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REVISTA BRASILEIRA DE FARMACOGNOSIABRAZILIAN JOURNAL OF PHARMACOGNOSYwww.sbfgnosia.org.br/revista

Editor-in-chiefCid Aimbiré de Moraes SantosUniversidade Federal do Paraná

Guest EditorsSuzana G. LeitaoUniversidade Federal do Rio de Janeiro, BrazilLucas RastrelliUniversity of Salerno, Italy

Editorial Board Aurea Elizabeth LinderUniversidade Federal de Santa CatarinaEmídio Vasconcelos Leitão da CunhaUniversidade Estadual da ParaíbaFernando Batista da CostaUniversidade de São PauloMaique Weber Biavatti Universidade Federal de Santa CatarinaMárcia do Rocio Duarte Universidade Federal do ParanáReinaldo Nóbrega de Almeida Universidade Federal da ParaíbaRúbia Maria Weffort Oliveira Universidade Estadual de Maringá

Universidade Federal do Paraná Laboratório de Farmacognosia

Rua Prof. Lothario Meissner, 632, Jardim Botânico 80210-170

Curitiba, PR, Brasil, Tel./Fax: + 55 41 3360 4062

[email protected]

Programa de apoio de publ icações cient í f icas

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy

21(5): Sep./Oct. 2011

Advisory BoardAlexander I GrayUniversity of Strathclyde, United Kingdom

Juceni Pereira DavidUniversidade Federal da Bahia, Brasil

Arnold J. VlietinckUniversity of Antwerp, Belgium

Leandros SkaltsounisUniversity of Athens, Greece

Elisaldo A. CarliniUniversidade Federal de São Paulo, Brasil

Mahabir GuptaUniversidad de Panamá, Panamá

Fernando de OliveiraUniversidade São Francisco, Brasil

Marcelo Sobral da SilvaUniversidade Federal da Paraíba, Brasil

Geoffrey A. CordellUniversity of Illinois at Chicago, USA

Matias MelzigUniversitst Berlin, Germany

Jairo Kenupp BastosUniversidade de São Paulo, Brasil

Nestor O. Caffi niUniversidae Nacional de La Plata, Argentina

Jean SassardUniversité de Lyon, France

Raimundo Braz FilhoUniversidade Estadual do Norte Fluminense, Brasil

José Carlos Tavares CarvalhoUniversidade Federal do Amapá, Brasil

Wagner Luiz Ramos BarbosaUniversidade Federal de Pará, Brasil

Brazilian Journal of Pharmacognosy is a bimonthly publication, distributed free to all active members of the Brazilian Society of Pharmacognosy. Indexing: Chemical Abstracts, International Pharmaceutical Abstracts, Medicinal and Aromatic Plants Abstracts (MAPA), Natural Products Alert (NAPRALERT), Natural Products Updates, CABI, LILACS, SCOPUS, SCIELO, EMBASE, Compendex, GEOBASE, EMBiology, FLUIDEX, World Textiles e Elsevier BIOBASE, Biological Abstracts, Index Copernicus, Science Citation Index Expanded (SciSearch®), Journal Citation Report/Science Edition, ISI, SIIC Data Bases.

Subscription 2011: Institutional R$ 385,00, Professional R$ 130,00, Student R$ 80,00

Editorial ManagerMárcio ChimelliUniversidade Federal do Paraná

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): Sep./Oct. 2011 Contents

869 Active caspase-3 detection to evaluate apoptosis induced by Verbena officinalis essential oil and citral in chronic lymphocytic leukaemia cellsLaura De Martino, Giovanni D’Arena, Maria Marta Minervini, Silvia Deaglio, Nicola Pio Sinisi, Nicola Cascavilla,Vincenzo De Feo

874 Phytochemical profile and analgesic evaluation of Vitex cymosa leaf extractsSuzana Guimarães Leitão, Tereza Cristina dos Santos, Franco Delle Monache, Maria Eline Matheus, Patrícia Dias Fernandes, Bruno Guimarães Marinho

884 Inhibition of human platelet aggregation in vitro by standardized extract of Wendtia calycinaMilagros Garcia Mesa, Anna Lisa Piccinelli, María Antonia Alfonso Valiente, Aldo Pinto, Alessia Fazio, Luca Rastrelli

889 Antispasmodic effects of Aloysia polystachya and A. gratissima tinctures and extracts are due to non-competitive inhibition of intestinal contractility induced by acethylcholine and calciumAlicia E. Consolini, Andrea Berardi, María A. Rosella, María G. Volonté

901 Inhibition of Saccharomyces cerevisiae Pdr5p by a natural compound extracted from Brazilian Red PropolisCinzia Lotti, Gabriellen M. Migliani de Castro, Leandro F. Reis de Sá, Beatriz dos Anjos Fonseca Sampaio da Silva, Ana Claudia Tessis, Anna L. Piccinelli, Luca Rastrelli, Antonio Ferreira-Pereira

908 Antileishmanial activity of nerolidol-rich essential oil from Piper claussenianumAndré Mesquita Marques, Anna Léa Silva Barreto, José Alexandre da R. Curvelo, Maria Teresa Villela Romanos, Rosangela M. de A. Soares, Maria Auxiliadora C. Kaplan

915 Antiviral activity of Ageratina havanensis and major chemical compounds from the most active fractionGloria del Barrio, Iraida Spengler, Trina García, Annele Roque, Ángel L. Álvarez, José S. Calderón, Francisco Parra

921 Assessment of estrogenic, mutagenic and antimutagenic activity of nemorosoneMariana S. Camargo, Soraya D. Varela, Ana Paula S. de Oliveira, Flávia Ap. Resende, Osmay Cuesta-Rubio, Wagner Vilegas, Eliana A. Varanda

928 Analgesic effect of leaf extract from Ageratina glabrata in the hot plate testGuadalupe García P, Edgar García S, Isabel Martínez G, Thomas R. F. Scior, José L Salvador, Mauro M Martínez P, Rosa E. del Río

Articles

786 How a Huottuja (Piaroa) community perceives genuine and false honey from the Venezuelan Amazon, by free-choice profi le sensory methodPatricia Vit, Rosires Deliza, Alfonso Pérez

793 Ethnopharmacological versus random plant selection methods for the evaluation of the antimycobacterial activityDanilo R. Oliveira, Gilda G. Leitão, Tatiane S. Coelho, Pedro E. Almeida da Silva, Maria Cristina S. Lourenço, ARQMO, Suzana G. Leitão

807 Cranberry in children: prevention of recurrent urinary tract infections and review of the literatureAngelica Dessì, Alessandra Atzei, Vassilios Fanos

814 Ethnopharmacology in Dublin: surveys on the medicinal plants use profileFrancis Cunningham, Fabio S. Menezes

818 HPTLC fingerprint: a modern approach for the analytical determination of Botanicals Marcello Nicoletti

824 Quinolizidine alkaloid composition in different organs of Lupinus aschenborniiEdith Montes Hernández, María L. Corona Rangel, Aidee Encarnación Corona, Jorge A. Cantor del Angel, Jesús Arnoldo Sánchez López, Frank Sporer, Michael Wink, Kalina Bermúdez Torres

829 Effect of the microfi ltration process on antioxidant activity and lipid peroxidation protection capacity of blackberry juiceGabriela Azofeifa, Silvia Quesada, Ana-Mercedes Pérez

835 The activity of flavones and oleanolic acid from Lippia lacunosa against susceptible and resistant Mycobacterium tuberculosis strainsAline Castellar, Tatiane S. Coelho, Pedro E. A. Silva, Daniela F. Ramos, Maria Cristina S. Lourenço, Celso L. S. Lage, Lisieux S. Julião, Ymira G. Barbosa, Suzana G. Leitão

841 Enantiomeric distribution of key volatile components in Citrus essential oilsIvana Bonaccorsi, Danilo Sciarrone, Antonella Cotroneo, Luigi Mondello, Paola Dugo, Giovanni Dugo

850 Phytochemical researches and antimicrobial activity of Clinopodium nubigenum Kunth (Kuntze) raw extractsGianluca Gilardoni, Omar Malagon, Vladimir Morocho, Riccardo Negri, Solveig Tosi, Maria Guglielminetti, Giovanni Vidari, Paola Vita Finzi

856 Data collection and advanced statistical analysis in phytotoxic activity of aerial parts exudates of Salvia spp. M. Giacomini, A. Bisio, E. Giacomelli, S. Pivetti, S. Bertolini, D. Fraternale, D. Ricci, G. Romussi, N. De Tommasi

864 Chemical composition and cytotoxic activity of the essential oil from the leaves of Casearia lasiophyllaMarcos José Salvador, João Ernesto de Carvalho, Alberto Wisniewski-Jr, Candida A. L. Kassuya, Élide P. Santos, Dilamara Riva, Maria Élida Alves Stefanello

The Italo-Latin American Society of Ethnomedicine (SILAE, www.silae.it) is an international non-profit organization dedicated to advancing science around the world by serving as an educator, leader, spokesperson and professional association. The fundamental objective of SILAE is to promote research and development into the use of medicinal and food plants in different countries of the World. SILAE welcomes and actively seeks opportunities to work cooperatively, activating and intensifying scientific relations between countries and between SILAE members. Since SILAE was founded (1990) its objective has been set to contribute to the close examination of the themes of great interest and actuality in the context of the relationships between Latin America and the European Union. In addition to this, SILAE aimed to individualize new ways of collaboration between its member countries and other European as well as Asiatic countries to sign accords with intergovernmental organizations. SILAE proposes to establish contacts with Scientific Communities, Universities, and Research Centres for the pursuit of medicinal and food plants knowledge. Moreover SILAE_live, the one-to-one live Chat and Messenger on our website (www.silae.it), is the first scientific chat on the web and is a developed tool to engage the interest and imagination of the public and for helping non-scientists to understand and enjoy scientific discoveries and the scientific processes. In addition to organizing membership activities, SILAE publishes the SILAE Special Issues, as well as many scientifi c newsletters, books and reports, and spearheads programs that raise the bar of understanding for science worldwide. Revista Brasileira de Farmacognosia - Brazilian Journal of Pharmacognosy is publishing a special issue that contains a selection of papers that were presented at the XIX SILAE Congress (Cagliari, Italy, September, 6-10, 2010). For the Conference, 292

SILAE Special Issue: Italo-Latin American Ethnoknowledge and Research on Medicinal Plants

Suzana G. Leitao,1 Luca Rastrelli2

1Departamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Brazil ([email protected]);2Dipartimento di Scienze Farmaceutiche e Biomediche, University of Salerno, Italy ([email protected]).


