyretic, tonic and stimulating. It shares an important place on the international herbal market due to the sensory and medicinal properties of it EOs. These attributes determine its use as a primary ingredient for infusions and nonalcoholic beverages as well as aromatic ingredient for the flavor and fragrance

industries. The pharmaceutical industry uses A. triphylla for its carminative, antispasmodic and sedative properties. There are several scientific studies that support the use of products obtained from A. triphylla. It has been found good antimicrobial activity of the methanolic and ethanolic extracts, as well as it has been described antimicrobial activity in the EOs (Akroum et al. 2009; Demo et al. 2005; Oskay et al. 2005; Sartoratto et al. 2004). The increasing interest in this specie has largely contributed to expanding A. triphylla crops in Argentina, Chile, Paraguay, Europe and Africa Mediterranean regions (Gil et al., 2007; Pascual et al., 2001, Sartoratto et al. 2004).

Cedrón is included in the Código Alimentario Argentino (CAA) as a corrective and coadjutant, in the section referred to vegetal condiments (Código Alimentario Argentino). “Cedrón” is recognized and described in the Farmacopea Nacional Argentina, VI Edición (FNA) as “the dried leaves with young stems, flowers and fruits of Aloysia triphylla (L´Hérit) Britt”. It is also described in the Pharmacopoeias of France, Spain, Mexico and Europe (Bandoni. 2000). It is included in the GRAS list (Generally Regarded as safe) and the Food and Drug Administration (FDA) has categorized it as a dietary supplement due to the wide use in America and Occidental Europe (Barboza y col. 2001).

Many EOs are known to exert antimicrobial activity (Schelz et al., 2006, Teixeira Duarte et al., 2007). Differences in the chemical composition of them related to variety, agronomic practice and processing are also likely to influence antimicrobial properties, since these factors contribute to both the profile and relative concentrations of active ingredients (Delaquis et al., 2002). The EOs content is influenced by genetic material, culture conditions, environment, season, crop and post-crop processing (Gil et al., 2007; Hussain et al., 2008).

The components commonly found in A. triphylla EOs are: neral, geranial limonene, geranyl acetate, betacaryophyllene, ar-curcumene, and spathulenol. Other compounds that could be founds in specific chemotypes are carvone, cedrol, 1,8-cineol, thujone isomers and citronellal (Gil et al., 2007).

Due to the differences described in the chemical composition of the EOs of a particular vegetable specie, the aim of this work was to study the chemical composition of EOs obtained from samples of A. triphylla collected in different regions



of Argentine and the relationship with the antimicrobial activity.


Plant material The plants of Aloysia triphylla were obtained

in March, 2005, from plants growing in farms (plantations) located in different regions of Argentina: from Córdoba Province: Río Primero and La Paz, from Salta Province: Las Viñas, from Mendoza Province, from San Luis Province and one sample from Paraguay Republic.

Essential oils obtention The EOs were obtained from dried vegetable

material, which was hydrodistilled in a Clevenger-like apparatus. The oil obtained was dried with anhydrous sodium sulphate and stored in the freeze until analysis (De Feo et al., 1998).

Gas Chromatography-FID The EO were analyzed with a Shimadzu GC-

R1A gas chromatograph equipped with a fused silica column (30 m x 0.25 mm) coated with CBP-1. The temperature of the column was programmed from 600C to 2400C at 40C/min. The injector and detector temperatures were at 2700C. The gas carrier was He, at a flow rate of 1 ml/min. Peak areas were measured by electronic integration. The relative amounts of the individual components are based on the peak areas obtained, without FID response factor correction. Programmed temperature retention index of the compounds were determined relative to n-alkanes. GC analysis was still performed using a column Supelcowax-10 with the same conditions as described above (Zunino et al., 1998).

Gas Chromatography-Mass Spectrometry GC-MS analyses were performed on a Perkin

Elmer Q-910 using a 30 m x 0.25 mm capillary column coated with CBP-1. The temperature of the column and the injector were the same than those from GC. The carrier gas was He, at a flow rate of 1ml/min. Mass spectra were recorded at 70 eV. The oil components were identified by comparison of their retention indices, mass spectra with those of authentic samples, by peak enrichment, with published data, mass spectra library of National Institute of Standards and Technology (NIST 3.0) and our mass spectra library which contains

references mass spectra and retention indices of volatile compounds. GC-MS analysis was still performed using a column Supelcowax 10 with the same conditions as describe above (Adams. 1989).

Microorganisms The activity of the EOs was tested against the

following microorganisms: Gram positive bacteria: Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis (milk), Micrococcus luteus ATCC 9341, Enterococcus faecalis ATCC 29212, Bacillus cereus (rice). Gram negative bacteria: Escherichia coli (water), Proteus mirabilis (urine), Klebsiella pneumoniae (composting of poultry), and Pseudomonas aeruginosa (water). The yeasts Candida albicans (mouth), Rhodotorula sp. (cereal) and Hansenula sp. (cereal) were used in order to probe antifungal activity.

Antimicrobial assays

Analysis of the antibacterial activity The antibacterial studies were carried out

according to De Pooter et al., (1995). The paper disc diffusion method was used to test the antimicrobial activity. Tubes containing Triptein Soy Broth (TSB) inoculated with the microorganisms were incubated during 18 h, at 37 ºC. From these tubes ten-fold dilutions were made, until an OD ≅ 0.04 (106 cfu/ml) was reached. The antifungal activity was determined with the same methodology but using that dilution with an OD ≅ 0.4 (106 cfu/ml). The inoculumm (200μl) was spread over plates containing Mueller-Hinton Agar and a paper filter disc (6mm) was impregnated with 10μl of the EO and placed on the surface of the media. The plates were left 30 minutes at room temperature to allow the diffusion of the oil in the agar; then they were incubated at 37°C during 24 hours. After this time the inhibition zone around the disc was measured with a caliper. Discs with gentamicine (10 μg) were used as positive control.

Analysis of the antifungal activity Antifungal experiments were performed in

the same way as those with bacteria using Sabouraud Agar (SA) for the plates. Discs with anfotericine B (2 μg/ml) were used as positive controls.



Minimum inhibitory concentration assay (MIC) The minimum inhibitory concentration

(MIC) was performed according to the method previously described by De Feo et al., (1998). It was determined by two-fold dilutions of EOs in dimethyl sulfoxide (DMSO), placing 10 μl of each dilution on a filter paper disc. The EOs concentration range was from 900 mg/ml to 7.03 mg/ml. The discs were placed on the surface of a TSA plate, previously inoculated with 200 μl of each inoculumm, and left at room temperature to allow the diffusion of the oil. Then they were incubated at 37 oC during 24 h. After this time the inhibition zone around the disc was measured with a caliper. MIC was defined as the lowest concentration that inhibited visible growth. The MIC with fungus was determined in the same way as with bacteria using Sabouraud Agar (SA) in the plates. The negative control consisted in a paper disc impregnated with 10 μl of DMSO. The positive control was a disc impregnated with the antibiotic gentamicine (10 μg) for bacteria. For yeasts, anfotericine B (2 μg/ml) was used.

Statistical analysis All the experiments were performed in

duplicate and statistical analysis of the data were performed using GraphPad Prism 4.0 program. A probability value of p<0.05 was considered statistically significant.

RESULTS

The chemical composition of the EOs from Aloysia triphylla collected from Rio Primero, La Paz, Las Viñas, Mendoza, San Luis and Paraguay has been investigated by means of gas chromatographic techniques. The EOs average yield in all the samples was 0.4% (w/v) and the components which were commonly found in all the EOs samples were: limonene, neral, geranial, spathulenol and caryophyllene oxide, with intrinsic variations in the quantity and quality of the resting terpenes in each sample (Table 1). The samples analyzed showed that the EOs from Mendoza had the biggest proportion of neral and geranial, followed by La Paz. Las Viñas had the biggest proportion of limonene and carvone, while neral and geranial were in the least proportion. Rio Primero EOs was the only one presenting camphor and borneol and the biggest proportion of α-thujone. The EOs from Paraguay had spathulenol and

caryophyllene oxide while San Luis had D germacrene and biciclogermacrene (Table1).

The relation between major terpenic components of an EO has been proposed as a criterion to identify chemotypes (Muñoz-Collazos et. al., 1993). In this work the rate between citral (neral + geranial) and limonene has been calculated. The EOs from La Paz showed the highest citral/limonene relation (16.9) and Las Viñas the least relation (0.73). The rest of the EOs relations were located between both of them (Table 1).

The antimicrobial activity of the EOs was assayed against Gram positive bacteria, Gram negative bacteria and yeasts. The yeasts resulted to be the most sensitive microorganisms to the effect of the EOs, followed by the Gram positive bacteria and lastly the Gram negative ones. It is interesting to note that the three yeasts were inhibited by all the EOs, with average inhibition zones diameters of 22 mm. (Table 2)

The EOs from La Paz showed the best antimicrobial activity, inhibiting the growth of all tested microorganisms, except P. aeruginosa. The inhibition zones obtained with this oil for B. cereus (38mm), M. luteus (33mm) and C. albicans are remarkable. Mendoza`s EOs presented good inhibition activity against microorganisms with average diameters of 29 mm against B. cereus. The EOs from Las Viñas, Paraguay and San Luis showed varied antimicrobial activity, with inhibition zones of 20 mm, 21 mm and 15 mm for B. cereus, respectively. (Table 2)

Previously, it had been reported good antimicrobial activity for the EOs obtained from Río Primero (Demo, et al. 2005). Taking into consideration these previous studies, La Paz and Rio Primero, both located in Córdoba Province, showed the best inhibition spectrum of all the oily samples. However there were differences in their chemical composition.

B. cereus, S. aureus and M. luteus were the most susceptible Gram positive bacteria to the EOs action. The Gram negative bacteria E. coli and K. pneumoniae were inhibited by the EOs from La Paz and Las Viñas.



Table 1: Components identified in the essential oils of A. triphylla (%) colleced in 6 different places. In bold data accounting for the main differences in composition.

Components Rio Primero La Paz Las Viñas Paraguay Mendoza San Luis α thujene 0,8 tr 0,6 tr tr tr α pinene 1,2 0,3 1,5 tr tr tr camphene 0,6 - 0,9 - - - 6-metil-5-hepten-2-one - tr 1,7 tr tr tr myrcene 1,7 tr 0,9 tr tr tr p-cymene 0,4 tr tr tr tr tr limonene 6,9 2,9 21,3 19,1 14,2 17,9 cis ocimene 0,5 tr 1,2 tr tr tr γ terpinene 0,7 tr 0,9 tr tr tr sabinene hydrate 0,5 - tr - - - camphenilone 0,7 tr 2,2 tr tr tr linalool 0,3 0,5 2,2 0,6 tr 0,3 α thujone 13,1 0,5 1,7 0,6 tr 0,2 2,2 dimetil-3,4 octadienal 0,4 1,3 - 0,5 tr 0,4 camphenol (6) 0,3 - tr - - - dihydrolinalool 0,1 - tr - - - cis verbenol 0,2 - tr - - - citronellal 0,1 tr 1,1 tr tr tr menthone 0,3 - - - - - isoborneol - tr 0,8 tr tr tr terpin-4-ol 0,3 - tr - - - α terpineol 0,5 - 2,4 - - - trans carveol 0,5 - 3,3 - - - cis carveol 0,4 - 1,1 - - - (E) ocimenone 0,4 0,5 tr 0,8 tr 0,4 neral 18,7 20 12,4 15,5 31,5 13 carvone 1,2 - 13,1 - - - carvotanacetone 0,1 - - - - - geranial 21,3 29,2 3,3 19,5 22,6 18,5 camphor 4,1 - - - - - borneol 1,2 - - - - - α copaene 0,8 0,5 tr 0,3 tr 0,8 β bourbonene 1 1 0,6 0,8 tr 0,9 β cubebene 0,2 tr 4,2 tr tr tr α cedrene 3,2 0,9 tr 2,8 tr 3,2 (E) caryophyllene 0,4 0,6 tr 0,7 tr 0,4 α humulene 0,6 1,1 tr 0,5 tr 0,6 Cisdihydroαterpineol - - tr - - - curcumene (ar) 0,1 tr 3,3 tr tr tr germacrene D 4,3 2,3 tr 5,3 tr 6,9 α zingiberene 0,4 0,5 tr 0,3 tr 0,4 bicyclogermacrene 3,8 4,2 tr 6,8 3,9 7,2 cubebol 0,1 0,9 tr 0,7 tr 1,3 β curcumene 0,1 0,4 0,5 tr tr 0,8 δ cadinene 0,2 0,3 4,2 tr 3,2 0,2 (E)nerolidol 0,5 0,6 tr 1,6 9,6 0,9 spathulenol 0,9 8,9 6,6 11,1 4,4 10,1 cariophyllene oxide 1 7 6,9 10,5 4,5 10 globulol 0,8 0,3 tr tr tr 0,3 viridiflorol 0,5 2,5 tr 0,6 tr 1,1 guaiol 0,3 0,3 0,8 tr tr 0,2 Total 95,3 87,5 99,7 98,6 93,9 96 Markers Citral (Neral + geranial) 40 49,2 15,7 35 54,1 31,5 Citral:Limoneno 5,8 16,9 0,73 1,83 3,8 1,76


Table 2: Antimicrobial activity of EOs of A. triphylla. Inhibition zones in (mm)


La Paz Las Viñas Paraguay Mendoza San Luis P(<0.05) MO δ δ δ δ δ

S. aureus 20 5.95 9 6.3 17 0.7 10 5.42 NI 0 0.0001 S. epidermidis 22 2.83 NI 0 13 3.53 11 2.82 9 2.12 0.0001 M. luteus 33 1.41 8 0.95 11 0.7 14 0.7 23 4.24 0.0001 E. faecalis 13.5 5.53 NI 0 NI 0 NI 0 NI 0 0.0001 B. cereus 38 11.2 20 11.54 21 5.65 29 6.38 15 0.7 0.0265 E. coli 8 4.33 7 7.68 NI 0 9 1.74 NI 0 0.0208 K. pneumoniae 10 0 4 4.92 NI 0 NI 0 NI 0 0.0169 P. mirabilis 10 0.7 NI 0 NI 0 4 4.94 NI 0 0.0001 P. aeruginosa NI - NI - NI - NI - NI - - C. albicans 39 18.18 14 4.76 9 12.72 27 8.81 9 12.02 0.0101 Hansenula sp 16 3.53 20 0 22 0 20 0 NI 0 0.0001 Rhodotorula sp 34 7.07 10 0.7 11 0 22 2.12 17 1.41 0.0054

NI: No inhibition; (-): Not done;

Table 3: Minimun Inhibitory Concentration of the EOs of A. triphylla (mg/ml)

MO Place/EOs Concentration (900-7 mg/ml)

La Paz Las Viñas Paraguay Mendoza San Luis

S. aureus 28.1 NI 900 56.25 NI S. epidermidis 28.1 NI 900 112.5 225 M. luteus 7 900 450 225 900 E. faecalis 56.25 NI NI NI NI B. cereus 7 900 7.03 7.03 56.25 E. coli 900 NI NI NI NI K. pneumoniae 900 900 NI NI NI P. mirabilis 450 NI NI NI NI P. aeruginosa NI NI NI NI NI C. albicans 28.1 7 56.25 56.25 14 Hansenula sp 7 56.25 7 225 NI Rhodotorula sp 7 900 7 28.1 112.5

The yeasts were the most sensitive

microorganisms, being inhibited by al the EOs. La Paz EOs showed the biggest inhibition zones, with diameters of 39 mm against C. albicans and 34 mm against Rhodotorula sp, while San Luis showed the smallest one. The others EOs samples showed different degrees of inhibition activity against the yeasts.

In order to analize if the differences found in the antimicrobial activity could be attributed to the origin and composition of the EOs, the statistical analysis was performed. This analysis showed significative differences in the antimicrobial activity of the EOs from all the places collected. These variability was present in all the tested microorganisms (Table 2).

For Gram positive bacteria the best values were obtained with La Paz EOs with values of 28.1 mg/ml for S. aureus and S. epidermidis and 7 mg/ml

for M. luteus and B. cereus (Table 3). Gram negative bacteria were inhibited by pure compounds with the exception of P. mirabilis that presented a CIM of 450 mg/ml with La Paz EOs.

The best MIC values for C. albicans were obtained with the EOs of Las Viñas (MIC: 7 mg/ml) and the best MIC values for Rhodotorula sp and Hansenula sp were obtained with La Paz and Paraguay EOs (MIC: 7 mg/ml) (Table 3).

DISCUSSION

Differences in the content and composition of the EOs of A. triphylla have been reported previously. In these reports, the EOs content ranged between 0.2 and 1% on dry weight (Gil et al., 2007, Sartoratto, et al. 2004). In this study, the average yield obtained with the EOs samples (0.4% (w/v)) was included between these values.



There were chemical differences in the quantity and quality of the EOs obtained from A. triphylla collected from different places. However, all the EOs samples shared the terpenic components limonene, neral, geranial, espathulenol and cariophyllene oxide, which were described by other authors as the characteristically constituents of A. triphylla EOs (Gil et al., 2007; Pascual et al., 2001; Stashenko y col., 2003). Each EO sample showed particular features in the rest of the constituents with variability in the composition of each one. The EOs content in vegetable species is influenced by genetic material, culture conditions, environment, season, extraction methods, crop and post-crop processing (Gil et al., 2007; Hussain et al., 2008; Tampieri et al.; 2005). The culture conditions, kind of soil and climate were particular for each one of the samples collected. Río Primero and La Paz are located in the central region of Argentina, Mendoza and San Luis are located in the central-west of Argentina while Las Viñas and Paraguay are in the north. Previous studies on A. triphylla have reported that the production and composition of the EOs vary according to the part of the plant, the stage of development and the harvesting locations of the plant (Gil et al., 2007). Other investigations support the fact that the differences in the quantity of the terpenes identified in an EO obtained from a vegetable collected in the same zone are due to the environment (Karaman, 2006; Merle et al., 2004). It was found variability in the terpenic composition of Ocimun basilicum EOs collected in different seasons, concluding that the growing season was affecting the chemical content (Hussain et al., 2008). It is worth to mention that it was analyzed an EO sample from La Paz collected the following year (2006) in order to analyze if variations in the chemical composition related to the harvesting year happened and it was found the same terpenic composition as in 2005 with variations in the quantity of them. (Data not shown)

Citral and limonene were the major components identified in these EOs. It has been reported antimicrobial activity of both of them (Demo y col. 2001, Di Pasqua et al., 2006; Wolken et al., 2002). The individual activity of citral and limonene could be suggesting that the relationship between them could be determining the antimicrobial activity. This justifies the study of the rate between them and the possible relation between this value and the antimicrobial activity of the EOs. In this work the EO of A. triphylla from La Paz showed the biggest rate citral/limonene and the best antimicrobial

activity, while Las Viñas EO showed the least terpenic relation and antimicrobial activity (Table 1 and 2). These results are suggesting that higher rates between both compounds could be determining a better antimicrobial ability and a broader microbial spectrum. Some authors suggested that the compounds present in the greatest proportions are not necessarily responsible for the greatest share of the total activity. The data on the activity of the essential oils, in some cases are not compatible with those of the pure constituents in higher percentages Thus, the involvement of the less abundant constituents should be considered (Cimanga et al., 2002; Tampieri et al., 2005; Zygadlo and Juliani. 2000).

Mono and sesquiterpenes and the mixture between them in the oil, could constitute a barrier to microbial infections (Cowan. 1999; Lambert et al., 2001; Tan et al., 1999; Vataru Nakamura et al., 2004). The biotic and abiotic factors (environment, specie, chemotype) of the places where specie are collected have influence on the quantity and quality of the terpenic composition of the EOs. Consequently, differences in the EOs yielding and in the biological activity are observed, being active against bacteria and fungi or only one of them (De Pooter et al., 1995; Hess et al., 2007; Zygadlo and Juliani. 2000). A study made with O. basilicum EOs with regard to seasonal variations, showed changes in the antimicrobial activity, attributing these variations to the different chemical composition of the oils. Some earlier reports showed that the changes in chemical composition of an EOs directly affected their biological activities (Hussain et al., 2008). The differences in the biological activity of each A. triphylla EOs could be attributed to the quantity and quality of the terpenic composition and the possible associations between them. In addition, it could be deduced that the antimicrobial activity is not only dependent on the quality and quantity of the EOs but, on the particular sensibility of each particular strain.

The inhibitory activity against microorganisms responsible for human and plant diseases of EOs from A. triphylla has been described in other investigations (Demo et al., 2005; Pascual et al., 2001; Sartoratto, et al. 2004). What is more, Sartoratto, et al. 2004, described MIC values of A. tryphilla (L´Hér.) Britton lower than chloramphenicol, when it was used as the positive control antibiotic, showing the antimicrobial potential of this EO. (Sartoratto, et al. 2004) But, a comparison of the chemical composition and the antimicrobial activity of the EOs obtained from A. triphylla



collected in different regions, and the relation between this two variables, have not been previously reported.

It is difficult to compare results with others reported in the literature because of the naturally varying composition of EOs even in the same species due to the presence of chemotypes, different harvest times, different extraction methods, etc. Furthermore, it is important to consider the different microbiological tests utilized and the different sensitivities of the strains (Tampieri et al., 2005). The information described here clearly shows the influence of the chemical composition of this EO on the antimicrobial activity. This is also suggesting that the genotypical composition of this specie should be taken into consideration, in order to obtain the ideal terpenic composition with the best antimicrobial activity.

The production of EOs and their utilization as potential therapeutic agents and natural food preservants could be of economical value. However, further investigations to establish how components interact to provide the biological activities are needed (Hussain et al., 2008).

ACKNOWLEDGEMENTS

María de las Mercedes Oliva, Mauro Nicolás Gallucci, Carina Caseros and Julio Alberto Zygadlo are researchers from CONICET. We are grateful to SECyT of Universidad Nacional de Río Cuarto for financial support. We thanks to Plantadroga S. A. Laboratories (Paraguay Republic sample) and Ing. Alvarez Toledo (Salta sample) for the provission of A. triphylla.

REFERENCES Adams RP. 1989. Identification of Essential Oils by Ion

Trap Mass Spectroscopy. Ac. Press. NY. Akroum, S, Satta, D, Lalaoui, K. 2009. Antimicrobial,

Antioxidant, Cytotoxic Activities and Phytochemical Screening of Some Algerian Plants. Eur J Sci Res. 31 (2):289-295

Bandoni A. 2000. Los recursos vegetales aromáticos en Latinoamérica, su aprovechamiento industrial para la producción de aromas y sabores. Editorial Universidad Nacional de La Plata.

Barboza GE, Bonzani, N, Filipa, E, Lujan M, Morero R, Bugatti M, Decolatti N, Ariza-Espinar L. 2001. Atlas Histo-Morfológico de plantas de interés medicinal de uso corriente en Argentina. Museo de Botánica. Fac. de Cs. Ex., Fcas. y Nat. y Fac. de Cs. Qcas. de la Universidad Nacional de Córdoba.