21(5): Sep./Oct. 2011

papers from authors coming from 19 different countries were accepted and published in the Proceedings of the SILAE 2010 (Abstract book ISBN: 88-8160-218-0). The most promising 30 submissions were proposed for publication in the special issue of Revista Brasileira de Farmacognosia - Brazilian Journal of Pharmacognosy in October 2010, each of which was reviewed by at least two anonymous referees. Following the review, 21 papers from different universities of Argentina (1), Brazil (7), Costa Rica (1), Cuba (1), Ireland (1), Italy (7), Mexico (2) and Venezuela (1) were selected for publication in this Special Issue. They are original papers on all aspects of natural products including ethnobotany/ethnopharmacology, isolation and characterization of natural products, biological activities, analytical methods and sensory analysis; several are collaborative works between two or more countries. This Revista Brasileira de Farmacognosia - Brazilian Journal of Pharmacognosy special issue provided an opportunity for publication of original, peer-reviewed, fulllength articles on new research on medicinal plants used in Latin America; this will serve to stimulate the studies in these areas that are extremely important for academia and industry. The Guest Editors would like to thank the contributors who gave so generously of their time and experience and who made this publication a valuable tool for scientists in the field of natural products chemistry and biology. Thanks are also due to the referees for their valuable comments and for the very detailed and accurate review of manuscripts; their comments certainly helped to improve the papers. We are also very grateful to Prof. Cid Santos Editor in Chief of Revista Brasileira de Farmacognosia - Brazilian Journal of Pharmacognosy for embracing this project with interest and enthusiasm, and for the opportunity to publish this Special Issue. I hope that this will be the first of a long series in this attractive and interesting Journal.

SOCIEDADE BRASILEIRA DE FARMACOGNOSIA2011 - 2013

The Brazilian Society of Pharmacognosy thanks Universidade Federal do Paraná by offering support to the Brazilian Journal of Pharmacognosy.

PresidentCid Aimbiré de Moraes SantosUniversidade Federal do Paraná

Vice presidentLaila Salmen Espíndola DarvenneUniversidade de Brasília

SecretaryJackson Roberto Guedes da Silva AlmeidaUniversidade Federal do Vale do São Francisco

TreasurerMaique Weber BiavattiUniversidade Federal de Santa Catarina

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy 21(5): Sep./Oct. 2011

786

Article

ISSN 0102-695Xhttp://dx.doi.org/10.1590/S0102-695X2011005000115

Received 27 Dec 2010Accepted 8 Mar 2011Available online 24 Jun 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 786-792, Sep./Oct. 2011 How a Huottuja (Piaroa) community perceives

genuine and false honey from the Venezuelan Amazon, by free-choice profi le sensory method

Patricia Vit,*,1 Rosires Deliza,2,3 Alfonso Pérez4

Abstract: Pot honey is the most abundant honey in the forest, produced by many species of stingless bees (Meliponini) of the Huottuja (Piaroa) community in Paria Grande, Venezuela. However, the commercialization of this honey is low, and false honeys, which are sold in labelled bottles, are easily found in the market. This study has investigated the ability of an untrained panel of Piaroa assessors to differentiate the genuine from the false pot honey using the Free-choice profile. This sensory method allows consumers to use their own words to describe and to quantify sensory attributes of a product. The genuine honeys, light amber Melipona fuscopilosa “isabitto” and dark amber Tetragona clavipes “ajavitte”, the false light and dark “angelita” honeys, and the amber Apis mellifera honey, were evaluated. Sensory attributes related to the appearance, color, odor, flavor and mouthfeel were elicited in a qualitative session and were quantified in 10-cm unstructured line scales using individual score sheets. The data were analyzed by Generalized Procrustes Analysis (GPA). The bidimensional plot successfully separated genuine from false pot honeys. The first dimension (39.50%) was represented by the low viscosity, fermented odor and sour taste, whereas the second dimension (24.69%) was related to fruity and honey odor and flavor. Huottuja assessors differentiated the five honey types in terms of the perceived sensory characteristics.

Keywords:Apisfree-choice profilehoneyHuottujaMeliponinisensory analysis

Introduction

Honey standards from Brazil (Ministério da Agricultura, 2000) and Venezuela (Covenin, 1984) were produced only for Apis mellifera, following the guidelines of international standards of the Codex Alimentarius Commission (1969, 1987, 2001). However, stingless bees also produce honey. They differ from A. mellifera at the subfamily level known as Meliponini (Michener, 2000). A proposal for quality standards for meliponine honey was presented few years ago (Vit et al., 2004; Souza et al., 2006) and was inserted for the first time in a honey standard by the Colombian regulations (Norma Técnica Colombiana, 2007). This honey is produced primarily by Melipona, Scaptotrigona and Tetragonisca (in America), Meliponula (in Africa), and Tetragonula (in Asia and Australia). There are around sixty genera of stingless bees (Rasmussen & Cameron, 2010), with 391 Neotropical species groups (Camargo & Pedro, 2007), and more than 500 are estimated worldwide.

Huottujas are an ethnic group of Amerindians in Venezuela (Melnyk & Bell, 1996) known as piaroas by the public. The Huottuja community in Paria Grande, located about 30 km to the South of the city Puerto Ayacucho, capital of the Amazonas State, has organized a cooperative of meliponiculture since 2005, and call its honey as “mayá”. Instead of honey hunting, they keep and multiply feral colonies in rational hives. Both local horizontal layout, and vertical hives that facilitate colony division, after the Brazilian experience (Villas Bôas, 2008) are used (Figure 1). Different stingless bee species exploit resources according to their honey, pollen or propolis yields (Kerr, 1987). Native people know that and use stingless bee products accordingly. After harvesting the honey, the cerumen pots are used to handcraft ceremonial Huottuja masks, and souvenirs. Honey was previously pressed but now is extracted with a syringe. Sensory attributes of honey stored in pots by Meliponini have been investigated by several authors in the past (Schwarz 1948; Gonnet et al., 1964). Kidder

1Departamento Ciencia de Los Alimentos, Facultad de Farmacia y Bioanálisis, Universidad de Los Andes, Venezuela, 2Embrapa Agroindústria de Alimentos, Brazil, 4Asociación Cooperativa de Meliponicultores Warime, Venezuela.

How a Huottuja (Piaroa) community perceives genuine and false honey from the Venezuelan Amazon, by free-choice profile sensory method

Patricia Vit et al.

Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 21(5): Sep./Oct. 2011 787

& Fletcher (1857) suggested that the sour honey of Brazilian stingless bees “will compensate for sweet lemons”. However, no study was carried out using any population from the Venezuelan Amazon to describe pot honey sensory attributes. This work aimed at investigating the genuine and false pot honeys from the Venezuelan Amazon as well as the comb honey by using the sensory Free-choice profiling (FCP) method by local people named Huottuja.

Materials and Methods

Honey

Two artificial honey samples (light and dark) were purchased at the local market in Puerto Ayacucho, Venezuela, named as “angelita”, which is the common name given to Tetragonisca angustula honey. Salesmen informed that the light honey came from yellow flowers and the dark honey from purple flowers. Both honeys were bottled in a recycled glass container from a Colombian aguardiente distilled drink, and had a label. These honeys had the thick syrup visual appearance of artificial honeys. One of them had an ant inside, pretending to be a stingless bee. The insect inside the bottle is a local practice in false honey, also seen in the Venezuelan Andes. Two genuine meliponine honeys were provided by a local stingless beekeeper. These samples were already harvested and kept refrigerated. Entomological samples of these bees were kindly identified in the past at Universidade de São Paulo in Ribeirão Preto, Brazil. The five honey samples are described in Table 1. The light amber honey was produced by “isabitto” Melipona fuscopilosa and the dark amber was produced by “ajavitte” Tetragona clavipes. The genuine and the false honeys are shown in Figure 2. Additionally, we used a reference honey of Apis

mellifera. The two bottles on the right are genuine M. fuscopilosa (isabitto) and T. clavipes (ajavitte) honeys. On the left side the two labeled angelita (name given to Tetragonisca angustula) false honeys, putatively originated from purple and yellow blooming. In the middle is the A. mellifera honey extracted from combs.

Table 1. Description of honey samples.

No. 3-digit Local name Entomological identification Origin

1 408 Angelita (dark honey)

False honey (not bee made)

Town market Puerto Ayacucho

2 335 Angelita (light honey)

False honey (not bee made)

Town market Puerto Ayacucho

3 120 Abeja Apis mellifera Nature shop

4 510 Isabitto (light honey)

Melipona fuscopilosa

Paria Grande forest

5 252 Ajavitte (dark honey)

Tetragona clavipes

Paria Grande forest

Figure 1. A meliponary from the Paria Grande, Amazonas State, Venezuela.