Burt, S. 2004. Essential oils: antibacterial properties and potential aplications in food – a review. Int. J. Food Microbiol. 94: 223-253

Cimanga K, Kambu K, Tona L, Apers S, de Bruyne T, Hermans N, Totté J, Pieters L, Vlietinck AJ. 2002. Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo. J. Ethnopharmacol. 79: 213-220.

Código Alimentario Argentino. Capitulo XVI Correctivos y Coadyuvantes. Articulo 1215

Cowan M. 1999. Plant Products as antimicrobial Agents. Clin. Microbiol. Rev. 12 (4): 564-582.

De Feo V, Ricciardi AI., Biscardi D, Senatore F. 1998. Chemical Composition and Antimicrobial Screening of the Essential Oil of Minthostachys verticillata (Griseb.) Epl. (Lamiaceae). J. Essent. Oil Res. 10:61-65.

Delaquis PJ, Stanich K, Girard B, Mazza G. 2002. Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. Int. J. Food Microbiol. 74: 101–109.

Demo MS, Oliva Ma de las M, Lopez ML, Zunino MP; Zygadlo JA. 2005. Antimicrobial activity of essential oils obtained from aromatic plants of Argentina. Pharm Biol. 43:129-134.

De Pooter H, Aboutabl E, El- Shabrawy O. 1995. Chemical Composition and Antimicrobial Activity of essential oils of leaf, stem and rhizome of Alpinia speciosa. Flavor Frag. J. 10:63-67.

Di Pascua R, Hoskins N, Betts G, Mauriello G. 2006. Changes in membrane fatty acids composition of microbial cells induced bu addiction of thymol, carvacrol, limonene, cinnamaldehyde, and eugenol in the growing media. J. Agr. Food Chem. 54: 2745-2749.

Geyid A, Abebe D, Debella A, Makonnen Z, Aberra F, Teka F, Kebede T, Urga K, Yersaw K, Biza T, Mariam BH, Guta M. 2005. Screening of some medicinal plants of Ethiopia for their anti-microbial properties and chemical profiles. J. Ethnopharmacol. 97: 421–427

Gil A, Van Baren CM, Di Leo Lira PM, Bandoni AL. 2007. Identification of the Genotype from the Content and Composition of the Essential Oil of Lemon Verbena (Aloysia citriodora Palau). J. Agric. Food Chem. 55: 8664–8669.

Hess SC, Peres MTLP, Batista AL, Rodrigues JP, Tiviroli SC, Oliveira LGL, Santos CWC, Fedel LES, Crispim SMA, Smania Júnior S, Smania EFA, Flach A, Pantaroto, S. 2007. Evaluation of seasonal changes in chemical composition and antibacterial activity of Elyonurus muticus (Sprengel) O. Kuntze (Graminae). Química Nova 30: 370-373.

Hussain AI, Anwar F, Hussain Sherazi ST, Przybylski R. 2008. Chemical composition, antioxidant and antimicrobial activities of basil (Ocimum basilicum)


Schelz Z, Molnar J, Hohmann, J. 2006. Antimicrobial and antiplasmid activities of essential oils. Fitoterapia 77:279–285

essential oils depends on seasonal variations. Food Chem. 108:986-995

Karaman, S. 2006. Morphogenetic Variation for Essential Oils in Salvia palestiana Bentham leaves and bracts from Turkey. Pakistan J. Biol. Sci. 9(14): 2720-2722

Stashenko EE, Jaramillo BE, Martinez JR. 2003. Comparación de la composición química y de la actividad antioxidante in vitro de los metabolitos secundarios volátiles de plantas de la família Verbenaceae. Rev. Acad. Colomb. Cienc. 27 (105): 579-597.

Koroch A., Juliani, H.R., Zygadlo J.A, 2007. Bioactivity of essential oils and their components. Ralph G Berger (Ed.). Flavours and Fragrances Chemistry, Bioprocessing and Sustainability. Berlin. Springer-Verlag. pp. 87-115 Tampieri MP, Galuppi R, Macchioni F, Carelle MS,

Falcioni L, Cioni PL, Morelli I. 2005. The inhibition of Candida albicans by selected essential oils and their major components. Mycopathologia. 159: 339–345

Lambert R, Skandamis P, Coote P, Nychas G. 2001. A study of the Minimun Inhibitory Concentracion and mode of accion of Oregano Essential Oil, Thymol and Carvacrol. J. Appl. Microbiol. 91: 453-462.

Tan RX, Lu H, Wolfender JL, Yu TT, Zheng WF, Yang L, Gafner S, Hostettmann K. 1999. Mono- and sesquiterpenes and antifungal Constituents from Artemisia Species. Planta Méd. 65:64-67.

Merle H, Morón M, Blazquez MA, Boira H. 2004. Taxonomical contribution of essential oils in mandarins cultivars. Biochem. Syst. Ecol. 32:491-497

Muñoz-Collazos S, Soriano-Ferrufino J, Collín GJ, Jean FI, Deslauriers H. 1993 .Variability in the composition of the essential oils of Minthostachys andina in Central Bolivia. Phytochemistry. 33:123-127

Teixeira Duarte MC, Leme EE, Delarmelina C, Soares AA, Figueira, GM, Sartoratto A. 2007. Activity of essential oils from Brazilian medicinal plants on Escherichia coli. J. Ethnopharm. 11: 197–201 Oskay, M.; Usame Tamer, A.; Ay, G.; Sari, D.; Aktas, K.

2005. Antimicrobial activity of the leaves of Lippia triphylla (L`Her) O. Kuntze (Verbenaceae) against on bacterial and yeast. J. Biolog. Sci. 5(5):620-622

Vataru Nakamura C, Ishida K, Faccin LC, Dias Filho BP, Garcia Cortez DA, Rozental S, de Souza W, Ueda-Nakamura T. 2004. In vitro activity of essential oil from Ocimun gratissimun L. against four Candida species Res. Microbiol. 155: 579-586.

Ozturk S, Ercisli S. 2007. Antibacterial activity and chemical constitutions of Ziziphora clinopodioides. Food Control 18: 535-540. Wolken WAM, Van Loo WJV, Tramper J, van der Werf

MJ. 2002. A novel, inducible, citral lyase purified from spores of Penicillium digitatum. Eur J Biochem. 269:5903-5910

Pascual ME, Slowing K, Carretero E, Sanchez Mata D, Villar A. 2001. Lippia: traditional uses, chemistry and pharmacology: a review. J. Ethnopharm. 76:201-214

Sartoratto, A.; Machado, A. L. M.; Delarmelina, C.; Figueira, G. M.; Duarte, M. C, Rehder, V. L. G. 2004. Composition and antimicrobial activity of esential oil from aromatic plants used in Brazil. Braz. J. Microbiol. 35:275-280.

Zunino MP, Newton MN, Maestri DM, Zygadlo JA. 1998. Planta Medica. 64: 86.

Zygadlo JA, Juliáni HR. 2000. Bioactivity of essential oil components. Curr topic in phytochemistry. 3:204-214.

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BLACPMA ISSN 0717 7917 Articulo original | Original article



Estudio farmacobotánico de hojas, cortezas y leños de Simaroubaceae sensu lato de Argentina. Parte I. Alvaradoa subovata Cronquist, Picramnia

parvifolia Engl., Picramnia sellowii Planch. y Castela coccinea Griseb. [Morphoanatomy of leaves barks and wood of Argentinean Simaroubaceae sensu latu. Part I: Alvaradoa

subovata Cronquist, Picramnia parvifolia Engl., Picramnia sellowii Planch. and Castela coccinea Griseb]

Adriana CORTADI1, Luisina ANDRIOLO1, María Noel CAMPAGNA1, María Laura MARTÍNEZ1, Osvaldo Di SAPIO1, Adriana BROUSSALIS2, Martha GATTUSO1, Susana GATTUSO1.

1Cátedra de Farmacobotánica. Departamento de Ciencias Biológicas. Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR. Suipacha 531. S2002 LRK. Rosario. República Argentina. 2Cátedra de Farmacognosia. Facultad de Farmacia y Bioquímica, UBA. Junín 956, 2º piso.

CP 1113 CABA.

Abstract

The Simaroubaceae (sensu lato) family is represented in Argentina by six genera and eight species, seven of which are native and only one non-autochthonous; some are used in folk medicine as tonic, insecticides and pesticides. Leaves were cut previous paraffin embedding and diaphanization. Longitudinal and cross-sectional cuts were made on cortex and leños and were cut and macerated. They were stained with Safranine-Fast-green and Cresyl Violet. Microscopic examination was performed by light microscopy and SEM. In the present work leaves, cortex and leños from Alvaradoa subovata, Picramnia parvifolia, Picramnia sellowii and Castela coccinea were morpho-anatomically analyzed in order to determine diagnosis characters to identify diagnostic characters to ensure the identity and quality of these resources. In leaves, namely, 1. presence or absence of gland hairs, mesophile type, presence of mucilage; 2. cortex: periderm, radii types, crystals, sclerenchyma elements; 3. leño size, pores location, radii, and axial parenchyma, among others. Complete the presentation with photomicrographs and keys in order to provide adequate differentiation between entities.

Keywords: Alvaradoa subovata, Picramnia parvifolia, Picramnia sellowii, Castela coccinea, Simaroubaceae, morphoanatomical characters

Resumen

Las Simaroubaceae (sensu lato) está representada en Argentina, por 6 géneros y 8 especies, de las cuales 7 son nativas y una introducida; algunas son utilizadas en medicina popular como tónicas, insecticidas y antiparasitarias. Las hojas se cortaron previa inclusión en parafina y se diafanizaron; las cortezas y leños se cortaron longitudinal y transversalmente y se maceraron. Se colorearon con Safranina-Fast-green y Violeta de Cresyl. Las observaciones se realizaron con microscopio óptico y microscopio electrónico de barrido. En este trabajo se han estudiado morfoanatómicamente las hojas, cortezas y leños de Alvaradoa subovata, Picramnia parvifolia, Picramnia sellowii y Castela coccinea a fin de determinar caracteres diagnósticos para garantizar la identidad y calidad de estos recursos. En hojas: presencia o ausencia de pelos glandulares, tipo de mesófilo, presencia de mucílagos; en cortezas: la peridermis, tipos de radios, cristales, elementos esclerenquimáticos; en leños: tamaño y disposición de los poros, radios, y parénquima axial, entre otros. Se completa la presentación con fotomicrografías y claves con el objeto de brindar una adecuada diferenciación entre las entidades.

Palabras Clave: Alvaradoa subovata; Picramnia parvifolia; Picramnia sellowii; Castela coccinea; Simaroubaceae; caracteres morfoanatómicos.

Recibido | Received: Agosto 4, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: Noviembre 30, 2009. Publicado en Línea | Published Online Diciembre 15 2009 Declaración de intereses | Declaration of interests: authors have no competing interests. Financiación | Funding: ANPCyT, proyecto PICT BID/2007-1494 y SECYT BIO/2008-200. This article must be cited as: Adriana Cortadi, Luisina Andriolo, María Noel Campagna, María Laura Martínez, Osvaldo Di Sapio, Adriana Broussalis, Martha Gattuso, Susana Gattuso. 2010. Estudio farmacobotánico de hojas, cortezas y leños de Simaroubaceae (sensu lato) de Argentina. Parte I. Alvaradoa subovata Cronquist, Picramnia parvifolia Engl., Picramnia sellowii Planch. y Castela coccinea Griseb. Bol Latinoam Caribe Plant Med Aromat 9(1):38 – 55. {EPub 15 December 2009 }.



Cortadi et al. Caracteres morfoanatómicos de especies de Simaroubaceae Part I


INTRODUCCIÓN

La familia Simaroubaceae incluye 30 géneros y 200 especies de climas tropicales y subtropicales.

Los estudios taxonómicos referidos a las Simaroubaceae de América fueron abordados por Cronquist (1944), Pirani (1987). Este mismo autor se ocupo de las especies de Picramnia de Brasil en un detallado trabajo (Pirani 1990).

En la Argentina las Simaroubaceae (sensu lato) están representadas por una especie introducida Ailanthus altissima (Mill) Swingle y siete especies nativas, Castela coccinea Griseb, Castela tweedii Planch., Picrasma crenata. (Vell.) Engl., Simaba glabra, Alvaradoa subovata Cronquist, Picramnia parvifolia Engl. y Picramnia sellowii Planch., (Xifreda 1999, Xifreda y Seo, 2006).

Existen datos anatómicos de la madera, (Webber, 1936; O´Donell, 1937; Heimsch 1942; Metcalfe y Chalk 1950, 1972), Wheeler et al. (1986, 1989) trabajaron con maderas duras, incluyendo al género Picramnia confeccionaron una base de datos con caracteres anatómicos; morfología del polen (Erdtman 1986; Moncada y Machado 1987, estudios fitoquímicos (Stuhlfauth et al. 1985; Da Silva y Gottlieb 1987; Simao et al. 1991) y de la estructura del pericarpio (Fernando y Quinn 1992).

Las hojas, cortezas y leños de algunas de estas especies de Simaroubaceae (sensu lato) son utilizadas en la medicina popular para el tratamiento de enfermedades de la piel: Picramnia sellowii (Balderrama et al. 2001); vermicida: Picramnia antidesma y Picrasma exelsa (Kelly y Dickinson 1985) y en trastornos del tracto gastrointestinal: Ailanthus altissima (Martínez Crovetto, 1981; Grieve, 1996); Castela tweedii y Picrasma crenata (Martínez Crovetto, 1981, Simao et al. 1991) y Castela coccinea (UMSA-Fundación, 2002, Bourdy et al. 2004). El leño de Alvaradoa amorphoides se usa como tónico estomacal y la corteza como antipruriginosa (Martinez, 1969; Toursarkissian, 1980).

Las Simaroubaceae se caracterizan por la presencia en todos sus representantes de cuasinoides, principios amargos, derivados de triterpenos degradados y alcaloides derivados del triptofano, β-carbolines, canthinones, clasificados en los tipos estructurales C1-C5, (Simao et al 1991). Los cuasinoides de acuerdo a su estructura química se dividen en 5 grupos C-18, C-19, C-20, C-22 y C-25. muchos de éstos tienen un amplio rango de

actividades biológicas in vivo y/o in vitro. Los cuasinoides de C-20 han sido extensamente estudiados por sus propiedades biológicas, dado que se han encontrado algunos compuestos con marcada actividad antileucémica (Guo et al. 2005); actividad antitumoral (Cuendet y Pezzuto 2004, Fukamiya et al. 1992, Guy Balansard y Hajime Ohigashi 2002, Ogura et al. 1977, Toyota et al. 1990), el modo de acción de los cuasinoides en ésta actividad fue encarada por Grieco et al. (1997) y Morre et al. (1998) entre otros; actividad antimalarica, (Bourdy et al. 2004, Castro et al. 2006, O´Neill et al. 1986, 1987, 1988, Ajaiyeoba et al. 1999, Bertani et al. 2007); actividad antiviral, (Grieco et al. 1997); actividad antifeedant-insecticida (Lidert et al. 1987); actividad antiparasitaria-antiprotozoaria (Camacho et al. 2003, 2004, Guy Balansard y Hajime Ohigashi 2002, Moncayo 2003, Martínez et al. 2009); actividad herbistática-herbicida (Grieco et al. 1997, Siwajinda et al 2001) y actividad citotóxica (Anderson et al 1991, Cordell et al. 1993, Tagahara y Kuo-Hsiung 1993).

Actualmente, el análisis micrográfico de aquellas especies biológicamente activas, es requisito indispensable para las Farmacopeas herbarias de todo el mundo, las cortezas y maderas de la flora de Argentina han sido poco estudiadas, en este contexto, el objetivo del presente trabajo es estudiar caracteres morfoanatómicos de hojas, cortezas y leños de Alvaradoa subovata, Picramnia parvifolia, Picramnia sellowii y Castela coccinea, de modo de tener no sólo una identificación morfológica sino también micrográfica de las mismas, permitiendo la identificación de cada especie cuando se encuentren molturadas.

MATERIALES Y MÉTODOS

Materiales estudiados Se emplearon materiales fresco y de los herbarios

BAA, MCNS, SF, SI y UNR, los que son citados conforme a las siglas respectivas (Holmgren et al., 1990). El material fresco fue coleccionado por los autores, en las provincias de Misiones, Corrientes, Chaco, Santiago del Estero, Tucumán, Jujuy, Salta y Santa Fe. Para los estudios anatómicos fueron utilizados siete ejemplares de cada especie.

Se utilizó material de herbario y fresco, a este se lo fijó en FAA (alcohol etílico 70º, ácido acético glacial, formaldehído y agua 50:5:30:15) y al material de herbario se lo hidrató. Para el estudio de



las hojas, las láminas, se cortaron transversalmente en la parte media de las mismas con micrótomo tipo Minot, previa inclusión en parafina (Gattuso y Gattuso 2002). Para el análisis de las epidermis, venación y micrografía cuantitativa, las láminas foliares se diafanizaron (Strittmatter, 1973) y se determinaron los siguientes parámetros: índice de estomas (Salisbury, 1927), estomas por milímetro cuadrado (Timmerman, 1927), índice de empalizada (Zorning y Weiss, 1925) y pelos simples por milímetro cuadrado; para todas estas medidas se trabajó con objetivo de 40x con un ocular de 10x. Para la descripción de la arquitectura foliar se utilizó la terminología de Hickey (1973) y para los pelos Üphof et al. (1962). Las cortezas y leños se cortaron con xilótomo en forma transversal y longitudinal (radial y tangencial), previo ablandado con agua hirviendo adicionada de una gotas de detergente comercial; y se maceraron aplicando la técnica de Boodle (1916) y se midió con ocular micrométrico la longitud y el diámetro de los elementos vasales como así también longitud y latitud de fibras.

Las coloraciones empleadas fueron Safranina alcohólica 80º, Safranina-Fast-green (Strittmatter, 1979) y Violeta de Cresyl (Strittmatter, 1980). La distribución de los cristales de oxalato de calcio fue analizada utilizando luz polarizada.

Para la descripción de los elementos del leño se usó IAWA Committee (1989).

Las ilustraciones son originales y fueron realizadas con microscopio óptico (MO) Nikon Alphaphot, con tubo de dibujo. Para los esquemas se siguió la simbología de Metcalfe y Chalk (1950). Las fotomicrografías fueron obtenidas con Microscopio Carl Zeiss Axiolab y equipo fotográfico MC 80. Los detalles de las epidermis en superficie, cortezas y leños fueron observados con microscopio electrónico de barrido (MEB) Leitz AMR 1000; en el caso de la láminas foliares las muestras fueron fijadas en glutaraldehído al 4 % deshidratadas en alcoholes ascendentes, se aplicó punto crítico y finalmente se metalizó con oro paladio (O´Brien y McCully, 1981). Las observaciones morfológicas se efectuaron con microscopio estereoscópico Nikon SMZ-U ZOOM1:1 con tubo de dibujo.

Para los caracteres anatómicos cuantitativos se calcularon las medias aritméticas (x) con su correspondiente desvío estándar sobre 10 campos.

Se acondicionaron los ejemplares para la incorporación a los herbarios UNR y de la Cátedra de Farmacobotánica de la Facultad de Ciencias

Bioquímicas y Farmacéuticas (UNR). Las preparaciones histológicas se hallan depositadas en la histoteca de dicha Cátedra.

RESULTADOS

La identificación de estas cuatro especies se realiza por una combinación de caracteres morfológicos y anatómicos de las partes utilizadas, los que se presentan en cuadros, figuras y láminas.

DESCRIPCIÓN MACROSCÓPICA DE LAS ESPECIES ESTUDIADAS

Alvaradoa subovata Cronquist. Brittonia 5 (2):134. 1944. Sinónimos: Alvaradoa amorphoides var. puberulenta Monach. Lilloa 8: 407. 1942. Nombres vulgares: “Pichi-blanco”, Sacha ruda” o “Chuquisaca”. Uso vernáculo: El leño se utiliza como tónico estomacal, y la corteza como antipurriginosa. (Toursarkissian, 1980). Hojas: compuestas, pecioladas, alternas, imparipinnadas con 16-24 folíolos, de 2-4 cm de long. x 0,4-1 cm lat., oblongos, alternos, borde entero, ápice retuso, base aguda (Fig.1 A, a). Corteza: color castaño grisáceo, escasas grietas longitudinales, numerosas lenticelas. Fractura entera. Leño: color amarillo pálido, poros apenas visibles, anillos pocos diferenciables, albura blanquecina.

Picramnia parvifolia Engl. Fl. Bras. 12(2): 242, pl. 49. 1874. Nombres vulgares: No conocidos. Uso vernáculo: No se registra en la bibliografía consultada ningún uso medicinal para esta especie. Hojas: compuestas, pecioladas, alternas, imparipinnadas de 7-15 folíolos, de 2-10 cm de long. x 0,8-2 cm lat., alternos a subopuestos, membranáceos, oblongo-elíptico a oblongo lanceolados, margen poco revoluto, ápice subacuminado o más raramente obtuso, base aguda y asimétrica, a veces oval obtusa (Fig.1 B, b). Corteza: color pardo rojizo superficie uniforme, escasas lenticelas. Fractura entera. Leño: color amarillo brillante, poros imperceptibles, anillos apenas diferenciables, albura y duramen de aspecto homogéneo


Fig.1. A-V: Caracteres morfo-anatómicos foliares diferenciales. A-D: tipos de hojas, observación con microscopio estereoscópico. A-C: compuestas pinadas: A, de 16-24 folíolos en A. subovata. B, de 7-15 folíolos en P. parvifolia. C, de 9-15 folíolos en P. sellowii. D, hoja simple en C. coccinea. E-V: observación con MO. E-M, V: vista superficial de las epidermis: E-F, adax. y abax respectivamente en A. subovata. G-H, adax. y abax. respectivamente en P. parvifolia. I-J, adax. y abax. respectivamente en P. sellowii. K-L, adax. y abax. respectivamente en C. coccinea. M, pelo glandular en P. sellowii. V, cavidad esquizógena en P. parvifolia. N-U, sección transversal de las láminas foliares: N-S, mesófilo dorsiventral: N-O, en A. subovata. P-Q, en P. parvifolia. R-S, P. sellowii. T-U, mesófilo céntrico en C. coccinea. En todos, esquemas y detalle de lo indicado. Caracter anatómico foliar común: a-c, D: arquitectura foliar camptódroma broquidódroma, a-c folíolos, D hoja. Escalas: 1 corresponde a O, Q, S, U. 2 corresponde a M, V. 3 corresponde a N, P, R, T. 4 corresponde a E-L.



Fig.2. A-G: Caracteres anatómicos foliares diferenciales. Observación con MO. A-C, sección transversal de la lámina foliar; A-B, mesófilo dorsiventral: A, A. subovata; B, P. parvifolia. C, mesófilo céntrico en C. coccinea. D-G: vista superficial: D-E en P. sellowii: D, pelos simples y glandulares (flechas), E, pelo glandular y estomas anomocíticos. F, C. coccinea, pelos simples (flechas) y estomas. G, A. subovata, pelos simples y estomas hundidos (flechas). ce, cavidad esquizógena; e, epidermis; ep, epidermis papilosa; et, estomas; h, hipodermis; m, mucílagos.



Fig.3. A-R: Caracteres anatómicos diferenciales de las cortezas. Observación con MO. A-H: Representación esquemática de la corteza y detalle de la sección transversal de las células del súber: A y E: C. coccinea. B y F: A. subovata. C y G: P. parvifolia. D y H: P. sellowii. Macerado de corteza: I: Células del súber en vista superficial. J: fibras libriformes. K: parénquima axial. L: células de radio. M y N: drusas, cristales poliédricos y estiloides de oxalato de calcio. O: braquiesclereidas. P: fibroesclereidas. Q: macroesclereidas. R: macroesclereidas con inclusión de cristales poliédricos. Escalas: 1 corresponde a J, O-R. 2 corresponde a L-M. 3 corresponde a E-I, K. 4 corresponde a A-D.



Fig.4. A-D: Sección transversal de los leños .a-d: detalle de los vasos. Observación con MO A y a: A. subovata. B y b: P. parvifolia. C y c: P. sellowii. D y d: C. coccinea



Fig.5. A-F: Caracteres anatómicos diferenciales y comunes del leño. Observación con MEB. A-D: Sección transversal del leño: A, A. subovata poros solitarios con distribución radial y oblicua. B, P. parvifolia poros solitarios. C, P. sellowii poros solitarios y múltiples radiales. D, C. coccinea poros solitarios, geminados, racemiformes y múltiples radiales. E-F: sección longitudinal: E, vasos. F, puntuaciones areoladas alternas. G-I: Cristales de oxalato de calcio en corteza. Observación con MEB. G, solitarios poliédricos. H, drusa. I, estiloides (flechas).




Picramnia sellowii Planch. London J. Bot. 5: 578. 1846. Sinónimos:Picramnia sellowii fo. glabrescens Chodat & Hassl. Bull. Herb. Boissier, sér.2, 3:800. 1903 Picramnia sellowii fo. hirsuta. Chodat & Hassl. Bull. Herb. Boissier, sér.2, 3:800. 1903 Picramnia sellowii fo. intermedia Chodat y Hassl. Bull. Herb. Boissier, sér.2, 3:800. 1903 Picramnia sellowii var. latifolia Engl. Fl. Bras. 12(2):232. 1874. Picramnia sellowii subsp. spruceana (Engl.) Pirani. Bol. Bot. Univ. Sao Paulo 12: 132. 1990. Nombres vulgares: “Cedrillo”, “Cedrillo-na” y “Tarirí” Uso vernáculo: Se utiliza como alterante

(Toursarkissian, 1980).

Hojas: compuestas, pecioladas, alternas, imparipinnadas de 9-15 folíolos, de 4-8 cm de long. x 1-3 cm lat., alternos a subopuestos en la misma hoja, membranáceos a cartáceos, oval lanceolados, margen poco revoluto, ápice obtuso, base asimétrica obtusa o raramente aguda (Fig.1 C, c). Corteza: color gris pardusco; estrías longitudinales y transversales poco profundas que delimitan pequeñas placas. Abundantes lenticelas con importante reborde y apertura en cruz. Fractura entera. Leño: color amarillo pardusco, poros no visibles, anillos apenas perceptibles, albura y duramen no diferenciables.

Castela coccinea Griseb. Abh. Konigl. Ges. Wiss. Gottingen 19: 107. 1874. Sinónimo:Ximenia americana var. purbens Griseb. Symb. Fl. Argent. 149. 1879. Nombres vulgares: “Espada”, “Granadillo”, “Meloncillo”, “Mistol del zorro”, “Mistol del chivo”, “Molle Colorado”, “Sacha melón” y “Sacha meloncillo”- Uso vernáculo: La corteza, hojas y raíz se utilizan en contra de la disentería, diarreas y fiebres intermitentes; también se reconoce como tónico gástrico (Xifreda y Seo, 2006) Hojas: simples, cortamente pecioladas, alternas, de 1,5-3 cm de long. x 0,5-1 cm lat., oblongas pinnatinervias, de margen liso, ápice redondeado, base cuneada (FIg.1 D, d). Corteza: color pardo grisáceo a pardo amarillento, muy rugosa, ligeras estrías longitudinales y

transversales cortas, lenticelas prominentes y notable depósito de líquenes. Fractura fibrosa. Leño: color castaño amarillento, brillante, anillos de crecimiento medianamente visibles, delimitados por una franja pardusca. Porosidad difusa, con tendencia a semicircular. Duramen y albura no diferenciables.

CARACTERES ANATÓMICOS COMUNES

Hojas 1. Lámina en vista superficial

Arquitectura: camptódroma, broquidódroma (Fig.1 a, b, c, D) En todas las especies hay de 4 a 5 órdenes de venas, las secundarias son pinnadas, mientras que las de orden superior son reticuladas. Las venas marginales forman ojales cerrados con terminaciones vasculares libres. Las areolas son poligonales dispuestas al azar, coexistiendo terminaciones vasculares simples y ramificadas y rectas o curvas. La red vascular es de densidad intermedia.

Epidermis: las células de la epidermis adaxial (Fig.1 E, G, I, K) son ligeramente más grandes que las de la epidermis abaxial (Fig.1 F, H, J, L), elongadas sobre los nervios. Los estomas están confinados a la epidermis abaxial. En ambas epidermis se observan escasos pelos simples, unicelulares, de paredes delgadas, que se ubican con mayor densidad sobre las nervaduras, siendo la longitud de los mismos diferente para cada especie (Fig.1 F, H, J, K, L. Fig.2 D, F, G).

2. Lámina en corte transversal Ambas epidermis son uniestratificadas. La hoja es

hipostomática (Fig.1 N, P, R, T). En posición subepidérrmica la vena media se halla reforzada por colénquima de tipo laminar del lado adaxial y abaxial. El nervio medio está constituido por 5 a 7 haces vasculares colaterales abiertos dispuestos en arco, acompañados por una vaina conspicua de fibras (Fig.1 N, P, R, T).

Cortezas Felodermis pluriestratificada.

Leños De porosidad difusa (Fig.4 A-D), la

disposición de los poros es variable según las especies. Los miembros de vasos son en su mayoría de contorno circular y se observan escasos elípticos, presentan placa perforada simple y oblicua, con



apéndices (Fig.4 a-d), las puntuaciones intervasculares son areoladas, de disposición alterna, con abertura interna elíptica e inclusa (Fig.5 E, F).

El parénquima axial se encuentra presente. Los radios son no estratificados, las células

no dejan espacios intercelulares y son de paredes medianamente engrosadas.

Las fibras son de dos tipo: 1- libriforme, dispuestas de manera no estratificada, algunas de ellas son septadas y 2- fibrotraqueidas de paredes moderadamente engrosadas.

CARACTERES ANATOMICOS DIFERENCIALES Tabla 1: Caracteres anatómicos diferenciales de las hojas de A. subovata, P. parvifolia, P. sellowii y C. coccinea.

Alvaradoa subovata Picramnia parvifolia Picramnia sellowii Castela coccinea

Epidermis abaxial Células de paredes

rectas (Fig.1 F. Fig.2 G)

Células de paredes ligeramente sinuosas (Fig.1 H, J, L. Fig.2 E, F)

Tipo Anomocíticos (Fig.1 F)

Anomo y paracíticos (Fig.1 H)

Anomocíticos (Fig.1 J. Fig.2 E)

Anomocíticos (Fig.1 L. Fig.2F) Estomas

Posición Hundidos (Fig.2 G) A nivel

Vis

ta su

perf

icia

l

Pelos glandulares Ausentes Pie uniseriado de

longitud variable y cabeza pluricelular

(Fig.1 M; Fig.2 D, E)

Ausentes

Cutícula en ambas epidermis Delgada y lisa Gruesa y lisa (Fig.2 C)

Uniestratificada

Epidermis adaxial Papilosa (Fig.1 O. Fig.2 A)

Con algunas células septadas;

mucilaginosas (Fig.2 B)

Uniestratificada Con hipodermis

mucilaginosa (Fig.1 T, U. Fig.2 C)

Dorsiventral (Fig.1 O, U, S) Céntrica (Fig.1 T. Fig.2 C)

2-3 hileras de empalizada (Fig.1 O.

Fig.2 A) 1-2 hileras de empalizada. (Fig.1 Q, S. Fig.2 B)

2-3 hileras de empalizada (Fig.1 U.

Fig.2 C) Mesófilo

Parénquima esponjoso sin mucílagos

Parénquima esponjoso con mucílagos (Fig.2

B) Parénquima esponjoso sin mucílagos

Estructuras secretoras internas Ausentes Cavidad esquizógena

con gomorresinas (Fig.1 V; Fig.2 B)

Ausentes

Cristales de oxalato de calcio Drusas y escasos estiloides

Drusas y abundantes cristales solitarios Drusas Drusas y escasos

cristales solitarios

Haces de nervios menores

La vaina parenquimática no

alcanza ambas epidermis

La vaina parenquimática alcanza ambas

epidermis (Fig.2 B)

La vaina parenquimática no alcanza ambas epidermis

Secc

ión

tran

sver

sal

Epidermis abaxial Uniestratificada papilosa

Uniestratificada mucilaginosa Uniestratificada



Tabla 2: Caracteres anatómicos diferenciales de las cortezas de A. subovata, P. parvifolia, P. sellowii y C. coccinea.


Células del súber

Dimensiones variables, paredes con engrosamiento mediano y homogéneo (Fig.3 F).

Paredes con engrosamiento mediano y heterogéneo, las basales lignificadas (Fig.3 G).

Dimensiones variables, paredes con engrosamiento mediano y heterogéneo, las basales en forma de “U” o lignificadas (Fig.3 H).

Homogéneas, no aplanadas, de paredes levemente engrosadas (Fig.3 E).

Parénquima cortical Abundante (Fig3 B). Escaso (Fig.3 C, D). Ausente (Fig.3 A).

Radios secundarios 1-3-seriados. 1-2-seriados, raro 3. 1-3-seriados con

ensanchamiento distal. 1-5-seriados con

ensanchamiento distal.

Elementos esclerenquimáticos

Braquiesclereidas aisladas en parénquima cortical; macro y braquiesclereidas en el límite con el floema (Fig.3 B).

Fibroesclereidas del sistema axial dispuestas en forma de estratos tangenciales discontinuos (Fig.3 C).

Braquiesclereidas aisladas en parénquima cortical. Macro, braqui y fibroesclereidas en el límite con el floema. Fibroesclereidas aisladas en el floema funcional (Fig.3 D).

Macro, braqui y fibroesclereidas en el límite con el floema. Fibras libriformes en el floema funcional (Fig.3 A).

Cristales de oxalato de calcio

Drusas y escasos poliédricos en parénquima cortical (Fig.5 G, H). Estiloides abundantes en el parénquima axial (Fig.3 B. Fig.5 I).

Poliédricos en serie de parénquima septado axial (Fig.3 C).

Drusas y poliédricos abundantes en parénquima cortical y escasos en el axial. Poliédricos incluidos en algunas macro y braquiesclereidas (Fig.3 D).

Drusas y escasos poliédricos en los radios secundarios (Fig.3 A).

Tabla 3: Caracteres anatómicos diferenciales de los leños de A. subovata, P. parvifolia, P. sellowii y C. coccinea.


Crecimiento en anillos

Poco marcados.

De transición gradual Marcados

Disposición de poros

Solitarios y múltiples con distribución radial

y oblicua (Fig.4 A; Fig.5 A).

Solitarios (Fig.4 B, Fig.5 B).

Solitarios (escasos) y múltiples radiales de 4-10 (Fig.4 C Fig.5 C).

Solitarios, geminados, racemiformes y

múltiples radiales cortos de 3-5 (Fig.4 D;

Fig.5 D).

Elemento de vaso con apéndice Apéndice pronunciado (Fig.4, a)

Apéndice medianamente

pronunciado (Fig.4, b)

Apéndice muy pronunciado

(Fig.4, c)

Apéndice apenas pronunciado (Fig.4, d)

Parénquima axial Escaso, difuso, apotraqueal.

Escaso, metatraqueal, de paredes engrosadas.

Casi ausente o apenas metatraqueal.

Abundante paratraqueal vasicéntrico, aliforme o en bandas diagonales.

Radios 1-3-seriados,

heterocelulares (Fig.4 A).

Uniseriados (Fig. 4 B, C), homocelulares, con células erectas cuadrangulares y 2-3 seriados,

heterocelulares.

1-7 seriados (Fig.4 D), homocelulares, con

abundantes cristales de oxalato de calcio.



CARACTERES CUANTITATIVOS

Los datos cuantitativos obtenidos del estudio anatómico de las partes utilizadas, de las especies analizadas, se expresan en las tablas 4, 5 y 6 Tabla 4: Resultados cuantitativos del estudio de las hojas de A. subovata, P. parvifolia; P. sellowii y C. coccinea.

Alvaradoa subovata Picramnia parvifolia Picramnia sellowii Castela coccinea Índice de estomas 9,02±0,91 12,81±1,76 11,04±1,28 13,36±2,65

Estomas (mm2) 4,50±0,85 9,70±2,06 8,20±2,10 13,70±2,50

Índice de empalizada 6,63±1,00 7,33±1,54 4,05±0,66 11,45±2,03

Pelos simple (mm2) 3,50±0,85 2,90±0,88 3,30±0,67 3,00±0,82

Longitud pelos simple (µm) 153,60±26,61 160,00±23,45 543,40±57,66 a 203,20±21,73

137,60±20,80 a 90,60±3,27

Tabla 5: Resultados cuantitativos del estudio de las cortezas de A. subovata, P. parvifolia; P. sellowii y C. coccinea.


Longitud fibras (µm) 359,60±45,19

a 656,00±69,64

338,40±50,74 a

720,00±61,51-

421,60±47,71 a

876,00±121,16-

439,30±64,29 a

1082,00±176,05 Latitud fibras (µm) 22,40±3,37 24,80±5,90 24,80±4,54 11,40±2,32

Tabla 6: Resultados cuantitativos del estudio de los leños de A. subovata; P. parvifolia; P. sellowii y C. coccinea.

Alvaradoa subovata Picramnia parvifolia Picramnia sellowii Castela coccinea Vasos (mm2) 26,50±6,13 11,60±1,51 89,60±10,71 104,20±12,26

Diámetro miembros de vasos (µm) 64,80±7,00 76,00±6,80 32,00±6,53 a 56,00±6,53

32,00±6,53 a 70,4±14,51

Longitud miembros de vasos (µm) 194,40±43,42 a 560,00±132,09

422,40±47,97 506,40±54,00 173,90±24,55

unicelulares 8,50±1,43 7,20±1,14 12,4±2,80 16,50±5,23 Altura radios (nº de células pluricelulares 23,50±3,37 16,00±1,94 29,30±6,75 31,90±5,97

Longitud fibras xilares (µm) 350,40±43,79 a 627,20±61,30

355,20±44,01 a 780,80±49,77

347,20±41,86 a 736,00±57,44

340,90±49,83 a 864,00±61,18

Latitud fibras xilares (µm) 26,40±5,40 24,00±5,33 24,80±4,54 11,60±1,57

DISCUSIÓN Y CONCLUSIONES

Las especies de la familia Simaroubaceae (sensu lato) aquí estudiadas, muestran caracteres morfo-anatómicos de valor diagnóstico que permiten su reconocimiento cuando se encuentran molturadas, que es la forma en que se utilizan y/o comercializan. De las especies estudiadas, P. parvifolia no cuenta, en la bibliografía consultada, con uso vernáculo reconocido; sin embargo por comunicaciones orales se sabe que se la comercializa indistintamente con las otras especies analizadas.

En la bibliografía consultada, las obras clásicas sobre anatomía de los órganos vegetativos de las Dicotiledóneas (Solereder, 1908; Metcalfe y Chalk, 1972), no se hallaron las entidades estudiadas en el presente trabajo, todas nativas de Argentina, si se mencionan los géneros, Alvaradoa, Picramnia y Castela.

El estudio morfoanatómico de las hojas ha aportado los siguientes caracteres diagnósticos: pelos glandulares presentes solo en P. sellowii acordando con Solereder (1908), quien señala que no ocurren



pelos glandulares en todos los miembros de esta familia. La estructura del mesófilo es en C. coccínea céntrica y dorsiventral en las tres restantes, coincidente con lo indicado por Metcalfe y Chalk (1972) para el género Castela; en esta misma especie se observa una hipodermis mucilaginosa, mientras que en P. parvifolia los mucílagos están presentes en las epidermis, carácter no mencionado por los autores consultados para estos géneros. Con respecto a las cavidades secretoras, solo se encontraron en los parénquimas de la lámina de P. parvifolia; Metcalfe y Chalk (1972) lo mencionan acompañando a los haces vasculares en numerosos géneros, indicando que son poco frecuentes en Picramnia y Alvaradoa, por lo que coincidimos en esta apreciación con respecto a Alvaradoa, no así con Picramnia. Los cristales de oxalato de calcio constituyen un caracteres valioso para diferenciar a estas cuatro especies, se presentan como cristales poliédricos solitarios y drusas en C. coccínea y P. parvifolia, como drusas y estiloides en A. subovata, acordando con lo mencionado por Solereder (1908) y Metcalfe y Chalk (1972), mientras que solo se presentan drusas en P. sellowii. El tamaño y cantidad de los cristales varía según las especies; así, en A. subovata los estiloides son escasos, en P. parvifolia son abundantes los cristales poliédricos y en C. coccinea abundan las drusas; estas observaciones son indicadas también por Solereder (1908) y Metcalfe y Chalk (1972). No es un dato menor la extensión de la vaina parenquimática de los haces vasculares menores las que se presentan solo en P. parvifolia.

Para cortezas, Solereder (1908) y Metcalfe y Chalk (1972), sólo se circunscriben a la especie Ailanthus altisssima, en base a las descripciones de Müller (1908). Di Sapio et al. (1997) analizan caracteres anatómicos de cortezas y leños de Ailanthus altisssima, Quassia amara y Castela tweedii a fin de contribuir al conocimiento y delimitación de las citadas especies.

Existe uniformidad respecto del número de peridermis excepto para Castella coccinea. Las células del súber son muy variables en la constitución de sus paredes ya que alternan notablemente estratos celulares con escaso o fuerte depósito de material graso o lignina en sus paredes, en algunos caso en forma de “U” como sucede en P. sellowii. La felodermis, es pluriestratificada.

La sección transversal de la corteza muestra, en la zona límite entre el parénquima cortical y el floema funcional en A. subovata, P. sellowii y C. coccinea

un anillo discontinuo constituido por tres tipos celulares: macro, braqui y fibroesclereidas. En P. sellowii, las macro y braquiesclereidas muestran incrustaciones de cristales poliédricos de oxalato de calcio.

En la corteza interna de C. coccinea y P. sellowii se observó un ensanchamiento distal de los radios secundarios. Roth (1981) en sus estudios acerca de la estructura anatómica de la corteza de árboles tropicales, reconoce la disposición de las fibras como el criterio diagnóstico de mayor valor, coincidiendo con esta observación, se ha encontrado que en C. coccinea, hay abundantes estratos formados por fibras libriformes, en P. parvifolia 3-5 estratos de fibroesclereidas y en P. sellowii aislados estratos de fibroesclereidas.

Los tipos de cristales de oxalato de calcio coinciden con los mencionados para los caracteres histofoliares; en cuanto a la distribución es típica para cada especie, así se observan estiloides en el floema funcional de A. subovata, para las dos especies de Picramnia los cristales poliédricos se ubican en el floema funcional siendo más abundantes en P. parvifolia y en C. coccinea las drusas y los poliédricos, que son escasos, están confinados a los radios secundarios.

La caracterización de las cortezas encaradas en este trabajo es la primera contribución al respecto, ya que las mismas no han sido descriptas con anterioridad.

Del análisis de los resultados obtenidos sobre las estructuras de los leños de las especies aquí estudiadas, se puede afirmar que existen escasas analogías entre ellas, lo que permite lograr una adecuada diferenciación de los mismos. Asimismo, y a pesar de algunas pequeñas discrepancias observadas, sobretodo en los caracteres referidos al tamaño y número de los elementos celulares, originadas por las distintas condiciones climáticas en sus hábitat, coincidimos con las observaciones realizadas por O’Donell (1937), Heimsch (1942) y Metcalfe y Chalk (1972), relativo a las descripciones del leño de las especies de los géneros Alvaradoa, Picramnia y Castela.



Clave para delimitar las especies por los caracteres morfo-anatómicos de la hoja

A- Hojas compuestas imparipinnadas. B- 16-24 folíolos.

C- Mesófilo dorsiventral, 2-3 hileras de células en empalizada, ambas epidermis papilosas, parénquima esponjoso sin mucílagos y sin cavidades secretoras, con drusas y estiloides de oxalato de calcio. A. subovata

BB- 7-15 folíolos. C- Epidermis adx. y parénquima esponjoso con mucílagos, cavidades secretoras, drusas y abundantes

cristales poliédricos de oxalato de calcio. P. parvifolia CC- Epidermis adx. y parénquima esponjoso sin mucílagos, sin cavidades secretoras y con drusas de

oxalato de calcio. P. sellowii AA- Hojas simples.

B- Mesófilo céntrico con 2-3 hileras de células en empalizada, parénquima esponjoso sin mucílagos y con drusas y escasos cristales solitarios de oxalato de calcio. Hipodermis mucilaginosa.