Figure 2. Stingless bee honeys from the Venezuelan Amazon, in Paria Grande.

How a Huottuja (Piaroa) community perceives genuine and false honey from the Venezuelan Amazon, by free-choice profile sensory methodPatricia Vit et al.

Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 21(5): Sep./Oct. 2011788

Assessors

A group of eight honey consumers, three females and five males, aged between 15 and 45 years old, from Paria Grande, a native Amerindian community took part in this study. They were selected based on their nutritional interest on honey, curiosity, commitment, availability and motivation. None of them had previous experience with sensory analysis, but all were familiar with forest pot honey. Their sense of smell was not altered by smoking, allergies, flues, or insomnia. The sessions took place in the morning, 2-3 h after breakfast. Their participation was voluntary and only rewarded with the pen they used to fill the forms. An informed consent form was filled prior to the sensory sessions.

Sensory evaluation

In the first session, assessors received a briefing of the FCP procedure and were instructed to provide a list of attributes to depict the honeys using their words to describe the appearance (color and visual consistency), odor, flavor (aroma and taste) and other sensations in their mouth and throat. Comparative and hedonic terms were avoided. About 5 g of each one of the five honeys showed in Table 1 were presented following a balanced presentation order, and participants were asked to list the sensory characteristics they perceived after tasting each honey. Honey samples were presented in capped plastic cups coded with three-digit numbers, in a day-light illuminated room. Appearance was evaluated first, then the odor, and finally half spoon of honey was tasted and any other sensation was also recorded. Mineral water was served to rinse the mouth between samples, as well as a piece of the local cassava cracker known as casabe, to reset the palate (Figure 3). For the second session, individual score cards were prepared to measure the intensities of each sensory attribute generated during the first session. The samples were evaluated monadically by using unstructured 10 cm line scales anchored with the words “weak” or “absent” at the left end, and “strong” at the right end. Each assessor crossed the intensity on the line scale in the position that best described his/her perception. Help was provided individually to facilitate the evaluation process without any induction.

Statistical analysis

The data acquired by FCP were analyzed by generalized Procrustes analysis, to generate an optimized consensus matrix by mathematical transformations, to reach a minimal overall deviation which was able to summarize the information about the samples, and replace

the panel mean (Williams & Langron, 1984). Correlations between the sample score of each sensory attribute and the corresponding sample score principal component, allowed the selection of the important attributes.

Figure 3. Presentation of honeys for sensory sessions.

Results

Elicited sensory attributes

The list of preliminary vocabulary elicited in the qualitative session was reduced to the 25 terms presented in Table 2, by the reduction of redundant and/or vague words. The eight assessors elicited on average thirteen sensory attributes (ranging from 10 to 16) for the five honey types produced by different species of Meliponini in different countries. For example fluid, thin, watery, viscous, thick etc. refer to low to high viscosity. The descriptor fermented was used to group alcohol, fermented and fermented pollen. Negative and positive correlations (<-0.7 and >0.7) between each attribute and the first two dimensions are given for each one of the eight assessors. As suggested by Guàrdia et al (2010) only agreed descriptors with correlation coefficients higher than 0.6 in at least one of the first two dimensions were used to visualize the relationships between samples and attributes.The first dimension was better explained by low viscosity (-0.8 to -1.0), fermented odor (0.9 to 1.0) and sour taste (0.8 to 1.0). However, brown color and the resin flavor also contributed to it. The second dimension coefficient’s was more related to the fruity and honey odor, sweet, fresh fruit, fruity sweet and honey flavor. The sensation of biting nose was in the first dimension but burns the throat in the second. The assessors used many common words to describe the five honeys, but some sensory descriptors were unique and very specific, related to local resins named artocha, and fruits which are known as manteco. As previously observed by Ferreira et al. (2009), the vocabulary developed to describe stingless bee honey by FCP, was similar to descriptors of appearance, odor, flavor, and trigeminal sensations used to describe A. mellifera honey by QDA (Vit 1993; Anupama et al. 2003; Persano Oddo & Piro 2004; Galán-Soldevilla et al., 2005).


Patricia Vit et al.


This is a good evidence for the Codex Alimentarius Commission, to show that stingless bees also produce honey, as recognized by untrained panels using simple words to describe pot honey. However, besides the similarities, differences between honeys of each stingless bee species may be somehow comparable to the diversity attributed to botanical origins of the honey produced by only one bee species, the A. mellifera. Melipona species produce light amber honeys, while Trigona tends to produce dark amber honeys, similarly to the characteristic light amber acacia honey and dark amber chestnut honey, widely documented and familiar to beekeepers and consumers from locations where these honeys are produced. Besides that, the fermented descriptors are somehow distinctive in pot honey.

Assessors

Eight assessors elicited an average of 13 attributes (ranging from 10 to 16) of five honey types produced by different species of Meliponini in different countries. The attributes they provided are listed in Table 2. The ability of each assessor seen from the combined attributes of all, is defined in the assessors’ plot by principal components of the consensus configuration. Individual performance could be checked by looking at the graphic of residuals (Figure 4). The graphic of residuals for each assessor by configuration after the transformations along with the consensus plot for the five honey types shows that assessor 5 has the highest residual, which means that he gave rates that do not match the consensus.

Residuals by configuration

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

Configuration

Res

idua

ls

Figure 4. Residual variance for each assessor.

Honeys

Figure 5 shows the consensus configuration of the five honeys. In Figure 5a, the first dimension explained 39.50% of the variability and separated genuine pot honeys (252, 510) from the other samples. The honeys are widely spread in space. Genuine pot honeys M. fuscopilosa

(510) and T. clavipes (252) produced by the Paria Grande Piaroa community are grouped on the right, separated from the genunie A. mellifera (120) and the false honeys (335 and 408) grouped in the left by the first dimension, with conspicuous contributions of a low viscosity, fermented odor and sour flavor. On the other hand, the second dimension explained 24.69% of the variability, and separated sample 510 from the others. The genuine Melipona honey 510 is set apart from the rest in the upper position of the dimension 2, which was explained by fresh fruit, honey, and burns the throat descriptors (Figure 5b). Despite the light and dark color of false honey to imitate genuine pot honey, the artificial dark amber honey 408 is close to the other artifical honey (335) revealing similar sensory characteristics between them. The genuine dark Tetragona honey 252 was positioned separately in dimension 1 showing that it was perceived by participants with specific sensory attributes. The color imitation of angelita honey samples (Figure 2) was surpassed by the other sensory attributes that positioned the honeys in opposite compartments. Indeed, the sour taste and intrinsic flavor of the light false honey angelita was more similar to Apis honey. Figure 6 shows the residuals by object after the transformations. We can see that the dark artificial honey 408 has the smallest residual. This indicates that there is most probably a consensus between assessors when evaluating this syrup honey. Whereas the highest residual is for the light artificial honey 335, which is a more elaborated syrup honey with sharper sour taste than traditional false honeys the author has been observing for more than twenty years in the Venezuelan market. The Melipona honey is characterized by the honey in the nose and the mouth, fresh fruit flavor, honey odor and aroma, and the sensation of burning the throat. The Tetragona honey is more complex, and was described as having brown color, sour and bitter taste, fermented flavor, peanuts, resin, biting nose. The Apis honey was perceived as having the caramel aroma, honey, sweet taste and medicinal aroma. The higher viscosity was attributed to the false honeys, the angelita dark amber had an odorless attribute, while the other angelita light amber was more elaborated medicinal, caramel and sweet. Figure 7 presents the consensus configuration showing the investigated honey sensory attributes. False honeys are closer to the A. mellifera honey, and not distinctive putative yellow and purple blooming was separated from both of them, so only one group of false honey. Pot honeys were spread in distinctive positions by the two first dimensions. The Melipona honey was characterized by sour taste, fresh fruit, honey and burns the throat attributes, while the Tetragona had more descriptors such as brown color, bitter and sour taste, fermented, fruity, resin, biting nose.



Table 2. Attributes better correlated with the first two dimensions (D1, D2), and attributes of honeys.Assessors 1 2 3 4 5 6 7 8

Dimensions D1 D2 D1 D2 D1 D2 D1 D2 D1 D2 D1 D2 D1 D2 D1 D2

ATTRIBUTES

Appearance

1. yellow color -0.8 -0.7

2. orange color

3. brown color 0.7 0.7 1.0 0.7

4. viscosity -0.9 -1.0 -0.9 -0.9 -0.9 -0.9 -0.8 -1.0

Odor

5. fermented 1.0 0.9 0.9 0.9 0.9 0.9 0.9

6. fruity 0.9 0.8 -0.7

7. fruity sweet 0.9

8. caramel

9. peanuts

10. honey 0.9 0.9

11. resin -0.7

12. medicinal

13. odorless

Flavor

14. sweet -1.0 0.8 0.9

15. sour 1.0 0.9 1.0 0.8 1.0 1.0

16. bitter 0.9

Aroma

17. hot

18. candy

19. fresh fruit 0.9 0.9 0.9

20. fruity sweet -0.7 -0.7

21. medicinal

22. resin 0.9 0.9 0.9

23. honey 0.9 0.9 0.9

Trigeminal sensations

24. biting nose 0.9 -0.7 0.9

25. burns the throat 0.9

Objects (axes F1 and F2: 64,19 %)

H o ne y 2 5 2

H o ne y 5 1 0

H o ne y 4 0 8

H o ne y 3 3 5

H o ne y 1 2 0

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-5

0

5

1 0

-1 5 -1 0 -5 0 5 1 0 1 5

F1 (39,50 %)

F2 (2

4,69

%)

Biplot (axes F1 and F2: 64,19 %)

H o ne y 2 5 2

H o ne y 5 1 0

H o ne y 4 0 8

H o ne y 3 3 5

H o ne y 1 2 0

s o ur t hr o a t

bi t ing no s e

ho ne y

r e s inme dic ina l

f r ui t y s we e t

f r e s h f r ui t

c a r a me lho t

bi t t e r

s o urs we e tno s me l l

me dic ina lr e s in

ho ne y

pe a nut s

c a r a me lf r ui t y s we e t

f r ui t y

f e r me nt e d

v is c o s i t y br o wn

o r a nge

ye l lo w

-4 0

-2 0

0

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6 0

-8 0 -6 0 -4 0 -2 0 0 2 0 4 0 6 0 8 0 1 0 0

F1 (39,50 %)

F2 (2

4,69

%)

(a) (b)

Figure 5. The five honeys, defined by the first two dimensions of the consensus configuration, showing (a) Position of honey samples, (b) Sensory attributes. See Table 1 for the identification of honey samples.