C. coccinea

Clave para delimitar las especies por los caracteres anatómicos de la corteza

A- Células del súber con engrosamiento homogéneo. B- Paredes de las células del súber levemente engrosadas, sin parénquima cortical, drusas de oxalato de calcio en parénquima de radios secundarios. C. coccinea BB- Paredes de las células del súber medianamente engrosadas, con abundante parénquima cortical y estiloides de oxalato de calcio en el parénquima axial. A. subovata

AA- Células del súber con engrosamiento heterogéneo. B- Células basales del súber lignificadas, radios secundarios 1-2 seriados (raro 3), cristales poliédricos en parénquima septado axial. P. parvifolia

BB- Células basales del súber engrosadas en U con lignina, radios secundarios 1-3 seriados con ensanchamiento distal, cristales poliédricos de oxalato de calcio incluidos en macro y braquiesclereidas. P. sellowii

Clave para delimitar las especies por los caracteres anatómicos del leño

A- Poros solitarios. B- Parénquima axial metatraqueal escaso. P. parvifolia

AA- Poros solitarios y múltiples, radiales. B- Poros geminados y racemiformes. Parénquima axial paratraqueal vasicéntrico, aliforme o

en bandas diagonales. C. coccinea BB- Parénquima axial metatraqueal, escaso. C- Radios 1-3 seriados heterocelulares. A. subovata CC-Radios uniseriados homocelulares y 2-3 seriados heterocelulares. P. sellowii



ANEXO 1:

Materiales estudiados Alvaradoa subovata Cronquist ARGENTINA. Prov. Jujuy: Dpto. Valle

Grande, R Jordán. 28-XII-1977, Kiesling y Ulibarry 1634 (SI); Dpto. Ledesma, camino de Río A. Negra a Abra de las Cañas, 21-III-1972, Legname et al 9153y 9155 (MCNS), Ruta provincial nº 83 Parque Nacional Calilegua Km 8-10 margen norte del río San Lorenzo 10-12 Km al NW del pueblo Ledesma 700-900 msm, 20-VI-1998, Novara et al 11083 (MCNS); Dpto. Santa Bárbara, Abra de los Morteros. 26-I-1975, Zuloaga y Deginiani 278 (SI). Prov. Salta: Dpto. Orán, ruta Prov.18 a 3-4 Km. del Puente Internacional Argentina-Bolivia. 22º 43´S 64º43´W, 1-V-2003, Morrone et al 4536 (SI); Dpto. Metan, Sierras de Metan Proyecto de Prospección Minera “León” Finca Cachari 18-20 Km al W de Lumbreras 1334 msm, 25° 12' 57,3" S 65° 06' 44,0" W, 10-IV-2006, Tolaba et al 4095 y 4116 (MCNS); Dpto. Capital, Río Vaqueros 5 Km al W del puente Ruta 9, 1-III-1982; Novara et al 2546 (MCNS); Dpto. Chicoana, Quebrada de Tilian 1300 msm, 13-II-1982, Novara et al 2390 (MCNS), 1400 msm, 2-XII-1983, Varela y Del Castillo 270 (MCNS), 5-XII-1986, Palaci 837 (A) (MCNS), 4-XII-1986, Del Castillo y Varela 970 (MCNS), 30-I-1987, Ortin 65 (MCNS); Quebrada de Scoipe Mal paso pasando Cañada de la Gotera, 19-XII-1980, Novara 1442 (MCNS), a 1 Km de Los Laureles 1400 msm, 27-III-1984, Varela et al 503 (MCNS), pasando Los Laureles 2-4 Km antes de Chorro Blanco 1600 msm, 15-XII-1989, Novara et al 9199 (MCNS), El Infienillo pasando El Nogalar antes de arroyo La Gotera 1600 msm, 16-XII-1995, Novara et al 10762 (MCNS),en la Quebrada Los Sauces al oeste de la Ruta 33 Km 16 1400 msm, 30-XI-1997, Novara et al 10955 (MCNS), ruta 33 pasando 1-2 Km Los Laureles antes de Finca Cerro Grande 1300-1400 msm, 28-II-2008, Lazaro et al 13074 (MCNS); Dpto. La Viña, 2-II-1951, Hunziker 1226 (SI). Prov. Tucumán: Dpto. burruyacú, Rio Nio al Alto de Medina 1500msm. 7-VII-2007, Ponesa s/nº (UNR)∗.

Picramnia parvifolia Engl. ARGENTINA. Prov. Misiones: Dpto. San

Pedro, Arroyo San Pedro, 1-XI-1958, Gamerro y Toursarkissian 69 (SI). Dpto. Oberá, Oberá, 5-V-2007, Oakley et al. s/nº (UNR). Prov. Corrientes: Dpto. Santo Tomé, Santo Tomé, 7-XII-1997, Múlgura et al. 1589 (SI)

Picramnia sellowii Planch. ARGENTINA. Prov.Formosa: s/f. Jorgensen

3260 (SI). Prov. Misiones: Dpto. Gral. Belgrano, Bernardo de Irigoyen 2 Km al S de B. de Irigoyen sobre naciente del río Pepirí Guazú, 26º16´S 53º38´W, 15-X-1996, Morrone et al 1426, (SI); Dpto. San Ignacio, Peñón del Teyucaré, 27º16´ S 55º35´W, 20-IX-2000, Múlgura et al 2157 (SI); Dpto. Posadas, Posadas, 17-I-1930, Rodríguez 27 (SI). Prov. Chaco: Dpto. Bermejo, arroyo Zapirán y ruta 11, 23-XI-2007, Oakley et al. 53 (UNR). Prov. Corrientes: Dpto. Loreto, Timbó Paso, 5-V-45, Huidobor 2179 (SI).

Castela coccinea Griseb. ARGENTINA. Prov. Jujuy: Dpto. San

Pedro, Cerritos de San Pedro a 700m, -X-1930, Venturi 19546 (SI). Prov. Salta: Dpto. Oran, Río Piedras, 13-XI-1911, Rodriguez 76 (SI). Prov. Chaco: Dpto. 1 de Mayo, Colonia Benítez, 30-IX-1971, Martínez et al 9479 (BAA); Dpto. Chacabuco, Charata, 24-I-2008, Oakley y Festa 73 (UNR); Dpto. Resistencia, A 5 Km. al S de Resistencia, 24-IX-1986, Pire 7707 (UNR). Prov. Santiago del Estero: Dpto. Robles, Ventura, 11-I-2008, Oakley 66 (UNR). Prov. Santa Fe: Dpto. Gral. Obligado, próximo a Berna, 17-XII-2004, Pensiero 6943 (SF); Dpto. Vera, Las Gamas, 12-I-1989, Pire 2641 (UNR); Dpto. 9 de Julio, al O de Sta. Margarita, 25-IX-1981, Lewis 3255 (UNR); Ruta 35 El Cuadrado, 25-IX-1981, Pire 3262 (UNR); Dpto. San Javier, Ruta 11 arroyo El Toba, 14-XII-1982, Pire 1177 (UNR).



BIBLIOGRAFÍA Ajaiyeoba E, Abalogu U, Krebs H, Oduola A. 1999. In

vivo antimalarial activities of Quassia amara and Quassia undulata plant extracts in mice. J. Ethnopharmacol. 67: 321–325.

Anderson M, O'Neill M, Phillipson J, Warhurst D. 1991. In vitro cytotoxicity of a series of quassinoids from Brucea javanica fruits against KB cells. Planta Med. 57: 62.

Balderrama L, Bracab A, Garcia E, Melgarejo Pizza C, De Tommasi N. 2001. Triterpenes and anthraquinones from Picramnia sellowii Planchon in Hook (Simaroubaceae). Biochem. Syst. Eco. 29: 331-333.

Bertani S, Houël E, Bourdy G, Stien D, Jullian V, Landau I, Deharo E. 2007. Quassia amara L. (Simaroubaceae) leaf tea: Effect of the growing stage and desiccation status on the antimalarial activity of a traditional preparation. J. Ethnopharmacol. 111: 40-42.

Boodle L. 1916. A method of macerating fibres. Bulletin Miscellaneous Inform. 5: 108-110.

Bourdy G, Oporto P, Gimenez A, Deharo E. 2004. A search for natural bioactive compounds in Bolivia through a multidisciplinary approach Part IV. Evaluation of the antimalarial activity of plants used by Isoceño-Guaraní Indians. J. Ethnopharmacol. 93: 269–277.

Camacho M, Phillipson J, Croft S, Solis P, Marshall S, Ghazanfar S. 2003. Screening of plant extracts for antiprotozoal and cytotoxic activities. J. Ethnopharmacol. 89: 185–191.

Camacho M, Phillipson D, Croft S, Yardley V, Solis P. 2004. In vitro antiprotozoal and cytotoxic activities of some alkaloids, quinones, flavonoids, and coumarins. Planta Med.. 70: 70-72.

Castro J, Montalto de Mecca M, Bartel L. 2006. Toxic side effects of drugs used to treat Chagas' disease (American trypanosomiasis). Hum. Exp. Toxicol. 25: 471- 479.

Cordell G, Kinghorn D, Pezzuto J. 1993. Separation, structure elucidation and bioassay of cytotoxic natural products. In: Bioactive Natural Products. Detection, Isolation and Structural Determination, Colegate, Molyneux (eds.), CRC Press: Boca Raton (USA) pp: 195-219.

Coster C. 1927. "Zür Anatomie und Physiologie der Zuwachszonen und Jahresringbildung in den Tropen". Ann. Jard. Bot. Buitenzorg (37): 49-160.

Cronquist A. 1944. Studies in the Simaroubaceae-IV. Resume of the American Genera. Brittonia 5(2): 128-147.

Cuendet M, Pezzuto J. 2004. Antitumor Activity of Bruceantin: An Old Drug with New Promise. J. Nat. Prod. 67: 269-272.

Da Silva M, Gottlieb O. 1987. Evolution of quassinoids and limonoids in the Rutales. Biochem. Syst. Eco. 15: 85-103.

Di Sapio O, Gattuso S, Gattuso M. 1997. Anatomía de Leños y Cortezas de Tres Especies de Simaroubaceae Empleadas en la Medicina Popular. VI Congreso Italo-latinoamericano de Etnomedicina. Antigua Guatemala, 6 – 9 Octubre 1997.

Erdtman G. (1986). Pollen morphology and plant taxonomy: angiosperms. EJ. Brill, Leiden, pp. 406-408.

Fernando E, Quinn C. 1992. Pericarp anatomy and systematics of the Simaroubaceae sensu lato. Austral. J. Bot. 40: 263-289.

Fukamiya N, Okano M y Miyamoto M. 1992. Antitumor agents, 127. Bruceoside C, a new citotoxic quassinoid glucoside, and related compounds from Brucea javanica. J. Nat. Prod. 55 (4): 468-475.

Gattuso MA, Gattuso SJ. 2002. Técnicas Histológicas en Material Vegetal. Facultad de Ciencias Bioquímicas y Farmacéuticas UNR, pp. 62-74.

Grieco P, Morré J, Corbett T. 1997. Valeriote F. Therapeutic quassinoids preparations with antineoplastic, antiviral, and herbistatic activity. United States Patent, (5,639,712) Jun. 17, 1997.

Grieve M. 1996. A Modern Herbal. Barnes & Noble, New York, p.166.

Guo Z., Vangapandu S., Sindelar R.W., Walker L. & Sindelar R. 2005. Biologically active quassinoids and their chemistry:potential leads for drug design. Curr. Med. Chem. 12: 173-190.

Guy Balansard E, Hajime Ohigashi F. 2002. In vitro anti-tumor promoting and anti-parasitic activities of the quassinoids from Eurycoma longifolia, a medicinal plant in Southeast Asia. J. Ethnopharmacol. 82: 55-/58.

Heimsch T. 1942. Comparative anatomy of the secondary xylem in the Gruinales and Terebinthales of Wettstein with reference to taxonomic grouping. Lilloa 8: 83-199.

IAWA 1989. List of microscopic features for hardwood identification. E.A. Wheeler, P. Baas & Grason Eds. 1989. IAWA Bull. 10: 219-332.

Hickey J. 1973. Classification of the architecture of Dicotyledons leaves. American Journal of Botany 60: 17-33, traducido por Zardini E. (1974). Bol. Soc. Argent. Bot. 16: 1-26.

Holmgren PK, Holmgren NH, Barnett LC. (eds.). 1990. Index Herbariorum. Ed. New York Botanical Garden, New York, U.S.A.

Jacobs H, Simpson D, Reynolds W. 2007. Quassinoids and a coumarin from Castela macrophylla (Simaroubaceae). Biochem. Syst. Ecol 35: 42-44.

Kelly D, Dickinson T. 1985. Local names for Vascular Plants in the John Crow Mountains, Jamaica. Econ Bot. 39(3): 346-362.



Lidert Z, Wing K, Polonsky J, Imakura Y, Okano M, Tani S, Lin Y, Kiyokawa H, Lee K. 1987. Insect antifeedant and growth anhibitory activity of forty-six Quassinoids on two species of agricultural pest. J. Nat. Prod. 50:442-448.

Martinez M. 1069. Plantas Medicinales de México. México, p. 656

Martínez ML, Campagna MN, Ratti MS, Nocito I, Serra E, Gattuso SJ and Gattuso MA. 2009. Trypanocide activity of Castela coccinea Griseb. Extracts. BLACPMA 8 (3), 211-218.

Martínez Crovetto R. 1981. Las plantas utilizadas en Medicina Popular en el NO de Corrientes (R.A.) Miscelánea Nº 69. Fundación Miguel Lillo, Tucumán, p. 84.

Metcalfe C, Chalk L. 1950. Anatomy of the Dicotyledons. Vol. I Clarendon Press, Oxford, London, pp. 317-326.

Metcalfe C, Chalk L. 1972. Anatomy of the Dicotyledons. Vol I. Clarendon Press. Oxford London, pp. 471-476.

Moncada M, Machado S. 1987. Los granos de polen de Simarubaceae. Acta Bot. Cub. 45: 1-7.

Moncayo A. 2003. Chagas disease: current epidemiological trends after the interruption of vectorial and transfusional transmission in the Southern. Mem Inst Oswaldo Cruz 98: 577-591.

Morre J, Grieco, Morre D. 1998. Mode of action of the anticancer quassinoids-inhibition of the plasma membrane NADH oxidase. Life Science 63(7): 595-604.

Müller R. 1908. Zur Anatomie der Ailanthus-Rinden. Pharm. Praxis 7: 261-263.

O´Brien T, McCully M. 1981. The study of plant structure, principles and selected methods. Termarcarphi Pty Ltd., Melbourne, Australia, p. 321.

O’Donell C. 1937. Anatomía comparada del leño de tres Simarubáceas argentinas. Lilloa 1: 263-282.

Ogura M, Cordeli G, Kinghorn A, Fransworth N. 1977. Potential anticancer agents VI. Constituents of Ailanthus excelsa. Lloydia 40: 579.

O’Neill M, Bray D, Boardman P, Phillipson D, Warhurst D, Peters W, Suffness M. 1986 Plants as sources of antimalarial drugs: In vitro antimalarial activities of some quassinoids. Antimicrob. Agents Chemother. 30(1): 101-104.

O'Neill M, Bray D, Boardman P, Chan K, Phillipson J, Warhurst D, Peters W. 1987. Plants as sources of antimalarial drugs, Part 4: Activity of Brucea javanica fruits against chloroquine-resistant Plasmodium falciparum in vitro and against Plasmodium berghei in vivo. J. Nat. Prod. 50: 41.

O'Neill M, Bray D, Boardman P, Wright C, Phillipson J, Warhurst D, Gupta M, Correya M, Solis P. 1988. Plants as sources of antimalarial drugs, Part 6: Activities of Simarouba amara fruits. J. Ethnopharmacol. 22(2): 183-190.

Pirani J. 1987. Simaroubaceae. Flora de Paraguay. Ed. Missouri Botanical Garden. Sant Louis, Estados Unidos, pp. 7-28. Pirani J. 1990. As Especies de Picramnia sw. (Simaroubaceae) do Brasil: uma sinopse. Bol. Botanica, Univ. Sao Paulo 12: 115-180. Roth I. 1981. Structural Patterns of Tropical barks.

Encyclopedia of Plant Anatomy. 609 pp. Salisbury EJ. 1927. Stomatal Frecuency Phil. Trans. Roy.

Soc. London 216 B: 1-65 Siwajinda S, Santisopasri V, Murakami A, Hirai N,

Ohigashi H. 2001. Quassinoids from Eurycoma longifolia as plant growth inhibitors. Phytochemistry 58: 959–962.

Simão S, Barreiros E, da Silva M, Gottlier O. 1991. Chemogeographical evalution of quassinoids in Simaroubaceae. Phytochemistry 30: 853-865.

Solereder HO. 1908. Systematic anatomy of the Dicotyledons, I. Clarendon Press, London, pp. 857-859.

Strittmatter C. 1973, Nueva técnica de diafanización. Bol. Soc. Argent. Bot. 15 (1): 126-129.

Strittmatter C. 1979 Modificación de una técnica de coloración Safranina-Fast green. Bol. Soc. Argent. Bot. 18 (3-4): 121-122.

Strittmatter C. 1980. Coloración con Violeta de Cresyl. Bol. Soc. Argent. Bot. 19 (12): 273-276.

Stuhlfauth T, Fock H, Huber H, Klug K. 1985. The distribution of fatty acids including petroselinic and taricic in the fruit and seed oils of the Pittosporaceae, Umbelliferae, Simarubaceae and Rutaceae. Biochem. Syst. Ecol. 13: 447-453.

Tagahara K, Kuo-Hsiung L. 1993. Bruceanols D, E, and F. Three new cytotoxic quassinoids from Brucea antidysenterica. J. Nat. Prod. 56(12): 2091-2097.

Timmerman HA. 1927. Stomatal numbers. Pharm. J. Ser. 4: 735-42.

Toursarkissian M. 1980. Plantas Medicinales Argentinas. Ed. Hemisferio Sur. Buenos Aires, p. 128.

Toyota T, Fukamiya N, Okano M. 1990. Antitumor agents, 118. The isolation and characterization of Bruceanic acid A, its methyl ether, and the new Bruceanics acids B, C, and D, fron Brucea antidyseterica. J. Nat. Prod. 53(6): 1526-1532.

UMSA-Fundación KAA-IYA-IRD, CABI-WCS Bolivia- HNB, CYTED-OEA. (eds). 2002. Plantas del Chaco II. Usos tradicionales Izoceño-Guaraní. Santa Cruz, Bolivia, pp. 44-46.

Üphof JC, Hummel K, Staesche K. 1962. Plant hairs. In K. Linsbauer (ed.), Hnadbuch der Pflanzenanatomie 4(5): 1-292. Gebrüder Borntraeger, Berlín, pp. 232, 238, 241.

Webber IE. 1936. Systematic anatomy of the woods of the Simarubaceae. Am. J. Bot. 23: 577-587.


Wheeler EA, Pearson R.G., LaPasha CA. Zack T., Hatley W. 1986. Computer-Aided Wood Identification. N. Carolina Agricult. Res. Service Bull. 474.

Wheeler EA, Bass P, Gasson PE. 1989. IAWA list of microscopic features for hardwood identification. IAWA Bull. 10: 219-332.

Xifreda C. 1999. Simaroubaceae. En Zuloaga F. y Morrone O. (eds.) (1999) Catálogo de las Plantas Vasculares de

la República Argentina. Syst. Bot. Missouri Bot. Gard. 74: 118.

Xifreda C, Seo M. 2006. 138. Simaroubaceae. Flora Fanerogámica Argentina, Fascículo 99, Proflora-CONICET. 1-13.

Zorning H, Weiss G. 1925. Anatomy of leaves of Compositae. Arch. Pharm. Berl. 263: 451-70.






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Composición química y efecto antibacteriano del aceite esencial de Aloysia triphylla (L’Hér.) Britton contra patógenos genito-urinarios

[Chemical composition and antibacterial effects of the essential oil of Aloysia triphylla against genito-urinary pathogens]

Luis B. ROJAS1, Judith VELASCO2, Tulia DÍAZ3, Ricardo GIL OTAIZA4, Juan CARMONA5, Alfredo USUBILLAGA1.

1Instituto de Investigaciones, 2Departamento de Microbiología y Parasitología, 3Departamento de Bioanálisis Clínico, 4Cátedra de Farmacognosia, 5Jardín de Plantas Medicinales “Dr. Luís Ruiz Terán”, Facultad de Farmacia y Bioanálisis. Universidad de Los Andes,

Mérida - Venezuela.

Abstract

The essential oil of Aloysia triphylla was obtained by hydrodistillation of the aerial parts of the plant and was analyzed by GC and GC-MS Twenty two components were identified. The main constituents were geranial (27.3%) neral (22.5%), geraniol (6.2%), biciclogermacreno (5.2%) and nerol (4.9%). Evaluation of antibacterial activity by the agar diffusion method with disks against clinical isolates from urinary tract infections and bacterial vaginosis revealed inhibition of development of all isolates (Escherichia coli, Klebsiella ozaenae, Enterobacter aerogenes, Proteus mirabilis, Staphylococcus aureus and Enterococcus sp.), with MIC values of 10-50 mg/ml. This is the first report about the antibacterial activity of this essential oil against genito-urinary pathogens. The low doses observed, suggested it may be used in pharmaceutical preparations for the treatment of infections caused by these microorganisms.

Keywords: Aloysia triphylla, Verbenaceae, essential oil, antibacterial activity.

Resumen

El aceite esencial de Aloysia triphylla fue obtenido por hidrodestilación de las partes aéreas de la planta y fue analizado por CG y CG-EM, se identificaron 22 componentes, siendo los mayoritarios geranial (27,3 %), neral (22,5 %), geraniol (6,2 %), biciclogermacreno (5,2 %) y nerol (4,9 %). La evaluación de la actividad antibacteriana del aceite esencial por el método de difusión en agar con discos contra aislados clínicos de infecciones del tracto urinario y de vaginosis bacteriana, reveló inhibición del desarrollo de todos los aislados (Escherichia coli, Klebsiella ozaenae, Enterobacter aerogenes, Proteus mirabilis, Staphylococcus aureus and Enterococcus sp.), con valores de CIM de 10-50 µg/ml. Este es el primer reporte sobre el efecto antibacteriano de este aceite esencial contra patógenos genito-urinarios y la baja dosis observada, sugiere que este aceite podría ser usado en preparaciones farmacéuticas para el tratamiento de infecciones causadas por estos micro-organismos.

Palabras Clave: Aloysia triphylla, Verbenaceae, aceite esencial, actividad antibacteriana.

List of Abbreviations GC: Gas Chromatography ; GC-MS: Gas Chromatography-Mass Spectrometry ; MIC: Minimal Inhibitory Concentration; CLSI: Clinical and Laboratory

Standars Institute; CG-EM: Cromatografía de Gases acoplada a Espectrometría de Masas ; CG: Cromatografía de Gases; CIM: Concentración Inhibitoria Mínima; ITU: Infección del Tracto Urinario ; VB: Vaginosis Bacteriana ; DMSO: Dimetilsulfóxido ;BLEE: β-Lactamasa de espectro extensor; SV: Secreción vaginal; URO: Urocultivo

Recibido | Received: September, 30, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: November 16, 2009. Publicado en Línea | Published Online 15 December 2009 Declaración de intereses | Declaration of interests: authors have no competing interests. Financiación | Funding: This work was financed by el Consejo de Desarrollo Científico Humanístico y Tecnológico (CDCHT Universidad de los Andes, Mérida-Venezuela) This article must be cited as: Luis B. Rojas, Judith Velasco, Tulia Díaz, Ricardo Gil Otaiza, Juan Carmona, Alfredo Usubillaga. 2009. Composición química y efecto antibacteriano del aceite esencial de Aloysia triphylla (L’Hér.) Britton contra patógenos genito-urinarios. Bol Latinoam Caribe Plant Med Aromat 9(1): 56 - 62. {EPub 15 Dec 2009}.

*Contactos | Contacts: Email: [email protected] ; Tel: 0058-2742403568; 00582742403410; Fax: 0058-2742403568.


Rojas et al. Composición química del aceite esencial de A. triphylla y efecto contra patógenos genito-urinarios


INTRODUCCIÓN

La infección del tracto urinario (ITU) es la invasión, colonización y multiplicación de microorganismos en el aparato urinario (Lizama et al., 2005, Ochoa et al., 2005). Es la segunda causa de infección más frecuente en humanos, constituye un importante problema de salud que afecta a millones de personas cada año (Echeverría et al., 2006).

La vaginosis bacteriana (VB) es una infección cervicovaginal, que resulta de alteraciones en la flora bacteriana aerobia y anaerobia, con disminución del número de bacilos de Döderlein con aparición de un flujo genital, lo cual se traduce en cambios fisicoquímicos de las secreciones vaginales. Es una de las dos infecciones genitales más frecuentes en las mujeres con vida sexual activa (Hillier & Holmes, 1999, Caballero et al., 2000, Mota et al., 2008).

A pesar de la amplia disponibilidad de antibióticos para el tratamiento de las ITU y VB, en ocasiones la sintomatología no desaparece, por un fenómeno creciente y que preocupa a la comunidad médica denominado resistencia bacteriana. Cada vez es más frecuente el aislamiento de bacterias multirresistentes como las productoras de β-Lactamasa de espectro extenso (BLEE) (Beigi et al., 2004, Karlowsky et al., 2006, Gobernado et al., 2007, Colodner et al., 2008, Gagliotti et al., 2008, Guneysel et al., 2009).

Frente a esta problemática, se destaca el importante papel que han desempeñado las plantas como fuente de sustancias con importante actividad farmacoterapéutica. En tal sentido, a la especie Aloysia triphylla se le ha descrito actividad antibacteriana y antifúngica (López et al., 2004, Teixeira et al., 2005, 2007, Sartoratto et al., 2004, Demo et al., 2005, Duarte 2006, Duarte et al., 2007).

Aloysia triphylla (L´Hér.) Britton (Aloysia citriodora [Lam.] H.B.K., es originaria de Sudamérica y pertenece a la familia Verbenaceae. La especie es ampliamente utilizada por los pobladores de la zona para resolver diversos problemas de salud. En trabajos anteriores se reporta la presencia de un flavonoide, la luteolin-7-diglucuronida y el compuesto fenólico verbascoside (Carnat et al., 1995; Skaltsa & Shammas, 1988). La composición del aceite esencial ha sido estudiada en diversos países, la mayoría presentan como componentes mayoritarios citral (neral + geranial) y limoneno (Montes et al., 1973, Özek et al., 1996, Carnat et al., 1999, Sartoratto et al., 2004, Gomes et al., 2006, Argyropoulou et al., 2007, ; Díaz

et al., 2007, Di Leo Lira et al., 2008), un trabajo realizado en Marruecos reporta 1,8-cineol y citral (neral + geranial) (Bellakhdar & ldrissi, 1994), otro en Argentina presenta a la Myrcenona y alfa-tujona (Zygadlo et al., 1994) y por último un estudio de Colombia muestra citral (neral + geranial), nerol y geraniol como componentes más abundantes (Jaramillo et al., 2003).

Motivado al incremento de patógenos genito-urinarios multirresistentes, en el presente estudio se evaluó la actividad antibacteriana del aceite esencial de A. triphylla contra 9 aislados asociados a ITU, 11 aislados asociados a VB y 5 cepas de referencia por el método de difusión en agar con discos, además se describe su composición química.

MATERIALES Y MÉTODOS

Material vegetal y extracción Las partes aéreas frescas (1 kg) de A. triphylla,

fueron recolectadas en junio de 2007 en la localidad de Cacute, Parroquia perteneciente al Municipio Rangel del Estado Mérida (Venezuela), una muestra (voucher N° Juan Carmona 788) se depositó en el Herbario MERF “Luís Ruiz Terán” de la Facultad de Farmacia y Bioanálisis de la Universidad de Los Andes. La muestra fue sometida a hidrodestilada durante 4 horas, usando un aparato tipo Clevenger. El aceite esencial obtenido se secó sobre sulfato de sodio anhidro y se guardó bajo refrigeración (4ºC) en la oscuridad.

Cromatografía de gases (CG) Un cromatógrafo de gases, modelo Autosystem,

marca Perkin Elmer, equipado con un detector de llama, fue usado para la determinación de los índices de Kováts, mediante et comparación con n-alcanos (C7-C22). Los análisis fueron llevados a cabo usando una columna capilar HP-5 (30 m x 0,25 mm id, con una película de 0,25 μm de espesor). La temperatura del bloque de inyección fue de 200 ºC. La temperatura del horno del GC fue programada como sigue: temperatura inicial 60 ºC, se aplicó luego un incremento de 4 ºC.min-1 hasta una temperatura final de 260 ºC. Se usó helio como gas portador con un flujo de 1.0 ml/min a volumen constante (Rojas et al., 1999).

Cromatografía de gases acoplada a espectrometría de masas (CG-EM)

El análisis se realizó en un cromatógrafo de gases Hewlett Packard 6890 serie II acoplado a un detector de masas Hewlett Packard 5973, equipado con un



inyector automático HP y una columna capilar HP-5MS de 30 m de largo (0.25 mm id; con una película de 0,25 μm de espesor). Se usó una energía de ionización de 70 eV. Se inyectó una muestra de 1.0 μl de 2% de solución de aceite en n-heptano con un reparto de 100:1. La identificación de los componentes de la esencia fue establecida utilizando la base de datos Wiley (6a Ed.) y comparación de los índices de kováts obtenidos con datos publicados en la literatura (Adams, 1995). La temperatura del inyector y el programa de temperatura fueron los mismos usados para la medición de los índices de Kováts.

Análisis Microbiológico

Cepas bacterianas Se sometieron al estudio las cepas bacterianas

descritas en la Tabla N° 3, que fueron proporcionadas por el Laboratorio Clínico “Lic. Ana Aparicio” y por el Departamento de Microbiología y Parasitología, Facultad de Farmacia y Bioanálisis de la Universidad de los Andes. Estas cepas fueron aisladas de pacientes con ITU y VB en Mérida - Venezuela. Además, se incluyeron las cepas de referencia que se mencionan en la Tabla N° 2.

Método antimicrobiano La evaluación de la actividad antibacteriana se

realizó de acuerdo al método de difusión en agar con discos descrita por Velasco et al., 2007 y la concentración inhibitoria mínima (CIM) solo contra los microorganismos que mostraron zonas de inhibición. La CIM se determinó por dilución del aceite con dimetilsulfóxido (DMSO) con un rango de 10-160 μg/ml, definida como la concentración más baja capaz de inhibir el desarrollo bacteriano (CLSI, 2009). Los ensayos se realizaron por duplicado.

RESULTADOS Y DISCUSIÓN

Caracterización Fitoquímica Por hidrodestilación de las hojas frescas de la A.

triphylla se obtuvo un aceite esencial de color ligeramente amarillo y olor penetrante, con un rendimiento del 0,2%. En la Tabla N° 1 se muestran los componentes identificados, que constituyen el 97 % del total del aceite, los cuales fueron identificados mediante búsqueda computarizada en la Librería Wiley y por comparación de los Índices de Kováts obtenidos experimentalmente, con los reportados en la literatura (Adams, 1995).

De los veintidós componentes identificados, 2,7 % resultaron ser compuestos alifáticos insaturados (de menos de 10 átomos de carbono), el 72,4 % monoterpenos y el 22,3 % sesquiterpenos.

Los componentes mayoritarios identificados fueron: geranial (27,3 %), neral (22,5 %), geraniol (6,2%), bicyclogermacrene (5,2 %) y nerol (4,9 %). Este aceite guarda estrecha relación con el aceite esencial de la misma especie estudiada en Colombia [geranial (38,1 %), neral (19,3 %), geraniol (5,4%), nerol (4,7 %) y biciclogermacreno (3,4 %)] (Jaramillo et al., 2003) y los componentes geranial y neral forman parte de los aceites esenciales de A. triphylla estudiadas en el ámbito mundial (Montes et al., 1973; Carnat et al., 1999; Sartoratto et al., 2004; Gomes et al., 2006; Argyropoulou et al., 2007; Díaz et al., 2007; Di Leo Lira et al., 2008). Siendo Colombia un país vecino al nuestro, se puede pensar que ambas especies están relacionadas botánicamente.

Evaluación de la actividad antibacteriana El aceite de A. triphylla mostró fuerte actividad

antibacteriana contra las cepas de referencia, observándose zonas de inhibición con diámetros entre 7 y 19 mm y valores de CIM entre 10 a 60 μg/mL, sin embargo, no presentó actividad contra P. aeruginosa ATCC 27853 (Tabla N° 2). También fue activo contra todos los patógenos genito-urinarios probados, con rangos de zona de inhibición de 9 a 30 mm y valores de CIM de 10 a 50 μg/mL (Tabla N° 3). En resumen, la actividad del aceite contra todas las bacterias ensayadas mostró valores de CIM de 10 a 60 μg/mL y la dosis de 20 μg/mL predominó en el 52 % del total de microorganismos.

La actividad biológica observada en el presente estudio coincide con los resultados obtenidos por Demo et al., 2005 (Argentina), quienes observaron actividad antibacteriana del aceite esencial de A. triphylla contra S. aureus, E. faecalis, E. coli, Klebsiella sp. y Proteus mirabilis y ausencia de actividad frente a P. aeruginosa. Sin embargo, difiere de los hallazgos de Sartoratto et al., 2004, en cuanto a la actividad contra E. coli.



Tabla N°1. Componentes volátiles del aceite esencial deAloysia triphylla en columna capilar HP-5

N° Compuesto % IKcala 1 1 – octen–3–ol 0,9 977 2 6–metil–5–hepten–2–ona 1,8 985 3 Limoneno 3,0 1030 4 Eucaliptol 1,4 1033 5 β– cis–ocimeno 1,5 1049 6 linalool 0,5 1099 7 cis-crisantenol 0,9 1169 8 mentol 1,3 1187 9 α -terpineol 1,0 1195 10 nerol 4,9 1235 11 neral 22,5 1250 12 geraniol 6,2 1261 13 geranial 27,3 1279 14 geranil acetato 1,9 1387 15 β-cariofileno 2,1 1427 16 germacreno-D 4,6 1494 17 α-zingibereno 0,6 1506 18 biciclogermacreno 5,2 1509 19 cis-gamma-bisaboleno 0,9 1522 20 trans-sesquisabinene-hidrato 2,1 1569 21 spatulenol 4,5 1583 22 cariofileno-oxido 2,3 1588 TOTAL 97,4 -

a La composición del aceite esencial fue determinada por comparación de los espectros de masas de cada componente con la base de datos Willey e índice de Kováts calculado (IKcal).

Tabla 2: Actividad antibacteriana del aceite esencial de Aloysia triphylla contra cepas de referencia

Zona de inhibición (mm)*

Microorganismos Aceite

esencial

Control positivo

SAM VA GM AZT CAZ

CIM

μg/ml

Staphylococcus aureus (ATCC 6538) 18,75±0,25 40,75±0,25 20

Enterococcus faecalis (ATCC 29212) 17,75±0,25 20,50±0,50 60

Escherichia coli (ATCC 25922) 12,50±0,50 21,00±0,00 10

Klebsiella pneumoniae (ATCC 23357) 7,00±0,00 29,50±0,50 30

Pseudomonas aeruginosa (ATCC 27853)

NA 28,00±0,00 NP

* Zona de inhibición en mm, discos 6 mm diámetro, media de dos ensayos, SAM: Sulbactam-Ampicilina® (10μg/10 μg), VA: Vancomicina® (30 μg), GM: Gentamicina® (10 μg), AZT: Aztreonam® (30μg), CAZ: Ceftazidima® (30 μg), NA: No activo, NP: No probado. CIM: Concentración inhibitoria mínima, rango de concentración 10-160 μg/ml.



Tabla 3: Actividad antibacteriana del aceite esencial de Aloysia triphylla contra patógenos genito-urinarios

Aceite esencial Microorganismos Zona de inhibición

(mm)*

CIM

μg/ml

Aislados clínicos de ITU:

Escherichia coli BLEE (URO 1) 9,75±0,25 20

Escherichia coli (URO 5) 11,00±0,00 20

Escherichia coli (URO 8) 9,50±0,50 20

Klebsiella ozaenae (URO 2) 9,00±0,00 30

Klebsiella ozaenae (URO 4) 10,75±0,25 10

Klebsiella ozaenae BLEE (URO 7) 9,00±0,00 50

Klebsiella ozaenae multirresistente (URO 11) 8,00±0,00 20

Enterobacter aerogenes (URO 6) 8,75±0,25 30

Proteus mirabilis (URO 9) 7,00±0,00 10

Aislados clínicos de SV:

Escherichia coli (SV 5) 12,75±0,25 20





Klebsiella ozaenae (SV 1) 9,75±0,25 20

Staphylococcus aureus (SV 4) 30,00±0,00 20

Enterococcus faecalis (SV 2) 14.50±0,50 20

Enterococcus faecalis (SV 3) 23,00±0,00 20

Enterococcus faecium (SV 7) 21,75±0,25 20

Enterococcus faecium (SV 9) 25,00±0,00 20

*Zona de inhibición en mm, discos 6 mm diámetro, media de dos ensayos, BLEE: β-Lactamasa de espectro extenso. ITU: Infecciones del tracto urinario; SV: Secreción vaginal. URO: Urocultivo.; CIM: Concentración inhibitoria mínima, rango de concentración 10-160 μg/ml.



Recientemente, Duarte et al., 2007 en una investigación sobre efecto inhibitorio de aceites esenciales obtenidos de varias plantas medicinales de Brasil contra cepas de E. coli diarreogénicas, señalan actividad inhibitoria del aceite de A. triphylla contra las diferentes categorías de esta especie bacteriana con valores de CIM de 400 – 1000 μg/ml.

De acuerdo a los valores de la CIM obtenidos se observa que el aceite esencial de A. triphylla de Mérida – Venezuela fue más activo que el aceite esencial de esta especie obtenido en Brasil y Argentina, contra E. faecium, S. aureus y E coli (Sartoratto et al., 2004, Demo et al., 2005, Duarte 2006, Duarte et al., 2007). Esta diferencia se puede atribuir a variación en la composición química del aceite, la cual está influenciada por las condiciones del medio ambiente donde se cultiva la planta, por otra parte, difieren en algunos casos las metodologías utilizadas para determinar la actividad antibacteriana.

El efecto antimicrobiano del aceite esencial de A. triphylla observado en este estudio se podría atribuir a los monoterpenos oxigenados, que son los compuestos más abundantes, geranial 27,3 % y neral 22,5 %, y a los cuales de manera individual se les ha demostrado actividad contra bacterias grampositivas y gramnegativas (Onawunmi et al., 1984, Kim et al., 1995).

Otro hallazgo importante es la actividad observada contra dos aislados productores de BLEE, Escherichia coli BLEE (URO 1) y Klebsiella ozaenae BLEE (URO 7), y una cepa de Klebsiella ozaenae multirresistente (URO 11), con valores de CIM de 20 μg/ml, 50 μg/ml y 20 μg/ml, respectivamente.

CONCLUSIÓN

Con base al efecto inhibitorio contra bacterias multirresistentes y la baja dosis de CIM mostradas por el aceite esencial de A. triphylla, se puede concluir que este aceite es una fuente importante de sustancias con alta actividad antibacteriana que podría ser usado en preparaciones farmacéuticas para el tratamiento de infecciones causadas por estos microorganismos. Por otra parte, este es el primer reporte de actividad antibacteriana del aceite esencial de A. triphylla contra patógenos genito-urinarios.

AGRADECIMIENTOS

Los autores agradecen al Consejo de Desarrollo Científico, Humanístico y Tecnológico (CDCHT

Universidad de los Andes, Mérida, Venezuela, Programa ADG, Grupo de investigación: Bacteriología Clínica) por el soporte financiero para el desarrollo de esta investigación.

REFERENCIAS Adams R. 1995. Identification of essential oils components

by gas chromatography/mass spectroscopy. Allured Publishing Corporation, Carol Stream IL, USA.

Argyropoulou C, Daferera D, Tarantilis P, Fasseas C, Polissiou M. 2007. Chemical composition of the essential oil from leaves of Lippia citriodora H.B.K. (Verbenaceae) at two developmental stages. Biochem Systemat Ecol. 35 (12): 831-837.

Beigi R, Austin M, Meyn L, Krohn M, Hillier S. 2004. Antimicrobial resistance associated with the treatment of bacterial vaginosis. American Journal of Obstetrics and Gynecology. 191(4): 1124-1129.

Bellakhdar J, ldrissi A. 1994. Composition of lemon verbena (Aloysia triphylla (L'Herit.) Briton) oil of Morococcan origin. J Essential Oil Res. 6: 523-526.

Caballero R, Batista R, Cué M, Ortega L, Rodríguez M. 2000. Vaginosis bacteriana. RESUMED. 13(2):63-75.

Carnat, A., Carnat, A.P., Chavignon, O., Heitz, A., Wylde, R., and Lamaison, J.L. 1995. Luteolin 7-diglucuronide, the major flavonoid compound from Aloysia triphylla and Verbena officinalis. Planta Med. 61 (5): 490.

Carnat A, Carnat AP, Fraisse D, Lamaison JL. 1999. The aromatic and polyphenolic composition of lemon verbena tea. Fitoterapia. 70 (1): 44-49.

CLSI. 2009. Clinical and Laboratory Standars Institute. Performance standars for antimicrobial susceptibility testing; nineteenth informational supplement. CLSI document M100-S19, Wayne, PA.

Colodner R, Kometiani I, Chazan B, Raz R. 2008. Risk factors for community-acquired urinary tract infection due to quinolone-resistant E. coli. Infection. 36(1): 41–45.

Demo M, Oliva MD, Lopez ML, Zunino MP, Zygadlo JA. 2005. Antimicrobial activity of essential oils obtained from aromatic plants of Argentina. Pharmaceut Biol. 43 (2): 129-134.

Di Leo Lira P, Van Baren C, Retta D, Bandoni A. 2008. Characterization of lemon verbena (Aloysia citriodora Palau) from Argentina by the essential oil. J Essential Oil Res. 20 (4): 350-353.

Díaz O, Duran D, Martínez J, Stashenko E. 2007. Estudio comparativo de la composición química de los aceites esenciales de Aloysia tryphylla L'Her Britton cultivada en diferentes regiones de Colombia. Scientia et Technica 13 (33): 351-353.

Duarte M. Atividade antimicrobiane de plantas medicinais e aromáticas utilizadas no Brasil. Multiciência: Revista interdisciplinar dos Centros e Núcleos da

http://apps.isiknowledge.com.saturno.serbi.ula.ve/WoS/CIW.cgi?SID=3DocgO@IjpBEO96nFmC&Func=OneClickSearch&field=AU&val=Demo+M&ut=000228466800006&auloc=1&curr_doc=1/1&Form=FullRecordPage&doc=1/1

http://apps.isiknowledge.com.saturno.serbi.ula.ve/WoS/CIW.cgi?SID=3DocgO@IjpBEO96nFmC&Func=OneClickSearch&field=AU&val=Oliva+MD&ut=000228466800006&auloc=2&curr_doc=1/1&Form=FullRecordPage&doc=1/1

http://apps.isiknowledge.com.saturno.serbi.ula.ve/WoS/CIW.cgi?SID=3DocgO@IjpBEO96nFmC&Func=OneClickSearch&field=AU&val=Lopez+ML&ut=000228466800006&auloc=3&curr_doc=1/1&Form=FullRecordPage&doc=1/1

http://apps.isiknowledge.com.saturno.serbi.ula.ve/WoS/CIW.cgi?SID=3DocgO@IjpBEO96nFmC&Func=OneClickSearch&field=AU&val=Zunino+MP&ut=000228466800006&auloc=4&curr_doc=1/1&Form=FullRecordPage&doc=1/1

http://apps.isiknowledge.com.saturno.serbi.ula.ve/WoS/CIW.cgi?SID=3DocgO@IjpBEO96nFmC&Func=OneClickSearch&field=AU&val=Zygadlo+JA&ut=000228466800006&auloc=5&curr_doc=1/1&Form=FullRecordPage&doc=1/1


Unicamp. 2006. Disponible en: http://www.multiciencia.unicamp.br/artigos_07/a_05_7.pdf. (Consulta: Febrero de 2009).


Duarte M, Leme EE, Delarmelina C, Soares AA, Figueira GM, Sartoratto A. 2007. Activity of essential oils from Brazilian medicinal plants on Escherichia coli. J Ethnopharmacol. 111 (2): 197-201.

Echevarría-Zarate J, Sarmiento E, Osores-Pleng F. 2006. Infección del tracto urinario y manejo antibiótico. Acta Med Per. 23(1): 26-31.

Gagliotti C, Buttazzi R, Sforza S, Moro ML, Romagna E. 2008. Antibiotic resistance study group. Resistance to fluoroquinolones and treatment failure/short-term relapse of community-acquired urinary tract infections caused by Escherichia coli. J Infection. 57(3): 179-184.

Gobernado M, Valdés K, Alós JI, García-Rey C, Dal-Ré R, García-de-Lomas J. 2007. Quinolone resistance in female outpatient urinary tract isolates of Escherichia coli: Age-related differences. Rev Esp Quimioterap. 20 (2): 206-210.

Gomes P, Oliveira H, Vicente A, Ferreira M. 2006. Production, transformation and essential oils composition of leaves and stems of lemon verbena [Aloysia triphylla (L'Herit.) Britton] grown in Portugal. Rev Bras Pl Med. 8: 130-135.

Guneysel O, Onur O, Erdede M, Denizbasi A. 2009. Trimethoprim/Sulfamethoxazole resistance in urinary tract infections. J Emerg Med. 36 (4): 338-341.

Hillier S, Holmes K. 1999. Bacterial vaginosis, p. 563-586. In Holmes K, Sparling P, Mardh P, Lemon S, Stamm W, Piot P, Wasserheit J (ed.), Sexually transmitted diseases. McGraw-Hill, New York, N.Y.

Jaramillo B, Stashenko E, Martínez J. 2003. Comparación de la composición química y de la actividad antioxidante in vitro de los metabolitos secundarios volátiles de plantas de la familia Verbenaceae. Rev Academ Colombiana Cs Exactas, 27 (105): 579-597.

Karlowsky J, Hoban D, DeCorby M, Laing N, Zhanel G. 2006. Fluoroquinolone-resistant urinary isolates of Escherichia coli from outpatients are frequently multidrug resistant: Results from the North American urinary tract infection collaborative alliance-quinolone resistance study. Antimicrob Agents Chemother. 50 (6):2251–2254.

Kim J, Marshall M, Cornell J, Preston J, Wei C. 1995. Antibacterial activity of Carvacrol, Citral, and Geraniol against Salmonella typhimurium in culture medium and on fish cubes. J Food Sci. 60 (6): 1364-1368.

Lizama M, Luco M, Reichhard C, Hirsch T. 2005. Infección del tracto urinario en un servicio de urgencia

pediátrico: Frecuencia y características clínicas. Rev Chil Infect. 22 (3): 235-241.