Patricia Vit et al.


Residuals by object

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

H o ne y 1 2 0 H o ne y 3 3 5 H o ne y 4 0 8 H o ne y 5 1 0 H o ne y 2 5 2

Object

Res

idua

ls

Figure 7. Distribution of the honey attributes along the two first dimensions.

Discussion

The separation of the bee genera observed in Figure 5 was also obtained by multivariate analysis of physicochemical factors in Venezuelan pot honeys (Vit et al. 1998). However, in this work A. mellifera and artificial honeys were incorporated, and the information derived from the FCP was promising for self control of honey authenticity by the Huottuja community. This experience could be wisely extended in schools, so children would learn how to discriminate a honey that is not genuine, from their traditional forest honeys, that are now produced in rational hives. The better knowledge of their products

could protect them with arguments of valorization. The first dimension was better explained by low viscosity (-0.8 to -1.0), fermented odor (0.9 to 1.0) and sour taste (0.8 to 1.0), also brown color and the resin flavor contributed to it. The second dimension was more related to the fruity and honey odor, sweet, fresh fruit, fruity sweet and honey flavor. The sensation of biting nose was in the first dimension but burns the throat in the second. Compared to the genus of Apis, which has eleven species, stingless bees have some sixty genera, and are the only group of social bees with a Cenozoic fossil record (Rasmussen & Cameron, 2010). Therefore, more variability is expected for the pot honey they produce due to the differences attributed to their entomological origin, additionally to the botanical and geographical origin. Local names of stingless bees in the Huottuja language, such as “isabitto” (Melipona fuscopilosa) and “ajavitte” (Tetragona clavipes) are unfamiliar to Spanish speakers. Melipona species have common names such as “erica" and “guanota, while Tetragonisca angustula is known as “angelita” (Vit et al., 2004). In the honey label, the name of the bee in the tropics could become as important as the name of the botanical source of unifloral honeys in Europe. However, local names of stingless bees in Venezuela have been more used in labels of artificial or false honey, than in the traditional industry of meliponiculture, due to several constraints, - especially the lack of official standards for Meliponini honey, and also the fact that this is a rural practice not organized in cooperatives, which are essential to handle the marketing. A better sensory knowledge of the forest honey harvested by the Huottuja people followed by the available information would aggregate monetary value to the product, and could contribute to the improvement of the community life conditions. Their cultural values would benefit by assessing the environmental and social roles of meliponiculture, but the suggested price-based and quantity-based techniques of valuation for indigenous cultural heritage, need further economical analysis to improve social welfare concepts (Venn & Quiggin, 2007). Pot honey hunting has been reported for bush honey or sugarbag used by Gunwinggu community (Trigona species named “bobitj” and “man.gung”) in Australia (Altman, 1984). In conclusion, this is the first time a Huottuja community has carried out a honey tasting of the traditional pot honey available in their forest and recently harvested from their stingless bee apiaries. The classic approach of Free-choice profile was successfully handled by the local people of the community area, with the honey we needed to test. Huottuja assessors were interested in the contribution they were giving, and their sensory skills were adequate to accomplish the differentiation of the honey types, as confirmed by the routine statistical test. A more knowledgeable consumer improves the protection

Figure 6. Residual variance for each honey.

Dimensions (axes F1 and F2: 64,19 %)

so ur t hr o at

bit ing no se

ho ney

r esin

medicinal

f r uit y sw eet

f r esh f r uit

car amel ho t

bit t er

so ursw eet no smell

medicinalr esin

ho ney

peanut s

car amelf r uit y sw eet

f r uit y

f er ment edvisco sit y

br o w n

o r ange

yello w

- 1

- 0 ,75

- 0 ,5

- 0 ,2 5

0

0 ,2 5

0 ,5

0 ,75

1

- 1 - 0 ,75 - 0 ,5 - 0 ,2 5 0 0 ,2 5 0 ,5 0 ,75 1

F1 (39,50 %)

F2 (2

4,69

%)



of customers against false honey. Sensory approaches like ours confirm the useful value of sensory evaluation to differentiate diverse entomological origins of honey.

Acknowledgement

The authors thank Mr. Fini Opa Carrasquel, who provided the genuine pot honey to carry on sensory analysis. To the late meliponine entomologists JMF Camargo, from the Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil, for the valuable identification of stingless bees. To CDCHT-ULA project FA-458-09-03-B.

References

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Camargo JMF, Pedro SRM 2007. Meliponini Lepeletier, 1836. In: Moure JS, Urban D, Melo GAR (org.). Catalogue of bees (Hymenoptera, Apoidea) in the Neotropical region. Curitiba: Sociedade Brasileira de Entomologia, p. 272-578.

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Norma Técnica Colombiana 2007. Miel de Abejas. NTC 1273. Instituto Colombiano de Normas Técnicas y Certificación, Bogotá, http://www.sinab.unal.edu.co/ntc/NTC 1273.pdf, accessed in June 2010.

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Villas-Bôas JK 2008. Meliponicultura e povos indígenas no Brasil. In: Vit P (ed). Abejas sin aguijón y valorización sensorial de su miel. Mérida, Venezuela: APIBA- Facultad de Farmacia y Bioanálisis, DIGECEX, Universidade de Los Andes, p. 31-33.

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*Correspondence

Patricia VitDepartamento Ciencia de Los Alimentos, Facultad de Farmacia y Bioanálisis, Universidad de Los Andes, Mérida 5101, [email protected] +58 274 2403565Fax: +58 274 2403475

793

Article

ISSN 0102-695Xhttp://dx.doi.org/10.1590/S0102-

695X2011005000084

Received 23 Dec 2010Accepted 8 Mar 2011

Available online 20 May 2011


21(5): 793-806, Sep./Oct. 2011Ethnopharmacological versus random plant selection methods for the evaluation of the antimycobacterial activity

Danilo R. Oliveira,*,1 Gilda G. Leitão,1 Tatiane S. Coelho,2 Pedro E. Almeida da Silva,2 Maria Cristina S. Lourenço,3 ARQMO,4 Suzana G. Leitão5

1Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco H, Brazil,2Laboratório de Micobactérias e Biologia Molecular, Faculdade de Medicina, Fundação Universidade Federal do Rio Grande, Brazil,3Instituto de Pesquisa Clínica Evandro Chagas, Serviço de Bacteriologia, Setor de Testagem de Drogas, Fundação Oswaldo Cruz, Brazil,4Associação de Comunidades Remanescentes de Quilombos do Município de Oriximiná, Brazil,5Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco A 2o andar, Brazil.

Abstract: The municipality of Oriximiná, Brazil, has 33 quilombola communities in remote areas, endowed with wide experience in the use of medicinal plants. An ethnobotanical survey was carried out in five of these communities. A free-listing method directed for the survey of species locally indicated against Tuberculosis and lung problems was also applied. Data were analyzed by quantitative techniques: saliency index and major use agreement. Thirty four informants related 254 ethnospecies. Among these, 43 were surveyed for possible antimycobacterial activity. As a result of those informations, ten species obtained from the ethnodirected approach (ETHNO) and eighteen species obtained from the random approach (RANDOM) were assayed against Mycobacterium tuberculosis by the microdilution method, using resazurin as an indicator of cell viability. The best results for antimycobacterial activity were obtained of some plants selected by the ethnopharmacological approach (50% ETHNO x 16,7% RANDOM). These results can be even more significant if we consider that the therapeutic success obtained among the quilombola practice is complex, being the use of some plants acting as fortifying agents, depurative, vomitory, purgative and bitter remedy, especially to infectious diseases, of great importance to the communities in the curing or recovering of health as a whole.