López A, Theumer M, Zygadlo J, Rubinstein H. 2004. Aromatic plants essential oils activity on Fusarium verticillioides Fumonisin B1 production in corn grain. Mycopathologia. 158(3): 343-349.

Montes P, Valenzuela L, Wilkomirsky T, Arrivé M. 1973. Sur la composition de l'essence d'Áloysia triphylla (Cedron). Planta Med. 23: 119-124.

Mota A, Di Pietrantonio K, Mota A. 2008. Vaginosis bacteriana: aspectos colposcópicos. Rev Obstet Ginecol Venez. 68(2): 87-91.

Ochoa C, Eiros J, Pérez C, Inglada L, Grupo de Estudio de los Tratamientos Antibióticos. 2005. Etiología de las infecciones del tracto urinario y sensibilidad de los uropatógenos a los antimicrobianos. Rev Esp Quimioterap. 18 (2): 124-135.

Onawunmi G, Yisak W, Ogunlana E. 1984. Antibacterial constituents in the essential oil of Cymbopogon citrates (DC.) Stapf. J Ethnophycol. 12: 279-286.

Özek T, Kirimer N, Baser K, Tumen G. 1996. Composition of the essential oil of Aloysia triphylla (L’Herit.) Britton grown in Turkey. J Essential Oil Res. 8, 581-583.

Rojas L, Usubillaga A, Galarraga F. 1999. Essential Oil of Coespeletia timotensis. Phytochemistry. 52 (8): 1483-1484.

Sartoratto A, Machado A, Delarmelina C, Figueira G, Duarte M, Rehder L. 2004. Composition and antimicrobial activity of essential oils from aromatic plants used in Brazil. Braz J Microbiol, 35 (4): 275-280.

Skaltsa H, Shammas G. 1988. Flavonoides from Lippia citriodora. Planta Med. 54 (5): 465.

Teixeira M, Figueira G, Sartoratto A, Garcia V, Delarmelina C. 2005. Anti-Candida activity of Brazilian medicinal plants. J Ethnopharmacol. 97:305–311.

Teixeira M, Leme E, Delarmelina C, Soares A, Figueira G, Sartoratto A. 2007. Activity of essential oils from Brazilian medicinal plants on Escherichia coli. J Ethnopharmacol. 111: 197–201.

Velasco J, Rojas J, Salazar P, Rodríguez M, Díaz T, Morales A, Rondón M. 2007. Antibacterial Activity of the Essential Oil of Lippia oreganoides Against Multiresistant Bacterial Strains of Nosocomial Origin. Nat Prod Comun. 2 (1):85-88.

Zygadlo J, Lamarque A, Maestri D, Guzman C, Lucini E, Grosso N, Ariza Espinar L. 1994. Volatile constituents of Aloysia triphylla (L’Herit.) Britton. J Essential Oil Res. 6: 407-409.

http://www.multiciencia.unicamp.br/artigos_07/a_05_7.pdf

http://www.multiciencia.unicamp.br/artigos_07/a_05_7.pdf

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BLACPMA ISSN 0717 7917 Artículo Original | Original Article

BLACPMA es una publicación de la Cooperación Latinoamericana y Caribeña de Plantas Medicinales y Aromáticas This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. (http://creativecommons.org/licenses/by-nc-nd/3.0/ ) which permits to copy, distribute and transmit the work, provided the original work is properly cited. You may not use this work for commercial purposes. You may not alter, transform, or build upon this work. Any of these conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights. Este es un articulo de Acceso Libre bajo los términos de una licencia “Atribución Creativa Común-No Comercial-No trabajos derivados 3.0 Internacional” (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.es) Usted es libre de copiar, distribuir y comunicar públicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los créditos de la obra de la manera especificada por el autor o el licenciador (pero no de una manera que sugiera que tiene su apoyo o apoyan el uso que hace de su obra). No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede alterar, transformar o generar una obra derivada a partir de esta obra. Al reutilizar o distribuir la obra, tiene que dejar bien claro los términos de la licencia de esta obra. Alguna de estas condiciones puede no aplicarse si se obtiene el permiso del titular de los derechos de autor. Nada en esta licencia menoscaba o restringe los derechos morales del autor.

Zederone from the rhizomes of Zingiber zerumbet and its anti-staphylococcal Activity

[Aislamiento de Zederona de los rizomas de Zingiber zerumbet y su actividad antiestafilocócica]

M. Golam KADER1, M. Rowshanul HABIB1, Farjana NIKKON1, Tanzima YEASMIN1, Mohammad A. RASHID2, M. Mukhlesur RAHMAN3*, Simon GIBBONS3

1Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh; 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh; 3Department of Pharmaceutical and Biological Chemistry,

The School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX, UK

Abstract A sesquiterpene, zederone (1), was isolated from the crude ethanolic extract of the rhizomes of Zingiber zerumbet (L.) Smith. It is the first time

report of isolation of this compound from the genus Zingiber. Its structure was established by a series of spectral data including high-field NMR (both 1D and 2D) and MS. The antibacterial activity of this compound was determined against a number of multi-drug resistant and methicillin-resistant Staphylococcus aureus strains (SA1199B, ATCC25923, XU212, RN4220 and EMRSA15) and minimum inhibitory concentration (MIC) values were found to be in the range of 64-128 μg/ml.

Keywords: Zingiber zerumbet; Zingiberaceae; Zederone; Antibacterial; Staphylococcus aureus

Resumen

Un sesquiterpeno, zederona (1), fue aislado del extracto crudo metanólico de los rizomas de Zingiber zerumbet (L.) Smith. Esta es la primera vez que se reporta este compuesto en el género Zingiber. Su estructura se estableció tras una serie de análisis espectrales incluyendo NMR de alto campo (1D y 2D) y espectrometría de masa. La actividad antibacteriana de este compuesto se determine frente a varias cepas multi-fármaco resistentes y meticilina-resistentes Staphylococcus aureus (SA1199B, ATCC25923, XU212, RN4220 and EMRSA15) y las concentraciones inhibidoras mínimas se encontraron en el rango de 64-128 μg/ml.

Palabras Clave: Zingiber zerumbet; Zingiberaceae; Zederona; Actividad antibacteriana; Staphylococcus aureus. Recibido | Received: December 01, 2009 Aceptado en Versión Corregida | Accepted in Corrected Version: December 15, 2009 Publicado en Línea | Published Online: December 17, 2009 Declaración de intereses | Declaration of interests: Authors have no competing interests. Financiación | Funding: none declared This article must be cited as:. M. Golam Kader, M. Rowshanul Habib, Farjana Nikkon, Tanzima Yeasmin, Mohammad A. Rashid, M. Mukhlesur Rahman, Simon Gibbons. 2010. Zederone from the rhizomes of Zingiber zerumbet and its anti-staphylococcal Activity. Bol Latinoam Caribe Plant Med Aromat 9(1):63 – 68. {EPub December 17, 2009}.

*Contactos | Contacts:. E-mail: [email protected]


Kader et al. Zederone from the rhizomes of Zingiber zerumbet and its anti-staphylococcal activity


INTRODUCTION

Zingiber zerumbet (L.) Smith (Fam. Zingiberaceae) locally known as ‘Bon Ada’ is a vigorous ginger with leafy stems growing to about 1.2 m in height that is widely cultivated throughout the tropics including Southeast Asia, Korea, India and Bangladesh for its medicinal properties (Kritikar and Basu, 1984; Fansworth and Bunyapraphatsara, 1992; Saadiah and Halijah, 1995). The most common use of Z. zerumbet is as a shampoo and conditioner for the hair (Burkill, 1966; Petard, 1986). Its rhizomes are used in traditional medicine for the treatment of inflammation, swelling, loss of appetite, lumbago, diabetes, chest pain, rheumatic pains, bronchitis, dyspepsia and sore throat (Burkill, 1966; Kritikar and Basu, 1984; Farnsworth and Bunyapraphatsara, 1992). The juice of the boiled rhizomes has also been used in indigenous medicine for worm infestation in children (Petard, 1986). Previous phytochemical investigations on this plant have revealed the isolation of several sesquiterpenes, flavonoids and aromatic compounds (Matthes et al., 1980; Masuda et al., 1991; Dai et al., 1997; Jang et al, 2004; Jang et al, 2005). The volatile oil of the rhizomes has been shown to contain zerumbone, humulene, camprene α-caryophyllene and camphene (Hasnah, 1991; Srivastava et al., 2000; Bhuiyan et al., 2009). Zerumbone, a predominant sesquiterpene from this plant, has been studied intensively for its use as anti-inflammatory, and in chemoprevention and chemotherapy strategies (Dai et al., 1997; Kitayama et al., 2001; Murakami et al., 1999; Murakami et al., 2002; Tanaka et al., 2001). From the pharmacological point of view, Z. zerumbet has been reported to inhibit colon and lung carcinogenesis in mice (Kim et al., 2009) and CXCL12-induced invasion of breast and pancreatic tumor cells (Sung et al., 2008), suppresses phorbol ester-induced expression of multiple scavenger receptor genes in THP-1 human monocytic cells and inhibits Epstein-Barr Virus activation (Vimala et al., 1999; Murakami et al., 1999). As a part of our research focused on bioactive compounds from indigenous medicinal plants, we here report the isolation and identification of a compound (1) from the ethanolic extract of Zingiber zerumbet (L.) Smith and its ant-staphylococcal activity against a series of multi-drug resistant (MDR) and methicillin resistant Staphylococcus aureus strains: SA1199B, ATCC25923, XU212, RN4220 and EMRSA15.


General experimental procedures NMR spectra (both 1D and 2D) were

acquired on a Bruker Avance 500 MHz NMR (500 MHz for 1H and 125 MHz for 13C) spectrometer using the residual solvent peak as internal standard. The IR spectra were recorded with a Perkin-Elmer Lambda spectrophotometer and the Mass spectra were recorded with HRTOF-MS in positive mode. The melting point were determined using a Digital Melting point Apparatus (model IA 8103, Electrothermal Engineering LTD, Southend-on-Sea, Essex, UK) and are uncorrected. All solvents used in this study were of analytical grade and purchased from BDH and Merck.

Plant materials Fresh rhizomes of Z. zerumbet were collected

from the hilly areas of Chittagong, Bangladesh in October 2007 and identified by a taxonomist, Dr. Mohammed Yusuf, BCSIR Laboratory, Chittagong, Bangladesh where a voucher specimen (No. 1061) of this collection has been maintained.

Extraction and isolation of compound (1) The powdered plant material (800 g) of Z.

zerumbet was extracted with ethanol (4 L) in an aspirator bottle for a week and then filtered and concentrated by using a rotary evaporator at 45oC under reduced pressure. The crude ethanol extract (2.0 g) was subjected to column chromatography over silica gel (Merck) eluting with petroleum ether and ethyl acetate of increasing polarity and finally with ethanol which yielded a total of 105 fractions. Based on TLC analysis, fractions 15-18 were combined together and then subjected to preparative TLC using the solvent system petroleum ether and ethyl acetate in a ratio of 20:1 to yield compound 1 (8.0 mg; white powder; Rf 0.45 in 5% EtOAc in petroleum ether). The structure of the compound (1) was confirmed by analysis of its IR, 1H-NMR, 13C-NMR and TOF- Mass spectral data at The School of Pharmacy, University of London, UK.

Compound 1: White powder; mp. 58-60oC;

IR (KBr): 1662, 1521, 1533, 1558, 914, 861 cm-1; 1H-NMR (500 MHz, CDCl3): δ 5.49 (1H, dd, J = 12.0, 4.0 Hz , H-1), 2.25 (1H, m, H-2a), 2.53 (1H, m, H-2b), 1.29 (1H, m, H-3a), 2.31 (1H, dt, J= 13.0, 3.5 Hz, H-3b), 3.81 (1H, s, H-5), 3.69 (1H, d, J= 16.0 Hz, H-9a), 3.77 (1H, d, J= 16.0 Hz, H-9b), 7.10 (1H,

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s, H-12), 1.61 (3H, s, H-13), 1.35 (3H, s, H-14), 2.12 (3H, s, H-15); 13C-NMR (125 MHz, CDCl3): 131.4 (C-1), 24.9 (C-2), 38.2 (C-3), 64.2 (C-4), 66.8 (C-5), 192.4 (C-6), 122.5 (C-7), 157.3 (C-8), 42.1 (C-9), 131.3 (C-10), 123.5 (C-11), 138.3 (C-12), 16.0 (C-13), 15.4 (C-14), 10.5 (C-15); HR-TOF-ESIMS [M+H]

+ m/z 247.0889.

Bacterial strains The antibacterial assay was performed

against a panel of multi-drug and methicillin- resistant strains of Staphylococcus aureus. S. aureus standard strain ATCC 25923 and tetracycline-resistant strain XU212 which possesses the TetK tetracycline efflux protein provided by Dr Edet Udo (Gibbons and Udo, 2000). Strain SA-1199B which overexpresses the norA gene encoding the NorA MDR efflux pump was provided by Professor Glenn Kaatz (Kaatz et al., 1993). Strain RN4220 which possesses the MsrA macrolide efflux protein was provided by Dr Jon Cove (Ross et al., 1989). EMRSA-15 (Richardson and Reith, 1993) was the generous gift of Dr Paul Stapleton.

Minimum inhibitory concentration (MIC) assay. All five S. aureus strains were cultured on

nutrient agar (Oxoid) and incubated for 24 h at 37°C prior to MIC determination. Norfloxacin was purchased from the Sigma Chemical Co. Mueller-Hinton broth (MHB; Oxoid) was adjusted to contain 20 and 10 mg/l of Ca2+ and Mg2+, respectively. An inoculum density of 5 x 105 cfu of each of the test organisms was prepared in normal saline (9 g/l) by comparison with a 0.5 MacFarland standard. MHB (125 μl) was dispensed into 10 wells of a 96 well microtitre plate (Nunc, 0.3 ml volume per well). A stock solution of norfloxacin was prepared by dissolving the antibiotic in DMSO (Sigma) and dilution in MHB to give a final concentration of 0.625%. A DMSO control was included in all assays.

Compounds were serially diluted into each of the wells followed by the addition of the bacterial inoculum and the microtitre plate was incubated at 37°C for 18 h. The MIC recorded as the lowest concentration at which no growth was observed. This was facilitated by the addition of 20 μl of a 5 mg/ml methanolic solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) to each of the wells and incubation for 20 minutes. A blue colouration indicated bacterial growth (Shiu and Gibbons, 2006).


Identification of compound (1) Compound (1) was isolated as an amorphous

off-white powder from the crude ethanol extract of the rhizomes of Zingiber zerumbet (L.) Smith. The high-resolution TOFMS showed the pseudo molecular ion, [M+H]+ at m/z 247.0889, corresponding to the molecular formula as C15H18O3. The 1H-NMR spectrum of compound 1 showed a downfield one proton singlet at δ 7.10, an olefinic proton signal at δ 5.49, another methine signal at δ 3.81, three methyl proton resonances at δ 1.35, 1.61, 2.12, and methylene proton resonances between δ1.29-3.77 integrating for four protons. The 13C-NMR spectrum displayed a total of 15 carbons while the DEPT-135 and HMQC experiments indicated that 9 out of 15 carbons had attached protons. Analysis of the 13C and DEPT135 spectra allowed discernment of the carbon resonances into three methyls (δC 10.5, 15.4, 16.0), three methylenes (δC 24.9, 38.2 and 42.1), three methines (δC 66.8, 131.4, 138.3), and six quaternary carbons, including a carbonyl group (δC 192.4). The assignment of all carbons and protons and thereby the structure of the compound was resolved by 2D experiments, notably COSY, HMQC and HMBC experiments. In the COSY experiment, the olefinic proton at δ 5.49 (H-1; δC 131.4 from HMQC) showed strong interaction with H2-2 protons at δ 2.25 and 2.53 along with a weak connectivity with the methyl singlet at δ 1.61 (H3-15). The methylene protons (H2-2) showed coupling with H2-3 protons at δ 1.29 and δ 2.31 in the COSY experiment. The presence of a methine (66.8; δH 3.81 from HMQC) and a quaternary (64.2) in the 13C experiment confirmed the presence of an epoxide in the molecule. The C-5 methine proton at δ 3.81 showed HMBC connectivities over two bonds (2J) to a quaternary carbon at δ 64.2 (C-4) and the carbonyl group at δ 192.4 (C-6). The methyl protons at 1.35 (δC 15.4 from HMQC) revealed 3J connectivities to δ 38.2 (C-3) and δ 66.8 (C-5) and thereby confirmed its linkage at C-4. The methylene protons at δ 3.70 and δ 3.77 (H2-9, δC 42.1 from HMQC) showed 2J correlations over two bonds to quaternary carbons at δ 131.3 (C-10) and δ 157.3 (C-8) and a 3J interactions to methyl (δC 16.0), olefinic (δC 131.4, C-1) and quaternary (δC 122.5, C-7) carbons. Furthermore, 3J connectivities from the methyl protons at δ 1.61 to δ 131.4 (C-1) and δ 42.1 (C-9) confirmed its placement


at C-10. The remaining methyl resonance at δ 2.12 (δC 10.5 from HMQC) showed connectivities to δC 122.5 (C-7) and a methine carbon at δ 138.3 (C-12) over three bonds. This allowed the placement of this methyl group at C-11. Accordingly, compound 1 was identified as zederone. Its NMR data were in agreement with those reported previously (Hikino et al., 1971). Although, it is a known natural product reported before from a number species of the genus Curcuma including C. zedoaria (Matthes et al., 1980), C. aromatic (Phan and Phan 2000), C. comosa (Qu et al., 2009), C. kwangsiensis ( Zhu et al., 2009), C. ochrorhiza. (Sirat et al. 2009), C. xanthorrhiza (Sukari et al., 2008), this is the first isolation from the genus, Zingiber.

The compound was tested for the antibacterial activity against a panel of five strains of Staphylococcus aureus: SA1199B, ATCC25923, XU212, RN4220 and EMRSA15 and showed weak activity with minimum inhibitory concentration (MIC) values in the range of 64-128μg/ml (Table 2). Figure 1. Structure of compound 1

O

14O

O

1

2

3 45

67

89

10

12

11

13

15

Table 1 1NMR (500 MHz), 13C NMR (125 MHz) and HMBC data of compound 1 in CDCl3.

HMBC Position δH δC2J 3J

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

5.49, dd, J= 12.0, 4.0 Hz 2.25, br d; 2.53, m 1.29, m; 2.31, dt, J= 13.0, 3.5 Hz - 3.81, s - - - 3.69, d, J= 16.0 Hz; 3.77, d, J= 16.0 Hz - - 7.10, s 1.61, s 1.35, s 2.12, s

131.4 24.9 38.2 64.2 66.8 192.4 122.5 157.3 42.1 131.3 123.5 138.3 16.0 15.4 10.5

C-2, C-10 C-3 C-2, C-4 - C-4, C-6 - - - C-8, C-10 - - C-11 - C-4 -

C-9, C-15 - - - C-14 - - - C-7, C-15 - - C-7, C-8, C-13 C-7, C-12 C-3, C-5 C-1, C-9

Table 2. MICs of 1 and standard antibiotic in μg/ml

SA1199B Xu212 ATCC 25943 RN 4220 EMRSA 15

Compound 1 128 64 128 64 128

Norfloxacin 16 4 0.5 0.5 0.5




REFERENCES Bhuiyan NI, Chowdhury JU, Begum J. 2009. Chemical

investigation of the leaf and rhizome essential oils of Zingiber zerumbet (L.) Smith from Bangladesh. Bangladesh J Pharmacol 4: 9-12.

Burkill H1. 1966. Dictionary of the economic products of the Malay Peninsula. Ministry of Agric and Coop, Kuala Lumpur, 2345.

Dai JR, Cardellina IIJH, McMahon JB, Boyd MR. 1997. Zerumbone, an HIV-inhibitory and cytotoxic sesquiterpene of Zingiber aromaticum and Z. zerumbet. Nat Prod Lett 10:115-118.

Fansworth NR, Bunyapraphatsara N. 1992. Thai Medicinal Plants. Prachachon, Bangkok, Thailand, 261-263.

Gibbons S, Udo EE. 2000. The effect of reserpine, a modulator of multidrug efflux pumps, on the in vitro activity of tetracycline against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) possessing the tet(K) determinant. Phytother. Research 14: 139–140.

Hasnah MS. 1991. Chemical constituents of some medicinal plants of zingiberaceae: Medicinal products from tropical rain forest. Proceedings of the Concerence, Forest Research Institute Malaysia, Kuala Lumpur 2: 299-304.

Hikino H, Tori K, Horibe I and Kuriyama K. 1971. Absolute configuration and conformation of zederone, a sesquiterpenoid of Curcuma zedoaria. J Chem Soc 37: 688-691.

Jang DS, Han AR, Park G, Seo EK. 2004. Flavonoids and aromatic compounds from the rhizomes of Zingiber zerumbet. Arch Pharm Res 27: 386-389.

Jang DS, Han AR, Park G, Seo EK. 2005. Potentially Bioactive Two New Natural Sesquiterpenoids from the Rhizomes of Zingiber zerumbet. Arch Pharm Res 28: 294-296.

Kaatz GW, Seo SM, Ruble CA. 1993. Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 37: 1086–1094.

Kim M, Miyamoto S, Yasui Y, Oyama T, Murakami A, Tanaka T. 2009. Zerumbone, a tropical ginger sesquiterpene, inhibits colon and lung carcinogenesis in mice. Int J Cancer 124:264-71.

Kirtikar RK, Basu BD. 1984. Indian Medicinal Plant. Lalit Mohan Basu MB. Allahabad, India, vol. 4, pp 2415-2419.

Kitayama T, Yamamoto K, Utsumi R, Takatani M, Hill RK, Kawai Y, Sawada S, Okamoto T. 2001.Chemistry of zerumbone. 2. Regulation of ring bond cleavage and unique antibacterial activities of zerumbone derivatives. Biosci Biotechnol Biochem 65:2193-2199.

Masuda T, Jitoe A, Kato S, Nakatani N. 1991.Constituents of Zingiberaceae. Part 3. Acetylated flavonol

glycosides from Zingiber zerumbet. Phytochemistry 30: 2391-2392.

Matthes HWD, Luu B, Ourisson G. 1980. Chemistry and Biochemistry of Chinese drugs. Part VI. Cytotoxic components of Zingiber zerumbet, Curcuma zedoaria and Curcuma domestica. Phytochemistry 19: 2643-2650.

Murakami A, Takahashi D, Kinoshita T, Koshimizu K, Kim HW, Yoshihiro A, Nakamura Y, Jiwajinda S, Terao J, Ohigashi H. 2002. Zerumbone, a Southeast Asian ginger sesquiterpene, markedly suppresses free radical generation, proinflammatory protein production, and cancer cell proliferation accompanied by apoptosis: The α,β-unsaturated carbonyl group is a prerequisite. Carcinogen 23: 795-802.

Murakami A. Takahashi M. Jiwajinda S. Koshimizu K. Ohigashi H. 1999 .Identification of zerumbone in Zingiber zerumbet Smith as a potent inhibitor of 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus activation. Biosci Biotechnol Biochem 63: 1811-2.

Phan MG, Phan TS. 2000. Isolation of Sesquiterpenoids from the Rhizomes of Vietnamese Curcuma aromatica Salisb. Tap Chi Hoa Hoc 38: 96-99.

Petard P. 1986. Quelques plantes utiles de la Polynesie et Ra'au Tahiti. Papeete, Haere Po no Tahiti, 1876.

Qu Y, Xu F, Nakamura S, Matsuda H, Pongpiriyadacha Y, Wu L, Yoshikawa M. 2009. Sesquiterpenes from Curcuma comosa. J Nat Med 63: 102-104.

Richardson JF, Reith S. 1993. Characterization of a strain of methicillin-resistant Staphylococcus aureus (EMRSA-15) by conventional and molecular methods, J. Hosp. Infect. 25: 45–52. Ross JI, Farrell AM, Eady EA, Cove JH, Cunliffe WJ. 1989. Characterisation and molecular cloning of the novel macrolide streptogramin B resistance determinant from Staphylococcus epidermidis. J. Antimicrob. Chemother. 24: 851–862.

Saadiah MS, Halijah I. 1995. Proceedings of the National Convention on Herbal medicine. 21, Kuala Lumpur, Forest Research Institute Malaysia, 205-07.

Shiu WKP, Gibbons S. 2006. Anti-staphylococcal acylphloroglucinols from Hypericum beanii. Phytochemistry. 67: 2568-2572.

Sirat, HM, Jamil, S, Rahman AA. 2009. Sesquiterpenes from the rhizomes of Curcuma ochrorhiza. Nat Prod Comm 4: 1171.

Srivastava AK, Srivastava SK, Shah NC. 2000. Essential Oil Composition of Zingiber zerumbet (L.) Sm. from India. J Essent Oil Res 12: 595-97.

Sukari M A, Rashid NY, Tang SW, Rahmani M, Lajis NH, Khalid K, Yusuf UK. 2008. Chemical constituents and bioactivity of Curcuma xanthorrhiza roxb. J Ultra Sci Phy Sci 20: 605-610.

Sung B. Jhurani S. AhnK. S. Mastuo Y Yi T. Guha S. Liu M. Aggarwal BB. 2008. Zerumbone down-regulates chemokine receptor CXCR4 expression leading to

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Kim%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Miyamoto%20S%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

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http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Jhurani%20S%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

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http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Mastuo%20Y%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Yi%20T%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Guha%20S%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Liu%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Liu%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Aggarwal%20BB%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus


inhibition of CXCL12-induced invasion of breast and pancreatic tumor cells. Cancer Res 68: 8938-44.

Vimala S. Norhanom AW. Yadav M. 1999. Anti-tumour promoter activity in Malaysian ginger rhizobia used in traditional medicine. Br J Cancer 80: 110-6.Tanaka T, Shimizu M, Kohno H, Yoshitani SI, Tsukio Y,

Murakami A, Safitri R, Takahashi D, Yamamoto K, Koshimizu K, Ohigashi H, Mori H. 2001.Chemoprevention of azoxymethane-induced rat aberrant crypt foci by dietary zerumbone isolated from Zingiber zerumbet. Life Sci 69:1935-1945.

Zhu K, Li J, Luo H, Li J, Qiu F. 2009. Chemical constituents from the rhizome of Curcuma kwangsiensis. Shenyang Yaoke Daxue Xuebao 26: 27-29.


http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Vimala%20S%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

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A protocol for evaluating the safety of herbal preparations in a rat model: the case of a supercritical fluid extract of Saw Palmetto

[Un protocolo para evaluar la seguridad de preparaciones herbales en un modelo de ratas: el caso de un extracto fluido supercrítico de Saw Palmetto]

María Eugenia LETELIER*, Paula ARACENA-PARKS, Liliana PEREDO-SILVA

Laboratory of Pharmacology and Toxicology, Department of Pharmacological and Toxicological Chemistry, School of Chemical and Pharmaceutical Sciences, Universidad de Chile, Sergio Livingstone Pohlhammer (ex-Olivos) 1007, Independencia, Chile.

Abstract

Herbal extracts must be evaluated for their efficacy and safety. In vivo acute toxicity studies must consider the different mechanisms by which active compounds may elicit toxicological outcomes. Thus, a methodology to test general parameters related to acute toxicity responses in a murine model was developed, using a Saw Palmetto extract (HiPower®): adult male Sprague-Dawley rats were treated orally with two doses of HiPower® (the recommended dose for humans and a dose 10-fold higher) for 10 days, to examine general homeostatic parameters (hemogram and clinical chemistry) as well as morphological features of tissues involved in the response to xenobiotics (liver, kidney, spleen, and lymphatic ganglia). None of the parameters analyzed underwent significant changes during treatment, suggesting that HiPower® displays a good safety profile for the period tested. This method may be adopted for testing the in vivo acute toxicity of herbal extracts.

Keywords: Saw Palmetto, safety profile, Sprague-Dawley rats, acute toxicity.

Resumen

Los extractos herbales deben ser evaluados en cuanto a eficacia y seguridad. Estudios de toxicidad aguda in vivo deben considerar los diferentes mecanismos por los cuales los principios activos pueden producir toxicidad. Por consiguiente, se desarrolló una metodología para examinar parámetros generales relacionados con las respuestas de toxicidad aguda en un modelo murino, utilizando un extracto de Saw Palmetto (HiPower®): ratas Sprague-Dawley macho fueron tratadas con dos dosis de HiPower® (la dosis recomendada para humanos y una dosis 10 veces mayor) durante 10 días, para ensayar parámetros generales homeostáticos (hemograma y perfil bioquímico), así como características morfológicas de tejidos involucrados en la respuesta a xenobióticos (hígado, riñón, bazo y ganglios linfáticos). Ninguno de los parámetros analizados sufrió cambios significativos durante el tratamiento, sugiriendo que HiPower® presenta un buen perfil de seguridad durante el periodo evaluado. Este método puede ser adoptado para ensayar la toxicidad aguda in vivo de extractos herbales.

Palabras Clave: Saw Palmetto, perfil de seguridad, ratas Sprague-Dawley, toxicidad aguda.

Recibido | Received: September, 16, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: December 16, 2009. Publicado en Línea | Published Online 17 December 2009 Declaración de intereses | Declaration of interests: Authors have no competing interests. Financiación | Funding: This work was supported by Madreselva Producción y Desarrollo Ltda. (Santiago, Chile). This article must be cited as: Names. 2009. Title. Bol Latinoam Caribe Plant Med Aromat 9(1):69 – 79. {EPub XX Month 200X }.

*Contactos | Contacts: E-mail: [email protected]; Tel: 56-2-9782885; Fax: 56-2-9782996;


Letelier et al. In vivo safety profile of a supercritical extract of Saw Palmetto


INTRODUCTION

Compounds occurring in plants have been used for millennia to treat diseases; this use has set the basis for the isolation of such compounds for therapeutic use. Only recently, however, scientific research on herbal extracts has begun to fully validate their therapeutic use and safety. Most of this research is focused on the therapeutic application of compounds found in herbal extracts. For instance, extracts from leaves of plants are rich in antioxidant compounds, such as polyphenols, which can be useful for the treatment of pathologies associated to oxidative stress (e.g. neurodegenerative diseases). Nevertheless, assessing the safety of herbal extracts is a challenging endeavour, due to their complex nature. In contrast to purified compounds, herbal extracts display a number of different molecules with potentially very different biological targets; interaction of herbal compounds with their targets may lead to beneficial and/or toxic consequences. Therefore, it becomes necessary to develop a methodology that evaluates the safety profile of herbal extracts containing more than one active compound.

The present work is aimed to establish a methodology for assessing the acute toxicity of a particular herbal extract of Saw Palmetto (Serenoa repens W. Bartram). Saw Palmetto belongs to the Arecaceae (Palmae or Palmaceae) family. It is also known as Serenoa serrulatum Schultes, Serenoa serrulata (Michaux) Nichols, or Sabal serrulata (Michaux) Nutall ex Schultes (Wilt et al., 2000)).

The therapeutic benefits of Saw Palmetto appear to be related to its fruits, rich in natural oils. Currently, Saw Palmetto extract is widely used for the treatment of benign prostate hyperplasia (BPH, prostate enlargement) (Bent et al., 2006; Gerber and Fitzpatrick, 2004; Hizli and Uygur, 2007; Wilt et al., 2000; Wilt et al., 1998). The type and relative abundance of characteristic compounds (phenolic compounds, phytosterols, flavonoids, polyprenoids, sugars, fatty acids, etc.) of the oily extract from Saw Palmetto depends on the extraction procedure. Currently, the most used procedures to obtain Saw Palmetto extracts are: 1) n-hexane (100%) extraction that produces a liposterolic extract (LESP) (Carraro et al., 1996); 2) ethanol (70-95% w/w) extraction (Derakhshani et al., 1997); or 3) supercritical fluid extraction with liquid CO2 (Cristoni et al., 1997).

Although there are several reports regarding the therapeutical applications of Saw Palmetto extracts (Bent et al., 2006; Carraro et al., 1996; Lowe and

Fagelman, 2004; Ulbricht et al., 2006; Vallancien and Pariente, 2001; Wilt et al., 2000), there are few classical toxicological studies in animals. Only a small amount of studies have shown significant toxic effects of a particular Saw Palmetto extract (PC-SPES), which has been removed from the market (de la Taille et al., 2000; de la Taille et al., 1999; Small et al., 2000; Sovak et al., 2002). More recent clinical studies in humans have found no serious adverse effects of Saw Palmetto extracts (Avins et al., 2008; Boyle et al., 2004; Ernst, 2002; Hizli and Uygur, 2007; Willetts et al., 2003). Some hepatotoxic effects have been associated to a n-hexane-based Saw Palmetto extract (Hamid et al., 1997); indeed, a classical toxicity study in rats has been reported, showing an increase in oxidative stress associated with the intake of 2X and 5X the maximum dose recommended for humans (480µl/day) for this type of preparation (Singh et al., 2007). Nonetheless, no toxicological studies have been reported using the supercritical fluid extract of Saw Palmetto. Therefore, to perform the first acute toxicological study in rats of a supercritical fluid Saw Palmetto extract (commercial name “HiPower®”), Sprague-Dawley rats were fed with 1X and 10X the recommended dose for humans of this product (doses adjusted for rat metabolism) for 10 days. Rat blood samples obtained at different time intervals were analyzed through hemogram and clinical chemistry parameters; biopsies from liver, spleen, thymus and lymphatic ganglia were also collected and analyzed for possible morphological changes indicative of tissue damage. This study showed no statistically significant changes compared to normal ranges of any of the parameters analyzed; in addition, no significant changes were found in the morphological profile of the studied tissues. This demonstrates the safety of this particular Saw Palmetto extract at the doses and periods tested.

The protocol followed for this study evaluates general homeostasis and the function of liver and kidney, organs responsible for xenobiotic biotransformation and excretion, respectively. Therefore, the type of study presented here may be adopted as a method for assessing the acute toxicity of herbal extracts.


Saw Palmetto extract HiPower® HiPower® is a supercritical fluid extract from Saw

Palmetto fruits and was provided by Madreselva



Desarrollo y Producción Ltda. (Santiago, Chile). This extract contains: 3.8% palmitic acid, 1.7% stearic acid, 14.8% oleic acid, 44.2% linoleic acid, and 34.3% linolenic acid.

Animals Male Sprague-Dawley rats (200-230g) were

maintained with normal pellet diet (Kimber), access to water ad libitum, in a 12:12 light/dark cycle at 21°C. Animals were maintained in the vivarium of the School of Chemical and Pharmaceutical Sciences (Universidad de Chile, Santiago, Chile). All procedures were performed according to the protocols approved by the Ethical Committee of the institution and to the “Guide for the Care and Use of Laboratory Animals” (NRC, USA).

Treatment of rats Dosage of HiPower® was calculated from the 1X

recommended dose for humans (480µl/day), and considering 70kg for normal human weight and that rats display a 4-fold higher metabolic rate than humans. Doses were diluted in sunflower oil for oral delivery. Rats were distributed in 3 experimental groups of 40 rats each: two groups that were given 28 (HiPower® 1X group) and 280µl (HiPower® 10X group) extract/Kg/day, in two oral (gavage) doses, respectively, and a control group that received sunflower oil alone in equivalent volumes (Sunflower oil group). Following 2, 4, 6, 8, and 10 days of daily treatment, 8 rats from each group were anaesthetized with ether and sacrificed by exsanguination through cardiac puncture. Blood samples were used for hemogram and biochemical profile, and biopsies of liver, thymus, spleen and lymphatic ganglia were collected for histopathology analysis.

Hemogram study It was performed by the Central Laboratory of the

Clinical Hospital of the Universidad de Chile (Santiago, Chile). Parameters analyzed were red blood cell count (RBC), hematocrit, haemoglobin, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and white blood cell, lymphocyte and platelet counts.

Clinical chemistry study It was performed by the Central Laboratory of the

Clinical Hospital of the Universidad de Chile (Santiago, Chile). Parameters analyzed were: calcium,

phosphorus, glucose, blood ureic nitrogen, cholesterol, total protein, albumin, total bilirubin, acid phosphatase (AP), lactate dehydrogenase (LDH), and glutamyl oxaloacetic transaminase (GOT).

Histopathology studies These studies were performed by the Cyto-

Histopathology Laboratory BiopsCyt (Santiago, Chile). Histomorphology studies were performed with haematoxylin-eosin on the biopsies obtained at the different intervals during treatment.

Statistical Analyses Data presented in this study correspond to mean ±

95% confidence intervals (95% CI). Statistical significances between means of hemogram and clinical chemistry data were obtained through ANOVA. Statistical significances between medians of data and reference values were obtained through Wilcoxon Signed Rank tests. Reference values for adult Sprague-Dawley rats were obtained from observations from the Institute of Public Health of Chile (Uribe et al., 1995) or according to Lillie et al., 1996. All statistical analyses were performed using GraphPad Prism 5.0. Significances were set at 95% confidence

RESULTS

1. Effect of HiPower® on hemogram parameters of Sprague-Dawley rats.

Adult male Sprague-Dawley rats were treated orally for up to 10 days with vehicle (Sunflower oil group), 1X (HiPower® 1X group), or 10X (HiPower® 10X group) the recommended dose of HiPower® for humans, as detailed in Material and Methods. Following 2, 4, 6, 8, and 10 days of each treatment, rats were sacrificed and blood samples were collected for hemogram analysis.

As shown in Figure 1, HiPower® treatment did not significantly alter the following parameters, compared to normal ranges: red blood cells count (RBC) or hematocrit (Figure 1); haemoglobin or mean corpuscular haemoglobin (MCHC, Figure 2); white blood cells count (WBC, Figure 3); and platelet count (Figure 4). We also found complete absence of bacilliform white cells, basophiles, eosinophiles, metamyelocytes, or myelocytes (not shown).


Figure 1. Effect of HiPower® acute treatment on red blood cell count (RBC) and hematocrit of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. RBC and hematocrit were analyzed at different time points of a 10-day treatment. Data points represent the mean of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for each parameter.

Figure 2. Effect of HiPower® acute treatment on red blood cell mean corpuscular volume (MCV) and hemoglobin-related parameters of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. Hemoglobin, MCV, mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were analyzed at different time points of a 10-day treatment. Data points represent the mean of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for each parameter. Stars indicate parameters with a median significantly different (p<0.05) than their corresponding reference range, according to Wilcoxon Signed Rank test.



Figure 3. Effect of HiPower® acute treatment on white blood cell count (WBC) and lymphocytes of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. WBC and lymphocytes were analyzed at different time points of a 10-day treatment. Data points represent the mean of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for each parameter. Stars indicate parameters with a median significantly different (p<0.05) than their corresponding reference range, according to Wilcoxon Signed Rank test.

Figure 4. Effect of HiPower® acute treatment on platelets of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. Platelets were analyzed at different time points of a 10-day treatment. Data points represent the mean of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for each parameter.

Figure 5. Effect of HiPower® acute treatment on blood levels calcium and phosphorus of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. Blood levels of calcium and phosphorus were analyzed at different time points of a 10-day treatment. Data points represent the mean of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for each parameter.



Figure 6. Effect of HiPower® acute treatment on blood levels of albumin and total protein of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. Blood levels of albumin and total protein were analyzed at different time points of a 10-day treatment. Data points represent the mean of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for each parameter.

Figure 7. Effect of HiPower® acute treatment on blood levels of glucose, cholesterol, bilirubin, and urea nitrogen of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. Blood levels of glucose, cholesterol, bilirubin, and urea nitrogen were analyzed at different time points of a 10-day treatment. Data points represent the mean of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for each parameter.



Figure 8. Effect of HiPower® acute treatment on blood levels of alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. Blood levels of alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase were analyzed at different time points of a 10-day treatment. Data points represent the mean of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for each parameter. Stars indicate parameters with a median significantly different (p<0.05) than their corresponding reference range, according to Wilcoxon Signed Rank test.



Figure 9. Effect of HiPower® acute treatment on morphological features of liver, spleen, lymphatic ganglia, and thymus biopsies from Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. Biopsies from liver, spleen, lymphatic ganglia, and thymus were collected at different time points of a 10-day treatment. Representative images from haematoxylin-eosin staining of samples are shown. A. 4X magnification showing a typical hepatic lobule (bracket) with a central vein (arrow). B. 10X magnification of a hepatic biopsy showing a better view of the central vein (arrow); hepatic trabecules (solid arrowhead) and sinusoids (open arrowheads) are also distinguishable. C. 20X magnification showing a portal triad, with the portal venule (a), the bile duct surrounded by a cuboid epithelium (b), and the hepatic arteriole (c). D. 20X magnification of a spleen biopsy showing white and red pulps, with a germinative center in the latter. E. Germinative center of a splenic red pulp at 40X magnification. F. Germinative center of a splenic red pulp depicting apoptotic bodies (arrows). All structures shown are representative of all groups and treatment intervals. G. 10X magnification of a lymphatic ganglion biopsy, in which cortex and part of the medulla can be distinguished. Two lymphoid follicles can be discriminated, with their respective germinative centers. H. 20X magnification of a germinative center of a ganglion lymphoid follicle, with numerous apoptotic bodies (arrows). I. 40X magnification of a germinative center of a ganglion lymphoid follicle, with a better view of apoptotic bodies (arrows). J. 4X magnification of thymus lobule structure, with clear discrimination of cortex (1) and medulla (2) K. 40X magnification of a Hassall body (black arrow), with normal architecture. L. 40X magnification of a medullar zone of a thymus lobule, displaying scarce apoptotic bodies (white arrows).




Wilcoxon Signed Rank tests showed that particular cohorts displayed medians significantly different from reference ranges in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and the % lymphocytes (stars in Figures 2 and 3). Two-way ANOVA tests, however, showed that these differences are not a reflection of significant deviations compared to the vehicle (p>0.05).

2. Effect of HiPower® on clinical chemistry parameters of Sprague-Dawley rats.

In addition to hemogram analysis, blood samples were also analyzed for several clinical chemistry parameters. As shown in Figures 5-8, HiPower® treatment did not change the following parameters: calcium or phosphorus (Figure 5); blood levels of albumin or total proteins (Figure 6); blood glucose, cholesterol, bilirubin, or urea nitrogen (Figure 7); and the activity of alkaline phosphatase (AP, Figure 8).

Wilcoxon Signed Rank tests showed that some cohorts displayed medians significantly higher than the upper limit of the normal range for aspartate aminotransferase activity (stars in Figure 8, middle panel). Two-way ANOVA tests, however, showed that these differences are not a reflection of significant deviations compared to the vehicle (p>0.05). Also, most cohorts showed medians significantly lower than the lower limit of the normal range for lactate dehydrogenase (LDH) activity (stars in Figure 8, bottom panel). Two-way ANOVA analysis demonstrated that most of these deviations were not significantly different from the vehicle cohort, except for the cohorts treated with 1X HiPower® (8 and 10 days of treatment, p<0.001) and 10X HiPower® (6 and 8 days of treatment, p<0.001).

3. Effect of HiPower® on morphological features of rat tissues.

We also obtained biopsies (hepatic, splenic, thymic, and lymphatic) from each group at different time intervals of the treatments. These biopsies were used to perform histopathology analysis of tissue sections with haematoxylin-eosin, as detailed in Material and Methods.

All liver biopsies, regardless of the group or the time of collection, showed a conserved histological architecture (Figure 9A-C). We observed classical structures, such as hepatic lobules with a

central vein (Figure 9A and 9B) and portal triads constituted by blood vessels and bile ducts (Figure 9C). Hepatocytes displayed a conserved structure, without noticeable degeneration or necrosis. Most liver biopsies displayed small hematopoietic loci, with the same occurrence regardless of the cohort analyzed or the collection time. Liver parenchyma displayed no mitosis count per mm2 in most samples, with a mitosis count of 1-4 per mm2 in a few biopsies, regardless of the cohort or collection time.

Spleen biopsies had also normal architecture of red or white pulp, regardless of the cohort or treatment interval analyzed (Figure 9D-F). All samples displayed hematopoietic activity and a mitotic count in germinative centers of 0-4 per mm2, regardless of the treatment or collection interval (Figures 9D and 9E). Vehicle cohort displayed virtually no apoptotic bodies, while 1X HiPower® and 10X HiPower® cohorts showed some apoptotic bodies in seldom cases (Figure 9F).

Lymphatic ganglia biopsies from all three cohorts displayed conserved histological structures (Figure 9G-I). We found lymphocytic elements and reticulo-histocytic cells in the lymphoid tissue. Most biopsies, regardless of the cohort or collection time, displayed secondary follicles with germinative centers (Figure 9G). With low frequency, some sinusal edema was found regardless of the cohort or the collection time. At the level of lymphoid follicles, some apoptotic activity was evidenced by the occurrence of apoptotic bodies in the cytoplasm of reticulo-histocytic cells (Figures 9H and 9I). The abundance of apoptotic bodies, however, was not associated to a cohort or collection time. On the other hand, at the level of germinative center, we observed a mitotic count of 2-25 per mm2. Variability of mitotic count was unchanged among cohorts or collection times.

Histological structures of the thymus were conserved in all biopsies tested (Figure 9J-L), regardless of the cohort or the collection time, with normal architectures of cortex and medulla (Figure 9J). Normal lymphoid and epithelial (Hassall bodies, Figure 9K) components were also visualized. Apoptotic scores did not reveal significant differences between cohorts or collection times (Figure 9L). Mitotic count in germinative centers was 1-2 per mm2, displaying no significant differences between cohorts or collection times.



DISCUSSION

Our study was aimed to develop a methodology that allows the evaluation of the safety profile of herbal extracts, using a supercritical fluid Saw Palmetto extract (HiPower®) as a start point. To this end, we evaluated possible changes in classical hemogram and clinical chemistry parameters (as a measure of general homeostasis) from Sprague-Dawley rats fed for 10 days with two different doses of HiPower®, compared with rats fed with vehicle (sunflower oil). We also evaluated potential gross morphological alterations in key organs involved in xenobiotic metabolism (to address possible toxicity associated with xenobiotic biotransformation) and immune system (to address potential toxic immunological response). With this purpose, blood samples and tissue biopsies were collected from rats at different intervals during the 10-day treatment.