Keywords:bioprospecting

quilombola communities traditional knowledge ethnopharmacological

random approach

Introduction

Tuberculosis (TB) is the main cause of death by infectious illnesses in the world. It is estimated that approximately 1/3 of the world population may be infected with the TB bacillus, Mycobacterium tuberculosis (Singh, 2004; Maher & Raviglione, 2005). According to data of the Brazilian Ministry of Health (MS/SVS, 2005), Brazil has an average incidence rate of 43,78 cases of TB per 100,000 inhabitants; and in the Pará state the average is of 50,15 cases per 100,000 inhabitants. In 2005, the municipality of Oriximiná presented an alarming number of TB cases, 95,98 cases per 100,000 inhabitants (MS/UFPA, 2009). This heterogeneous geospatial distribution of TB cases in different regions of the country keeps a close relationship to their socioeconomic conditions. In the Amazon region,

major part of the population suffers from chronic problems of malnutrition, precarious habitation and sanitation. In addition, the forest people who live in remote areas, far from civilization, have great diffi culty in accessing primary health care (Basta et al., 2004). Another factor that has favored the higher incidence of TB in the North is the fact that there live about 60% of the indigenous population and that they have suffered real epidemics of the disease with an incidence 10 times higher in relation to TB average of the Brazilian population (Coimbra Jr. & Basta, 2007). The progression of TB is mainly governed by the integrity of the host immune response, which may be effective in the microbial control shown by the action of macrophages, CD4 lymphocytes, interferon gamma and tumor necrosis factor. But in many cases, the bacillus cannot be destroyed and can remain in dormant state

Ethnopharmacological versus random plant selection methods for the evalua-tion of the antimycobacterial activityDanilo R. Oliveira et al.


for years. Between 90 to 95% of those infected people fail to express the active form of the disease. However, others manifest the disease, usually in a condition of immunosuppression (Ducati et al., 2006; Barker, 2008). The inflammatory and pathologic processes produce symptoms typical of TB such as fever, weakness, weight loss, night sweats, chest pain, dyspnea, and cough. The pathology in advanced stages can also lead to the rupture of blood vessels causing hemoptysis (Baker, 2008). TB has been considered a neglected disease, with little investment in research to develop new drugs. This is changing recently, according to the gravity and danger that TB represents, with its high rate of morbidity and mortality and the unavailability of new drugs to control and cure patients with resistant strains to multiple drugs (MDR) and extensively resistant to multiple drugs (XDR) (Rivers & Mancera, 2008). Medicinal plants have attracted great interest as a source for finding new efficient drugs or prototypes to TB treatment, and the literature is covered with many examples of plant selection methods based on random or ethnopharmacological approaches (Cantrell et al., 1998; Newton et al., 2002; Leitão et al., 2006; Gautam et al., 2007; McGaw et al., 2008; Ramos et al., 2008; Mohamad et al., 2011). The vast Amazonian biodiversity together with the traditional knowledge of the forest people could represent a potential source for the discovery of new therapeutic agents, including against TB. The city of Oriximiná, Pará State, Brazil, has 33 quilombola communities (Oliveira et al., 2010). These communities are ethnic groups with historical background, specific territorial relations, and presumption of black ancestry related to resistance to oppression suffered historically. In the late 18th and early 19th centuries the slaves that were used on cocoa, coffee, and cotton plantations as a labor force, fled to remote areas, especially to regions of lakes and waterfalls that were difficult to access. Many of these communities are still in full contact with the natural biodiversity of regions far from the urban area of Oriximiná. Their close contact with nature over centuries, the knowledge formed from an Indian-Black-Portuguese complex, and their geographic isolation, have brought to the members of these communities a vast knowledge of medicinal plants (Oliveira et al., 2011a). In December 2007, the Federal University of Rio de Janeiro obtained the first approval in Brazil to access the traditional knowledge for bioprospecting purposes in quilombola communities from Oriximiná (Oliveira et al., 2010). This work reports part of the vast knowledge of the quilombola people from Oriximiná (Oliveira, 2009) and aimed at the bioprospection of the medicinal plants used by them for TB-related diseases by ethnopharcological approach and a comparison between random plant selection methods for the evaluation of the

antimycobacterial activity.

Material and Methods

Characterization of the search area

The municipality of Oriximiná is located in northern Brazil, in the State of Pará, and has an area of 107,603 km2, being the second largest municipality in the Brazilian territory. It borders Suriname, Guyana, and French Guiana to the north, the cities of Faro, Juruti, and Óbidos to the South and East, and the States of Amazonas and Roraima to the West. According to data from the 2010 census, Oriximiná has 62,963inhabitants, being 40,182 in urban areas and 22,781 in rural areas (IBGE, 2010). Currently, there are 33 known “quilombola” communities in the municipality of Oriximiná, which are divided into eight territories (Água Fria, Boa Vista, Trombetas, Erepecuru, Alto Trombetas, Jamari/Último Quilombo, Moura, and Ariramba) that, together, encompass more than 600,000 hectares (Figure 1). The “quilombolas” were represented by their association called Associação de Comunidades Remanescentes de Quilombos do Município de Oriximiná – ARQMO (Association of the Remaining of “Quilombo” Communities from the Municipality of Oriximiná). In this work, five communities, representing two “quilombola” areas were chosen: Bacabal and Arancuã-de-Cima from the Trombetas region, as well as Serrinha, Jauari, and Pancada, from the Erepecuru region (Figure 1).

Ethnopharmacological data collection

This work has received authorization for access to the traditional knowledge associated with bioprospecting purposes by the Directing Council of Genetic Heritage (Conselho de Gestão do Patrimônio Genético), through the Resolution no. 213 (6.12.2007), published in the Federal Official Gazette of Brazil on 27 December 2007 (Oliveira et al., 2010). The selection of the interviewees began with the search for key informants who were respected people in the community, such as the community coordinator, matriarch or patriarch, and/or community health agent. Eventually, they led to the local specialists, who were “quilombolas” with wide experience in the use of medicinal plants, such as extractivists, woodsmen, healers, faith healers, prayer ladies, midwives, and “puxadores” or “puxadoras”, who are like traditional chiropractors. For data acquisition four ethnobotanical field trips were performed between the period of June 2006 (after signing the Prior Informed Consent between UFRJ and ARQMO), until the month of September 2008. Each field work had a residence period of 30 to 60 days in the communities studied. Ethnobotanical data was collected through

Ethnopharmacological versus random plant selection methods for the evalua-tion of the antimycobacterial activity

Danilo R. Oliveira et al.


Free-List and Salience Index (S)

The free-list technique can identify items within an emic category or a cultural domain, and it offers a direct method to obtain data easily and simply. It is also used as an exploratory technique (Oliveira et al., 2011a,b). Using this technique, a direct list of the medicinal plants known and used by the informers was obtained, with the aim of searching for specific information on this cultural domain within the communities and its diffusion. Additionally, an ethnodirected inquiry regarding the plants used against TB and lung problems was conducted employing a glossary with the emic illnesses or symptoms like cough, dry cough, “whooping cough” (“tosse braba”), pneumonia, “weakness”, tuberculosis, lung problem or illness, and infectious diseases of chest These therapeutic terms used locally were obtained in a previous study carried out in Oriximiná city (Oliveira, 2004) and through participant observation and ethnographic techniques. Taken together, these approaches suggest the most important cultural elements and the order of their importance (Albuquerque & Lucena, 2004). The S was calculated using the program ANTHROPAC 4.0 (Analytic Technologies, USA).

semi-structured interviews, participating observation and walk in the woods (Albuquerque & Lucena 2004). The formularies applied contain socio-economic data (sex, age, professional, level of schooling, monthly family income, number of residents) and the medicinal plants information (common name, therapeutic indications, doses, preparation methods, counterindications and the local to obtain it). Thirty five quilombolas from the five communities studied, twenty women and fifteen men, were interviewed. Participants included four women and two men from Arancuã-do-meio, nine women and two men from Bacabal, three women and five men from Jauari, one women and four men from Pancada and three women and two men from Serrinha. They were between 19 and 87 years old. They survive mainly by fishing, hunting, and subsistence farming, and their only source of income is the extraction of the Pará nut (“Brazil nut”), which is available for only a few months of the year. Quantitative data analysis techniques, such as the free-list salience index (S), and corrected major use agreement (MUAc), were also applied.

Figure 1. Map representing the “quilombola” area and the studied communities of Oriximiná, Pará State, Brazil.



Major Use Agreement (MUA):

The Major Use Agreement (MUA) is a quantitative technique used to evaluate the agreement between the main uses cited by various informants (Oliveira et al., 2006). It is determined by the ratio between the number of informers who independently cited the species for a major use (MU) and the total number of informers who mentioned that species for any use (total uses, TUs).

MU TUs

A correction factor (CF) was applied to calculate the corrected MUA (MUAc), given by the formula: MUAc = MUA x CF. The correction factor is the ratio between the number of informers citing each species for any use (TUs) and the highest number of informers citing the most-cited species. In this study, Dipteryx odorata was the most-cited species and was cited by 24 informers (TUs+) (Oliveira et al., 2011a).

TUs TUs +

Collection and identification of plant material

Plants were collected by one of the authors (DRO). with informants and woodsmen of the “quilombola” communities. Specimens were deposited at the Herbarium of the Amazonia National Institute of Research (INPA), where the parataxonomist Mr. José Ferreira Ramos, and specialists in specific botanical families, such as Dr. Fátima Salimena (Verbenaceae), Dr. Washington Marcondes-Ferreira (Apocynaceae), and Dr. Kátia Calago (Asteraceae), provided botanical identifications. Voucher numbers of plants collected by ethnopharmacological approach are listed in Table 1. Voucher number for plants collected by random approach: Acrocomia aculeata (Jacq.) Lodd. ex Mart. (INPA 224624), Ampelozizyphus amazonicus Ducke (INPA 224161), Anacardium giganteum W.Hanc.exEngl. (INPA 224700), Aspidosperma excelsum Benth. (INPA 224704), Aspidosperma rigidum Rusby (INPA 224692), Bauhinia platycalyx Benth. (INPA 223277), Brosimum potabile Ducke (INPA 224693), Brosimum sp. (without voucher number), Couepia paraensis (Mart.&Zucc.) Benth. (INPA 224627), Cucurbita moschata (Duch.) Duch.e xPoir. (INPA 223273), Endopleura uchi (Huber) Cuatrec. (INPA 224690), Machaerium ferox Mart. (INPA 233440), Operculina alata (Ham.) Urb. (INPA 223281), Simaba cedron (Rol) Ward (INPA 223283), Spondias mombin L. (INPA 224699), Trichilia quadrijuga Kunth (INPA 224620), Uncaria guianensis (Aubl.) Wild. (INPA 224608), Virola surinamensis (Rol) Ward. (INPA 224143).