Essentially none of the parameters tested underwent any statistically significant changes following this treatment, as assessed by Wilcoxon Signed Rank test. Although these analyses showed some deviations of medians from the normal ranges, most of such deviations were not significantly different from those of the vehicle cohort. This may be the reflection of parameters with high inter-individual dispersion. Noteworthy, lactate dehydrogenase activity values were lower than the lower limit of normal ranges for this parameter in all cohorts, especially at the beginning of the treatment. Although this parameter reached the normal interval in the case of the vehicle cohort, it remained under the lower limit for the 1X HiPower® and 10X HiPower® cohorts. Since lactate dehydrogenase activity is a classical marker for liver damage, these data suggest that HiPower® may display hepatoprotective activity.

In summary, hemogram and clinical chemistry data show that the treatment of Sprague-Dawley rats with a supercritical fluid Saw Palmetto extract led to undetectable alterations of red blood cells integrity, haematopoiesis, immune or inflammatory responses, coagulation, metabolism of glucose, nucleic acids, proteins, fatty acids or steroids, and function of liver, spleen, kidney or parathyroid gland. The latter was also corroborated by the absence of gross morphological changes in liver, spleen, lymphatic ganglia or thymus. Therefore, we concluded that this acute treatment did not lead to any classic toxicological outcomes, even with doses that differ in one order of magnitude.

It has been shown that some Saw Palmetto extracts can be toxic in acute toxicological studies (Hamid et al., 1997; Singh et al., 2007). On the other hand, several studies have demonstrated the safety of these extracts in humans (Avins et al., 2008; Boyle et al., 2004; Ernst, 2002; Hizli and Uygur, 2007; Willetts et al., 2003). It is very difficult to address the safety of a Saw Palmetto extract due to the diversity of extraction methods (Habib and Wyllie, 2004). Although this extract appears to be more concentrated than other commercial Saw Palmetto extracts, we postulated that the absence of organic solvents in the supercritical fluid extraction of Saw Palmetto fruits, leading to the preparation of an extract with a good safety profile. Due to their oily nature, it is possible that Saw Palmetto extracts used for the treatment of benign prostate hyperplasia contain lipoperoxides; these substances must be controlled due to their potential for promoting lipid peroxidation in tissues. Noteworthy, the particular Saw Palmetto extract used in this study did not lead to an increase in basal lipoperoxidation of rat liver microsomes (data not shown), a system routinely used by our lab to test oxidative damage (Letelier et al., 2007). The absence of pro-oxidant activity corroborates the good safety profile of this Saw Palmetto extract. In light of our data, we can conclude that HiPower® displays a safety profile of at least one order of magnitude in dosage.

The general strategy used for the present study indeed allowed the assessment of potential alterations in general homeostasis and morphological features of key organs in xenobiotic biotransformation (liver) and excretion (kidney). Therefore, we conclude that this type of studies may be adopted for future evaluations of safety profiles of herbal extracts.

REFERENCES Avins AL, Bent S, Staccone S, Badua E, Padula A,

Goldberg H, Neuhaus J, Hudes E, Shinohara K, Kane C. 2008. A detailed safety assessment of a saw palmetto extract. Complement Ther Med 16: 147-154.

Bent S, Kane C, Shinohara K, Neuhaus J, Hudes ES, Goldberg H, Avins AL. 2006. Saw palmetto for benign prostatic hyperplasia. New Eng J Med 354: 557-566.

Blumenthal M, Busse WR, Goldberg AD, Gruenwald J, Hall T, Riggins CW, Klein S, Rister RS. 1998. The complete German Commission E monographs: Therapeutic guide to herbal medicines. Ed. Lippincott Williams & Wilkins, Austin, Texas, pp.74, 201, 432.


Boyle P, Robertson C, Lowe F, Roehrborn C. 2004. Updated meta-analysis of clinical trials of Serenoa repens extract in the treatment of symptomatic benign prostatic hyperplasia. BJU Int 93: 751-756.

Carraro JC, Raynaud JP, Koch G, Chisholm GD, Di Silverio F, Teillac P, Da Silva FC, Cauquil J, Chopin DK, Hamdy FC, Hanus M, Hauri D, Kalinteris A, Marencak J, Perier A, Perrin P. 1996. Comparison of phytotherapy (Permixon) with finasteride in the treatment of benign prostate hyperplasia: a randomized international study of 1,098 patients. Prostate 29: 231-240.


Cristoni A, Morazzoni P, Bombardelli E. 1997. Chemical and pharmacological study on hypercritical CO2 extracts of Serenoa repens fruits. Fitoterapia 68: 355-358.

de la Taille A, Buttyan R, Hayek O, Bagiella E, Shabsigh A, Burchardt M, Burchardt T, Chopin DK, Katz AE. 2000. Herbal therapy PC-SPES: in vitro effects and evaluation of its efficacy in 69 patients with prostate cancer. J Urol 164: 1229-1234.

de la Taille A, Hayek OR, Buttyan R, Bagiella E, Burchardt M, Katz AE. 1999. Effects of a phytotherapeutic agent, PC-SPES, on prostate cancer: a preliminary investigation on human cell lines and patients. BJU Int 84: 845-850.

Derakhshani P, Geerke H, Böhnert KJ, Engelmann U. 1997. Beeinflussung des Internationalen Prostata-Symptomen-Score unter der Therapie mit Sägepalmenfrüchteextrakt bei täglicher Einmalgabe. Der Urologe B 37: 384-391.

Ernst E. 2002. The risk-benefit profile of commonly used herbal therapies: Ginkgo, St. John's Wort, Ginseng, Echinacea, Saw Palmetto, and Kava. Ann Intern Med 136: 42-53.

Gerber GS, Fitzpatrick JM. 2004. The role of a lipido-sterolic extract of Serenoa repens in the management of lower urinary tract symptoms associated with benign prostatic hyperplasia. BJU Int 94: 338-344.

Habib FK, Wyllie MG. 2004. Not all brands are created equal: a comparison of selected components of different brands of Serenoa repens extract. Prostate Cancer Prostatic Dis 7: 195-200.

Hamid S, Rojter S, Vierling J. 1997. Protracted cholestatic hepatitis after the use of prostata. Ann Intern Med 127: 169-170.

Hizli F, Uygur MC. 2007. A prospective study of the efficacy of Serenoa repens, tamsulosin, and Serenoa repens plus tamsulosin treatment for patients with benign prostate hyperplasia. Int Urol Nephrol 39: 879-886.

Letelier ME, Entrala P, López-Alarcón C, González-Lira V, Molina-Berríos A, Cortés-Troncoso J, Jara-

Sandoval J, Santander P, Núñez-Vergara L. 2007. Nitroaryl-1,4-dihydropyridines as antioxidants against rat liver microsomes oxidation induced by iron/ascorbate, nitrofurantoin and naphthalene. Toxicol in Vitro 21: 1610-1618.

Lillie LE, Temple NJ, Florence LZ. 1996. Reference values for young normal Sprague-Dawley rats: weight gain, hematology and clinical chemistry. Hum Exp Toxicol 15: 612-616.

Lowe FC, Fagelman E. 2004. Permixon: A review. Curr Prostate Rep 2: 133-136.

Singh YN, Devkota AK, Sneeden DC, Singh KK, Halaweish F. 2007. Hepatotoxicity potential of saw palmetto (Serenoa repens) in rats. Phytomedicine 14: 204-208.

Small EJ, Frohlich MW, Bok R, Shinohara K, Grossfeld G, Rozenblat Z, Kelly WK, Corry M, Reese DM. 2000. Prospective trial of the herbal supplement PC-SPES in patients with progressive prostate cancer. J Clin Oncol 18: 3595-3603.

Sovak M, Seligson AL, Konas M, Hajduch M, Dolezal M, Machala M, Nagourney R. 2002. Herbal composition PC-SPES for management of prostate cancer: identification of active principles. J Natl Cancer Inst 94: 1275-1281.

Ulbricht C, Basch E, Bent S, Boon H, Corrado M, Foppa I, Hashmi S, Hammerness P, Kingsbury E, Smith M, Szapary P, Vora M, Weissner W. 2006. Evidence-based systematic review of saw palmetto by the Natural Standard Research Collaboration. J Soc Integr Oncol 4: 170-186.

Uribe M, Mariné L, Catán F, Capetillo M, Cavallieri S, Bianchi V, Pizarro F, Romero S, Carvajal C, Contreras R, Valdés P. 1995. Valores hematológicos, serológicos y peso de órganos en la rata Sprague-Dawley adulta [Hematological, serological values, and organs weight in adult Sprague-Dawley rats]. Rev Med Chil 123: 1235-1242.

Vallancien G, Pariente P. 2001. Treatment of lower urinary tract symptoms suggestive of benign prostatic obstruction in real life practice in France. Prostate Cancer Prostatic Dis 4: 124-131.

Willetts KE, Clements MS, Champion S, Ehsman S, Eden JA. 2003. Serenoa repens extract for benign prostate hyperplasia: a randomized controlled trial. BJU Int 92: 267-270.

Wilt T, Ishani A, Stark G, MacDonald R, Mulrow C, Lau J. 2000. Serenoa repens for benign prostatic hyperplasia. Cochrane Database Syst Rev CD001423.

Wilt TJ, Ishani A, Stark G, MacDonald R, Lau J, Mulrow C. 1998. Saw palmetto extracts for treatment of benign prostatic hyperplasia: a systematic review. JAMA 280: 1604-1609.



Comunicación | Communication



Volatile Components from the Leaves of Solanum hypomalacophyllum Bitter.

[Componentes volátiles de las hojas de Solanum hypomalacophyllum Bitter.] Alida PÉREZ COLMENARES1, Luis B. ROJAS1*, Alfredo USUBILLAGA1

1Instituto de Investigaciones, Facultad de Farmacia y Bioanálisis. Universidad de Los Andes, Mérida - Venezuela.

Abstract

The essential oil from the leaves of Solanum hypomalacophyllum (Solanaceae) collected in May 2007 at Páramo La Culata (Mérida State, Venezuela) was obtained by hydrodistillation and its composition was determined by GC and GC/MS. Eleven compounds, representing 92.4 % of the oil, were identified 5-octen-1-ol (39.7 %), cis-3-hexenol (20.2 %), 1-hexanol (12.0%) and hexadecanoic acid (5.2 %) were the most abundant components.

Keywords: Solanum hypomalacophyllum; Solanaceae; essential oil; 5-octen-1-ol; GC-MS analysis.

Resumen

El aceite esencial de las hojas de Solanum hypomalacophyllum (Solanaceae) recogidas en mayo 2007 en el Páramo La Culata (Estado de Mérida, Venezuela) fue obtenido por hidrodestilacion y fue analizado por GC y GC/MS. Se identificaron 11 componentes que representan el 92.4% del total del aceite. Los componentes mayoritarios fueron identificados como 5-octen-1-ol (39.7 %), cis-3-hexenol (20.2 %), 1-hexanol (12.0 %) y ácido hexadecanoico (5.2 %).

Palabras Clave: Solanum hypomalacophylllum; Solanaceae; aceite esencial; 5-octen-1-ol; GC-MS análisis.

Recibido | Received: November, 18, 2009. Aceptado en Versión Corregida | Accepted in Corrected Version: January, 10, 2010. Publicado en Línea | Published Online January 21, 2010. Declaración de intereses | Declaration of interests: Authors have no competing interests. Financiación | Funding: This work was supported by: Consejo de Desarrollo Científico, Humanístico y Tecnológico (CDCHT–Mérida–Venezuela, project FA-380-06-03-A), FONACIT-PCP (46.255), University of Los Andes-Plan II. This article must be cited as: Pérez Colmenares A, Rojas L B., Usubillaga A. 2010. Volatile Components from the Leaves of Solanum hypomalacophyllum. Bol Latinoam Caribe Plant Med Aromat 9(1):80 – 83. {EPub 21 Jan 2010 }.

*Contactos | Contacts: E-mail: [email protected]; Tel: 0058-2742403958 ; Fax: 0058-2742403455.


Pérez Colmenares et al. Essential Oil of Solanum hypomalacophyllum


INTRODUCTION

The Solanaceae family comprises about 90 genera and 3.000 species which are widely distributed in the world. They are a rich source of active secondary metabolites (Coletto da Silva et al., 2004). Within this family, the genus Solanum is the largest and most complex with more than 1500 species (Chowdhury et al., 2007) which yield a great variety of steroidal saponins and glycoalkaloids of interest from ecological and human health viewpoints

(Roddick et al., 2001). Numerous species of Solanum are known to possess a variety of biological activities including antimycotic (Gonzales et al., 2004; Singh et al., 2007), antiviral (Arthan et al., 2002), molluscicidal (Silva et al., 2006), teratogenic (Keeler et al., 1990), and cytotoxic properties (Nakamura et al., 1996; Lu et al., 2009). Previous studies reported the chemical composition of the essential oil of several species of Solanum, like S. verbascifolium (Ma et al., 2006), S. lyratum (Xu et al., 2006), S. aculeastrum (Koduru et al., 2006) and of S. pseudocapsicum (Aliero et al., 2006, 2007), but no reports have been published on the composition of the volatile constituents of S. hypomalacophyllum Bitter. This species is a small tree native to the Venezuelan Andes where it grows wild in humid places at altitudes between 2150 and 3200 meters. Previous studies reported the isolation of 4-keto steroidal alkaloids solaphyllidine, deacetoxysolaphyllidine, desacetylsolaphyllidine, spirosolaphyllidine and solamaladine from green berries and leaves (Usubillaga, 1970, 1984; Usubillaga et al., 1982; Alarcon et al., 2006). Therefore, in continuation of our study of the Venezuelan Solanaceae, we report here the first study on the essential oil of S. hypomalacophyllum in order to obtain knowledge on its chemical composition.


Plant Material Leaves of Solanum hypomalacophyllum were

collected in May 2007, in the area of El Páramo de la Culata, Mérida State, Venezuela at 2800 m above sea level. A voucher specimen has been deposited at the MERF Herbarium (Herbarium of the Faculty of Pharmacy and Bioanalysis in Mérida, Venezuela). The botanical identification was made by Forest Engineer Juan Carmona, MERF Herbarium.

Extraction and analysis of the essential oil Fresh leaves (2000 g) were cut into small

pieces and submitted to hydrodestillation for 3 h using a Clevenger-type apparatus. 0.1 mL of essential oil was obtained that corresponds to a yield of 0.005% v/w.

The composition of the essential oil was determined by comparison of the mass spectrum of each component with Wiley GC/MS library data and also from retention index (RI) data (Morteza et al., 2004).

Gas chromatography GC analyses were performed using a Perkin-

Elmer Autosystem gas chromatograph equipped with a FID detector and data-handling system. A 5% phenylmethylpolysiloxane fused-silica capillary column was used (30 m x 0.25 mm i.d., film thickness 0.25 μm; HP-5, Hewlett-Packard, CA, USA). The oven temperature was programmed from 60 ºC to 260 ºC at 4 ºC/min. The injector and detector (Flame ionization detector, FID) temperatures were 200ºC and 280 ºC, respectively. The carrier gas was helium at 0.8 mL/min. The sample (1.0 μL) was injected using a split ratio of 10:1. Retention indices were calculated by comparing the retention times of the eluting peaks with those of standard C8-C24 n-alkanes. The percentage composition of the oil was calculated by the normalization method from the GC peak areas.

Gas chromatography – mass spectrometry GC-MS analyses were carried out on a Model

5973 Hewlett-Packard GC-MS system fitted with a HP-5MS fused silica column (30 m x 0.25 mm i.d., film thickness 0.25 μm, Hewlett-Packard). The oven temperature program was the same as that used for the HP-5 column for GC analysis; the transfer line temperature was programmed from 150ºC to 280ºC; source temperature, 230ºC; quadrupole temperature, 150ºC; carrier gas, helium, adjusted to a linear velocity of 34 cm/s; ionization energy, 70 eV; scan range, 40:500 amu; 3.9 scans/s. Sample (1.0 μL) was injected using a Hewlett-Packard ALS injector with a split ratio of 50:1. The identity of the oil components was established from their GC retention indices, by comparison of their MS spectra with those of standard compounds available in the laboratory, and by a library search (NIST 05 and Wiley)(Adams, 1995).




Hydrodistillation of the leaves yielded a yellowish liquid oil in 0.005 % v/w yield. The chemical composition of the oil was investigated using both GC and GC-MS techniques. It was possible to identify 11 components, which represent 92.4% of the total oil. These compounds with their retention indices (RI) and relative percentage concentrations are listed in Table 1 according to the elution order on the HP-5 column by GC and by comparison of the fragmentation pattern. The oil was found to contain alcohols (78.2%), fatty acid (5.2%) and aldehydes (4.8%) as major components. The dominant compounds were 5-octen-1-ol (39.7 %), cis-3-hexenol (20.2 %), 1-hexanol (12.0 %) and hexadecanoic acid (5.2 %). Table 1. Percentage composition of the essential oil of the leaves of Solanum hypomalacophyllum.

Peak Nº Compounds Area (%) RI

1 Trans-3-hexenol 1.4 844 2 Cis-3-hexenol 20.2 850 3 1-Hexanol 12.0 862 4 Cis-4-heptenal 4.8 898 5 1-heptanol 1.6 967 6 5-octen-1-ol 39.7 1071 7 1-nonanol 1.4 1174 8 1-decanol 1.9 1276 9 β-ionone 1.8 1497

10 Butylated-hydroxytoluene 2.4 1523 11 Hexadecanoic acid 5.2 1961

Total 92.4

RI: retention index

CONCLUSION

The chemical composition of the essential oil from leaves the Solanum hypomalacophyllum contain 5-octen-1-ol (39.7 %), cis-3-hexenol (20.2 %), 1-hexanol (12.0 %) and hexadecanoic acid (5.2 %). as major components. These compounds are reported for the first time in this species.

ACKNOWLEDGMENTS

The authors would like to thank the financial support of Consejo de Desarrollo Científico, Humanístico y Tecnológico (CDCHT–Mérida–Venezuela, project FA-380-06-03-A), FONACIT-PCP (46.255), University of Los Andes-Plan II and Forest Engineer Juan Carmona (MERF Herbarium, Facultad de Farmacia y Bioanálisis) for the identification of the botanical material.

REFERENCES Adams R. 1995. Identification of Esential Oil Components

by Gas Chromatography/Mass Spectrometry. (USA). Alarcon L, Velasco J, and Usubillaga A. 2006.

Determinación de la Actividad Antibacteriana de los Alcaloides presentes en los Frutos Verdes del Solanum hypomalocophyllum Bitter. Rev Latinoam Quím 34: 13-21.

Aliero A, Asekum O, Grierson D, Afolayan A. 2006. Chemical Composition of the Esentail Oil from Solanum pseudocapsicum. J Biol Sci 9: 1175-1177.

Aliero A, Asekun O, Grierson D, Afolayan, A. (2007). Volatile Components from the roots Solanum pseudocapsicum. J Med Food 10: 557-558.

Arthan D, Svasti J, Kittakoop P, Pittayakhachonwut D, Tanticharoen M, Thebtaranonth Y. 2002. Antiviral Isoflavonoid Sulfate and Steroidal Glycosides from the Fruits of Solanum torvum. Phytochemistry 59: 459-463.

Coletto da Silva A, Kinupp V, Absy M, and Kerr W. 2004. Pollen Morphology and Study of the Visitors (Hymenoptera, Apidae) of Solanum stramoniifolium Jacq. (Solanaceae) in Central Amazon. Acta Bot Bras 18: 653-657.

Chowdhury N, Bhattacharjee I, Laskar S, Chandra G. 2007. Efficacy of Solanum villosum Mill. (Solanaceae: Solanales) as a Biocontrol Agent against Fourth Instar Larvae of Culex quinquefasciatus Say. Turk J Zool 31: 365-370.

Gonzales, M., Zamilpa, A., Marquina, S., Navarro, E., and Alvarez, L. 2004. Antimycotic Spirostanol Saponins from Solanum hispidum Leaves and their Structure-Activity Relationships. J Nat Prod 67: 938-941.

Keeler R, Baker D, Gaffield W. 1990. Spirosolane-Containing Solanum Species and Induction of Congenital Craniofacial Malformations. Toxicon 28: 873-884.

Koduru S, Aseku, O, Grierson D, Afolayan A. 2006. Isolation of Volatile Compounds from Solanum acueslastrum (Solanaceae). J Essent Oil-Bear Plant 9: 65-66.

Lu Y, Luo J, Huang X, Kong L. 2009. Four New Steroidal Glycosides from Solanum torvum and their Cytotoxic Activities. Steroids 74: 95-101.

Ma R., Guo S., Zhu H., Zhang T. 2006. Chemical Constituents of Esential Oil from Leaves of Solanum verbascifolium (Solanaceae). J Trop Subtrop Bot 14: 526-529.

Morteza K, Azadbkht M, Goodarzi A. 2004. The Essential Oils Composition of Phlomis herba-venti L. Leaves and Flowers of Iranian Origin. Flavour Fragance J 19: 29-31.

Nakamura T, Komori A, Lee Y, Hashimoto F, Yahara S, Nohara T, Ejima A. 1996. Cytotoxic Activities of Solanum Steroidal Glycosides. Biol Pharm Bull 19: 554-566.


Usubillaga A. 1970. Structure of solaphyllidine, a novel 4-ketosteroidal. J Amer Chem Soc 92:700-701.

Roddick J, Weissenberg M, Leonard A. 2001. Membrane Disruption and Enzyme Inhibition by Naturally-Occurring and Modified Chacotriose-Containing Solanum Steroidal Glycoalkaloids. Phytochemistry 56: 603-610.

Usubillaga A. 1984. Alkaloids from Solanum hypomalacophyllum. J Nat Prod 47: 52-54.

Usubillaga A, Zabel V, Watson W. 1982. The Revised Structure of Solamaladine, 3-hydroxy-22-(4-methyl-1-pyrrolin-2-yl)-23,24-dinor-5-cholane-4,22-dione. Acta Crystallogr B 38: 966-969.

Silva T, Camara C, Agra F, de Carvalho M, Frana M, Brandoline, S, da Silva L, Braz-Filho R. 2006. Molluscicidal Activity of Solanum Species of the Northeast of Brazil on Biomphalaria glabrata. Fitoterapia 77: 449-452.

Xu S, Wang L, Li R, Liu H. 2006. Analysis of Chemical Constituents of the Volatile Oil from Solanum lyratum Thunb. J Chin Integr Med 17: 1390-1391. Singh O, Subharani K, Singh N, Devi N, Nevidita L 2007.

Isolation of Steroidal Glycosides from Solanum xanthocarpum and Studies on their Antifungal Activities. Nat Prod Res 21: 585-590.


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