Selection the plant species and the preparation of the extracts

The ETHNO plants (ten species) were selected from the free-listing for TB; and related diseases and the RANDOM pants (eighteen species) were selected by the other important medicinal uses in the communities, but not used for lung disturbances. Plant materials used for the preparation of extracts (Table 2) were air-dried, powdered, and macerated with ethanol to make ethanolic extracts. Aqueous extracts were obtained by decoction prepared at 1% (w/v), approaching the traditional method of use. Saps and latex were obtained directly from incisions or perforations of the plants. Teas, saps, and latex were freeze-dried.

Isolates and strain preparation of Mycobacterium tuberculosis

Mycobacterium tuberculosis H37Rv (ATCC-27294) pan-susceptible strain was used for all experiments in both microbiology laboratories (FURG and Fiocruz). Additional assays with a rifampicin-resistant strain (ATCC 35338, His-526-Tir) were performed at the FURG laboratory. The isolates were maintained in Ogawa-Kudoh medium for ca. 14 days. The bacterial suspensions were prepared in sterile water containing 3-mm glass beads. The suspensions were homogenized by vortex agitation and turbidity was adjusted in agreement with tube one of the McFarland scale (3.2 x 106 colony-forming units/mL). The inoculums were prepared by diluting the bacterial suspension 1:20 in Middlebrook 7H9 OADC medium (4.7 g Middlebrook 7H9 base; Difco, Becton Dickinson USA) enriched with 10% (v/v) oleic acid-dextrose-albumin-catalase (BBL).

In vitro evaluation of antimycobacterial activity

Plant extracts selected by ethnodirected method (ETHNO) obtained with the free-list and by random method (RANDOM) were screened against M. tuberculosis H37Rv strain by microdilution method using resazurin as indicator of cell viability. Initially, a fixed concentration of 200 μg/mL was used. Media plus bacteria, with and without rifampicin, were used as positive and negative controls, respectively. For the active samples and the positive control, the minimum inhibitory concentration (MIC) was determined.

Data analyses

The Chi-square Test and Fisher Exact Test were used to analyze differences between the positive and negative results obtained by the ETHNO and RANDOM,

MUA = x 100

CF =




employing the statistical package SigmaStat 3.0 (Jandel Scientific, USA).

Results and Discussion

Thirty four individuals were interviewed, who reported 254 ethnospecies and a total of 2,508 use indications. Among these, 233 plant species were identified, belonging to 211 genera and 72 botanical families (Oliveira et al., 2011a). Another survey was conducted to assess the plants used by the “quilombolas” to treat TB and TB-related diseases and symptoms. In the communities, there isn’t a clear perception and clinical diagnosis of TB. In this way, an ethnodirected method of free-list focusing on cough, dry cough, “whooping cough” (“tosse braba”), pneumonia, “weakness”, tuberculosis, lung problem or illness, and infectious diseases of chest was employed, which led to the survey of 43 ethnospecies, displayed in Table 1, listed in order of their Salience (S). They are distributed into 29 botanical families, being Fabaceae the most well represented family (18.3%), principally for subfamily Caesalpinioideae.

Out of these 43 ethnospecies, only seven were cited by more than 20% of the interviewees (Frequency - FR≥20%) for TB-related diseases; seven displayed a Salience value (S) above 0.1; and eight had a MUAc value higher than 25%. So, at least five ethnospecies - represented by seven identified plant species have potential for TB bioprospecting by ETHNO. They are: Dipteryx odorata (S=0,541; MUAc=79,2%; Fr=64,5%), Chenopodium ambrosioides (S=0,24; MUAc=45,8%; Fr=45,2%), Hymenaea courbaril, H. intermedia, H. oblongifolia (S=0,179; MUAc=41,7%; FR=29%), Plectranthus amboinicus (S=0,163; MUAc=37,5%; Fr=25,8%) and Cereus sp (S=0,12; MUAc=41,7%; Fr=22,6%). The symptoms of TB appear confused with other bronchial and respiratory diseases as well as malnutrition and weakness. Moreover, older people in these communities refer to TB as the "weakening". The body weakness as a factor for the development of TB, in the view of traditional communities, is reported by other authors (Maués, 1990; Santos & Muaze, 2002), and is extremely well discussed globally that TB is a disease that manifests itself often in cases of disability or low immune

Table 1. Ethnospecies listed in order of the Salience values for diseases and symptoms related to TB and the respective MUA and MUAc.

Species Ethnospecies Voucher number

Diseases and symptoms related to TB* S TI MUA

(%)MUAc

(%) FR (%)

Dipteryx odorata (Aubl.) Willd. Fabaceae-Faboideae

cumaru INPA 224607 pneumonia (19), cough (11), TB (2)

0.541 24 79.2 79.2 64.5

Chenopodium ambrosioides L. Chenopodiaceae

Mastruz INPA 224606 cough (11), pneumonia (7), TB (6)

0.24 20 55.0 45.8 45.2

Hymenaea courbaril L., Hymenaea intermedia Ducke, Hymenaea oblongifolia Huber Fabaceae-Caesalpinioideae

jutaí/jatobá INPA 224658, INPA 223296, INPA 223297

cough (10), hoarseness (2), TB (3), pneumonia

(1)

0.179 17 58.8 41.7 29

Himatanthus sucuuba (Spruce ex Müll. Arg.) Woodson Apocynaceae

sucuuba INPA 224151 cough (6), pneumonia (2), illness of chest (2), TB (2)

0.174 16 37.5 25.0 22.6

Plectranthus amboinicus (Lour.) Spreng. Lamiaceae

hortelã-grande

INPA 224639 cough (9), pneumonia (3) 0.163 13 69.2 37.5 25.8

Jatropha curcas L. Euphorbiaceae

peão-branco INPA 224670 pneumonia (4), TB (1), cough (2)

0.123 23 17.4 16.7 22.6

Cereus sp. Cactaceae Jaramacaru INPA 224611 cough (10), TB/weakness (1), illness of chest (1)

0.12 11 90.9 41.7 22.6

Anacardium occidentale L. Anacardiaceae

cajueiro INPA 224167 pneumonia (2), cough (1) 0.085 9 22.2 8.3 9.7

Eryngium foetidum L. Apiaceae chicória INPA 223295 cough (4) 0.082 12 33.3 16.7 16.1

Ruta graveolens L.Rutaceae arruda INPA 224600 pneumonia (3), cough (2), illness of chest (1)

0.081 19 15.8 12.5 9.7

Parahancornia amapa Ducke Moraceae

amapá-amargo

INPA 224693 cough (3), TB (3) 0.075 7 42.9 12.5 16.1

Mangifera indica Wall. Anacardiaceae

mangueira INPA 224636 cough (5), pneumonia (1) 0.073 8 62.5 20.8 9.7

Ocimum americanum L. Lamiaceae

esturaque INPA 224614 cough (6), pneumonia (1) 0.063 10 60.0 25.0 16.1

Bertholletia excelsa Kunth. Lecythidaceae

castanheira/castanha-do-

pará

INPA 224171 pneumonia (2), cough (1) 0.057 16 12.5 8.3 9.7



Portulaca pilosa L. Portulacacea

amor-crescido INPA 223286 pneumonia (2), TB (1) 0.053 14 14.3 8.3 6.5

Bryophyllum calycinum Salisb. Crassulaceae

diabinho INPA 224141 TB (3), cough (3) 0.05 9 33.3 12.5 9.7

Luffa operculata Cogn. Curcubitaceae

cabacinha INPA 224139 pneumonia (3), cough (2) 0.036 9 33.3 12.5 9.7

Orthopappus angustifolius (Sw.) Gleason Asteraceae

língua-de-vaca

INPA 223284 TB (1), cough (1) 0.036 3 33.3 4.2 6.5

Carapa guianensis Aubl. Meliaceae

andiroba INPA 223282 cough (7), chest pain (1), pneumonia (1)

0.036 16 43.8 29.2 6.5

Allium sativum L.Liliaceae alho Without Voucher Number

cough (5) 0.032 16 31.3 20.8 9.7

Justicia pectoralis Jacq. Acanthaceae

trevo-cumaru INPA 224671 pneumonia (2) 0.032 4 50.0 8.3 3.2

Piper cf. dactylostigmum Yunck. Piperaceae

pau-de-angola INPA 224137 pneumonia (1) 0.032 2 50.0 4.2 3.2

Dalbergia riedelii (Benth.)Sandw. Fabaceae- Faboideae

verônica INPA 224158 pneumonia (1) 0.032 6 16.7 4.2 3.2

Pouteria sp. Sapotaceae cramuri INPA 224628 TB (1) 0.032 7 14.3 4.2 3.2

Physalis angulata L. Solanaceae

gamapú INPA 224149 pneumonia (1) 0.032 2 50.0 4.2 3.2

Sesamum indicum L. Pedaliaceae

gergelim INPA 224675 illness of chest (1) 0.032 11 9.1 4.2 3.2

Zingiber officinale Roscoe Zingiberaceae

mangarataia INPA 224604 cough (4) 0.028 10 40.0 16.7 3.2

Cedrela odorata L. Meliaceae cedro INPA 223380 pneumonia (1) 0.027 5 20.0 4.2 6.5

Eupatorium triplinerve Vahl. Asteraceae

japana INPA 224678 cough (3) 0.026 10 30.0 12.5 6.5

Citrus limon (L.) Burm. f. Rutaceae

limão INPA 224613 cough (5) 0.024 21 23.8 20.8 3.2

Inga bourgoni (Aubl.) DC. Fabaceae-Mimosoideae

ingá-xixica INPA 223279 cough (1) 0.024 5 20.0 4.2 3.2

Campsiandra comosa Benth. Fabaceae-Caesalpinioideae

manaiara INPA 223288 illness of chest (1) 0.022 11 9.1 4.2 3.2

Aniba canelilla (Kunth) Mez. Lauraceae

preciosa INPA 224705 pneumonia (1) 0.022 8 12.5 4.2 6.5

Copaifera sp. Fabaceae-Caesalpinioideae

copaíba Without Voucher Number

cough (3) 0.016 13 23.1 12.5 3.2

Caesalpinia ferrea Mart. Fabaceae-Caesalpinioideae

jucá INPA 224643 catarrh (1), cough (1) 0.016 7 14.3 4.2 3.2

Allium cepa L. Liliaceae cebola Without Voucher Number

cough (2) 0.012 3 66.7 8.3 3.2

Davilla kunthii A. St.-Hil. Dilleniaceae

cipó-d’água INPA 224163 whooping cough (1) 0.011 3 33.3 4.2 3.2

Leucas martinicensis R.Br. Lamiaceae

catinga-de-mulata

INPA 233361 TB (1), cough (1), pneumonia (1)

0.008 8 12.5 4.2 3.2

Psidium guajava L. Myrtaceae goiabeira INPA 224145 cough (1) 0.008 6 16.7 4.2 3.2

Lippia origanoides Kunth Verbenaceae

salva-de-marajó

CESJ 39532 lung (1) 0.007 10 10.0 4.2 3.2

Cinnamomum zeylanicum Blume Lauraceae

canela INPA 224156 TB (1) 0.006 7 14.3 4.2 3.2

Polypodium decumanum Willd. Polypodiaceae

guaribinha INPA 223280 whooping cough (4) 0.005 4 100.0 16.7 3.2

Cissus sicyoides L. Vitaceae cipó-pucá INPA 223271 pneumonia (1) 0.004 4 25.0 4.2 3.2S=Salience Index; TI= total of the interviewed that cited the ethnospecies; MUA= Major Use Agreement; MUAc= Corrected MUA; *In the column “diseases and symptoms related to TB” the symptom used to calculate the Major Use Agreement and Corrected MUA is highlighted in bold, while between parentheses is the number of times that the plant was cited for this indication.




system, especially in the case of individuals with Acquired Immune Deficiency Syndrome (AIDS) and those who suffer from chronic malnutrition. Therefore, many times, the indicated remedies are the same one to treat respiratory problems in general and to treat anemia and to fortify the body. Beyond the medicinal plants, a curious question is the use of the “queixada” tooth for treatment of pneumonia or TB in the quilombola communities of Oriximiná. This use has also been reported also in Amazonian communities of the valley of the rivers Purus and Acre (Santos & Muaze, 2002). “Queixada” (Tayassu pecari Link.) it is an Amazonian wild boar frequently used in the feeding of the communities studied. They can be very aggressive when confronted. When angry, it snaps its teeth in order to warn the intruder that it is about to attack. The use of teeth in the treatment of "weakening" probably gives the strength and stiffness of the teeth of the species, as well as the dry and shrill sound emitted by queixada when it is angry or threatened. The tooth should be dried (or toasted) and ground to then be used alone or in combination with some of the plants listed in Table 1. As a result of the plant use information for the lung disturbances and related diseases, ten species were tested by ethnodirected method (ETHNO) and eighteen by random method (RANDOM) for antimycobacterial activity. In the screening eight species gave positive results

with MIC varying between 12,5-200 µg/mL, being five selected by ETHNO, and three by RANDOM (Table 2). The best result was obtained by ETHNO, because 50% of the species tested had some activity, while by RANDOM only 16.7% of species had some activity (Table 2). Most of the natural products from plant sources do not display strikingly potent activities (Newton et al., 2000), and for most of them, published MIC ranges of substances considered to show modest activity lay between 50 to 216 µg/ml (Okunade et al., 2004). Among all positive samples Dipteryx odorata (ETHNO) and Aspidosperma spp. (RANDOM) showed MIC below 25 μg/mL, which can be considered a good activity for ethanolic crude extracts when compared with the antimycobacterial literature for medicinal plants. Five plant extracts (Dipteryx odorata, Campsiandra comosa, Machaerium ferox, Aspidosperma excelsum and A. rigidium) were active against both strains of M. tuberculosis indicating the absence of a cross-resistance to RMP when the molecular basis of resistance is rpoB His-526-Tir mutation (Table 2). Importantly, this mutation is one of the most common in RMP resistan strains. In this study the target and possible antimicrobial mechanism of action these extracts was not identified, although it is possible that the antimicrobial activity is due to a single molecule or could also be the result of a synergic action among various compounds.

Table 2. Antimycobacterial activity and Minimum Inhibitory Concentration (MIC) of plant samples selected by ETHNO (E) and RANDOM (R) methods against Mycobacterium tuberculosis.

Species Selection method Plant part Extract

H37Rv strain 35338 strain

200 µg/mL MIC (µg/mL) 200 µg/mL MIC (µg/mL)

Cumaru Dipteryx odorata (Aubl.) Willd. E

bark ethanol (+) 12.5 (+) 12.5

seed water (-) ----- (-) -----

seed oil (-) ----- (-) -----

Jutaí Hymenea courbaril L. E bark ethanol (-) ----- (-) -----

Jutaí Pororoca Hymenea intermedia Ducke E

bark ethanol (-) ----- (-) -----

sap - (-) ----- (-) -----

Mangueira Mangifera indica Wall. E stalk

ethanol (+) > 200 (+) >200

water (-) ----- (-) -----

Amapá-amargo Parahancornia amapa Ducke E bark ethanol (-) ----- (-) -----

Manaiara Campsiandra comosa (Benth.) Cowan

Eseed ethanol (+) 200 (+) 100

bark ethanol (-) ----- (-) -----

Amor-crescido Portulaca pilosa L. E leaves ethanol (-) ----- (-) -----

Andiroba Carapa guianensis Aubl. E seed oil (+) > 200 (-) -----

Castanheira Bertholletia excelsa Kunth.

E

bark ethanol (-) ----- (-) -----

collumela ethanol (-) ----- (-) -----

fruit bark ethanol (-) ----- (-) -----

bark juice (-) ----- (-) -----



Amapá-doce Brosimum potabile Ducke R bark latex (-) ----- (-) -----

Saratudo Machaerium ferox Mart. R

bark ethanol (+) 200 (+) 100sap - (-) ----- (-) -----

Cajuaçú Anacardium giganteum W.Hanc.ex Engl.

R barkethanol (-) ----- (-) -----water (-) ----- (-) -----

Mururé Brosimum sp. R bark latex (-) ----- (-) -----

Ucuuba Virola surinamensis (Rol) Ward R bark latex (-) ----- (-) -----

Saracuramirá Ampelozizyphus amazonicus Ducke

R bark water (-) ----- (-) -----

Pau-paratudo Simaba cedron (Rol) Ward R bark water (-) ----- (-) -----

Escada-de-jabuti Bauhinia platycalyx Benth. R

bark ethanol (-) ----- (-) -----root ethanol (-) ----- (-) -----

Jamaru Cucurbita moschata (Duch.) Duch.e xPoir.

R leaves ethanol (-) ----- (-) -----

Uxirana Couepia paraensis (Mart.&Zucc.) Benth.

R bark ethanol (-) ----- (-) -----

Mucajá Acrocomia aculeata (Jacq.) Lodd. ex Mart.

R bark ethanol (-) ----- (-) -----

Taperebá Spondias mombin L. R bark ethanol (-) ----- (-) -----

Uxi-Liso Endopleura uchi (Huber) Cuatrec. R bark water (-) ----- (-) -----

Batatão Operculina alata (Ham.) Urb. R potate water (-) ----- (-) -----

Unha-de-gato Uncaria guianensis (Aubl.) Wild. R bark water (-) ----- (-) -----

Grajió Trichilia quadrijuga Kunth R bark water (-) ----- (-) -----

Carapanaúba Aspidosperma excelsum Benth. R Bark

ethanol (+) 50 (+) 25water (-) ----- (-) -----

Carapanaúba Aspidosperma rigidum Rusby R bark

ethanol (+) >200 (+) 25(-) ----- (-) -----

Rifampicin Positive control (+) 0.03 (+) 8g/mL(+) = active/sensible; (-) = not active/resistant

The ethnopharmacological data show the importance of Dipteryx odorata (cumarú = tonka been) in the treatment of pulmonary illnesses, by the highest salience index (S=0.541), corrected major use agreement (MUAc=79.2%) and frequency of citation (RF=64.5%). These data are consistent with the good antimycobacterial activity obtained for the crude ethanol extract from their barks (MIC 12.5 µg/mL). However, the seeds did not show positive results at the concentrations tested. From the barks of Dipteryx odorata were isolated the triterpene lupeol, a mixture of fatty acid methyl esthers,

the flavonoid luteolin, plus several isoflavonoids (Nakano & Suárez, 1969; Hayashi & Thompson, 1974; Nakano et al., 1979; Nakano & Yoshimura, 1979; Socorro et al., 2003). The MIC of 12.5 μg/mL obtained for cumarú barks, against both strains of M. tuberculosis, may be related to the presence of diterpenes, triterpenes, fatty acids and isoflavones, since these natural product classes have several representatives with known antimycobacterial activity described in the literature (Cantrell et al., 2001; Copp, 2003; Okunade et al., 2004; Carballeira, 2008). Considering that the cumarú positive sample was a crude




ethanolic extract, this result becomes very promising for the identification of bioactive substances. Aspidosperma rigidum and Aspidosperma excelsum, known as carapanaúba, are rich in indole alkaloids (Vieira et al. 2010) and antimycobacterial activity has already been recently demonstrated for several substances of this class (Copp, 2003; Okunade et al., 2004), as well as for other species of the Apocynaceae family (Case et al., 2006; Gautam et al., 2007; Ramos et al., 2008; Mohamad et al., 2011). Some species such as Allium cepa, Anacardium occidentale, Cynnamomum zeylanicum, Jatropha curcas, Psidium guajava and Zingiber officinale in Malaysian (Mohamad et al., 2011), besides Chenopodium ambrosioides, Ruta graveolens, Ocimum americanum, Allium sativum and Mangifera indica in Índia (Gautam et al., 2007) are also indicated as a possible source of new antimycobacterial agents, and in South Africa, Ruta graveolens has also highlighted its potential (McGaw et al., 2008). It is expected that the traditional knowledge about medicinal plants indicates the presence of biologically active substances. The collection of plants for biological testing from its traditional use can be a great advantage, or a shortcut, increasing the chances of discovering new drugs (Elisabetsky & Shanley, 1994; Soejarto, 1996). Table 3 shows the enormous potential of the ethnopharmacological approach found in several studies, compared with the randomized.

Table 3. Comparison between Ethnopharmacological and Random approaches in the search for different biological activities.

Biological Activities RANDOM (%)

ETHNO (%) References

Antineoplastic 6 25 Elisabetsky & Shanley, 1994

Antihypertensive 31 44 Adsersen & Adsersen, 1997

Antihelmintic 9.8 29.3 Bourdy et al., 2008

Icthyotoxicity 9.6 38.6 Bourdy et al., 2008

Toxic/venoms 10.5 52.2 Bourdy et al., 2008

Anti-HIV 8.5 71.4 Slish et al., 1999

Antimicrobial 22 37 Boily & van Puyvelde, 1986

Antiplasmodial 0.7 18 Carvalho & Krettli, 1991

Acetylcholinesterase inhibition 8 42.3 Oliveira et al.,

2011a In this study, the best results for antimycobacterial activity were also obtained with the ethnopharmacological approach - 50% ETHNO x 16,7% RANDOM (Table 2), however, because of the limited number of the samples, it was not found significant difference (p>0,05) between these approaches by the

use of the Chi square and Fisher exact tests. Dipteryx odorata is the species with the highest salience index and showed a good antimycobaterial activity (MIC 12.5 µg/mL). This result points out a preference towards the ethnopharmacological information. In recent work published by our group (Oliveira et al., 2011a), the ethnodirected approach using the salience index and the major use agreement also improved the probability of finding activity species for acetylcholinesterase inhibition. In contrast, the study of Case et al. (2006) that used the informant consensus model (informant agreement ratio) to select plants used in traditional medicine for persistent respiratory symptoms among the Manus (Papua New Guinea), was inaccurate in predicting antimycobacterial activity plants. However, these authors related that “Due to the complexity of the human body, it cannot be assumed that in vitro bioassay results translate to human systems, for either a positive or negative result. Consequently, the species identified in the survey may be beneficial in the treatment of TB for many reasons apart from direct antimycobacterial activity, e.g., they may provide symptomatic relief from cough or have immunostimulatory effects. It must also be noted that the in vitro test may not be predictive of activity in vivo. With infinite resources, samples could be submitted to other relevant assays such as immune-modulating assays to better understand and assess the biological activity of traditional medicine” (Case et al., 2006). Oliveira et al. (2011a,b) discuss that some plants could have an adaptogen effect that contribute to quilombola health by an unspecific way. Some plants have the role of panacea, being used to cure all ills. In the “quilombola” communities of Oriximiná TB is often called the “weakening”. So, the ETHNO's good results can be even more significant considering that the therapeutic practices among the “quilombolas” from Oriximiná are complex. The use of fortifying agents, depuratives, vomitory agents, purgatives, bitter remedies, as well as curing infectious diseases, weakness, and memory loss, play an important role in the processes of curing diseases and/or health recovery, acting as order to restore overall health (Oliveira et al., 2011a,b). Similar data were surveyed by Rodrigues & Carlini (2004; 2006) in the Sesmaria quilombola community in the State of Mato-Grosso, in a transition area between Cerrado and Pantanal biomes, where certain species are characteristic for their versatility, or nonspecific therapy, is also employed for rejuvenation, to energize, to muscle building and to fortify the brain. Another study at the Pará State also showed a substantial number of general “cure alls” or panaceas, fortifiers, tonics, nerve tonics and aphrodisiacs, as a reflex of the caboclo culture (Branch & Silva, 1983; Berg, 1984; Amoroso & Gély, 1988). A broad literature review about the 43 cited ethnospecies, used against TB and TB-related diseases and



symptoms, revealed that 93% of them have been described as useful for the treatment of respiratory diseases and 86%

were indicated as tonic and stimulant, corroborating with the ETHNO information (Table 4).

Table 4. Literature data review about the 43 ethnospecies employed in “quilombola” communities to treat TB and TB-related diseases and their publicated indications for use as a tonic/stimulant/fotifier, or to treat TB and TB-related diseases.

Ethnospecies Tonic/fortifier/stimulant TB and TB-related diseases

Alho Allium sativum Almeida, 1993; PDR, 2007 Cruz, 1982; Martins, 1989; Vieira, 1992; Almeida, 1993; Ming, et al. 1997; Maciel & Cardoso, 2003; Oliveira, 2004; Maciel & Guarim-Neto, 2006; PDR, 2007; Pinto & Barbosa, 2009; Mosca & Loiola, 2009.

Amapá Parahancornia amapa

Figueiredo, 1979; Branch & Silva, 1983; Berg & Silva, 1986; Amorozo & Gèly, 1988; Rodrigues, 1989; Schultes & Raffauf, 1990; Vieira, 1992; MEB, 1993; Berg, 1993; Tenório et al., 1991; Revilla, 2002; Pinto & Barbosa, 2009.

Figueiredo, 1979; Berg & Silva, 1988; Rodrigues, 1989; Martins, 1989; MEB, 1993; Tenório et al., 1991; Vieira, 1992; Revilla, 2002; Oliveira, 2004; Rodrigues, 2006; Pinto & Barbosa, 2009.

Amor Crescido Portulaca pilosa

Corrêa, 1926; Cid, 1978; Ducke & Martinez, 1994;

Andiroba Carapa guianensis Cid, 1978; Corrêa, 1926; Matta, 2003; Rodrigues, 2006 Branch & Silva, 1983; MEB, 1993; Berg & Silva, 1986; Berg, 1993; Revilla, 2002; Rodrigues, 2006; Pinto & Barbosa, 2009

Arruda Ruta graveolens Corrêa, 1926; Cruz, 1982; Branch & Silva, 1983; Martins, 1989; Oliveira et al. 2011a

Teixeira et al., 1991; MEB, 1993; Di Stasi & Hiruma-Lima, 2002; Oliveira, 2004

Batatão Operculina alata Branch & Silva, 1983; Cruz, 1982; Matos, 2007 Matos, 2007

Cabacinha Luffa operculata ------------------------------ Tenório et al., 1991; Gupta, 1995; Revilla, 2002; De La Cruz, 2008; Mosca & Loiola, 2009

Cajueiro Anacardium occidentale

Corrêa, 1926; Cruz, 1982; Rodrigues, 1989; Vieira, 1992; Almeida, 1993; Gupta, 1995; Revilla, 2002; Matta, 2003; Matos et al., 2004

Corrêa, 1926; Cruz, 1982; Gupta, 1995; Revilla, 2002; Matta, 2003; Ramos et al, 2005

Canela Cinammomum zeylanicum

Corrêa, 1926; Cid, 1978; Figueiredo, 1979; Martins, 1989; Amorozo & Gèly, 1988; Tenório et al., 1991; Vieira, 1992; Schardong & Cervi, 2000; Maciel & Cardoso, 2003; Oliveira et al. 2011a

Cid, 1978; Tenório et al., 1991; Vieira, 1992; Souza et al., 2004; PDR, 2007

Castanheira Bertholletia excelsa

Oliveira, 2004; Pinto & Barbosa, 2009; Oliveira et al. 2011a

Ming, 1997; Oliveira, 2004

Catinga-de-mulata Leucas martinicensis

Corrêa, 1926; Cid, 1978; Tenório et al., 1991; Vieira, 1992; Di Stasi & Hiruma-Lima, 2002; Lorenzi & Matos, 2002

Di Stasi & Hiruma-Lima, 2002; Revilla, 2002; Pinto & Barbosa, 2009

Cebola Allium cepa Vieira, 1992; PDR, 2007 Corrêa, 1926; Vieira, 1992; Almeida, 1993; PDR, 2007; Monteles & Pinheiro, 2007; Pasa et al., 2008

Cedro Cedrela odorata Cid, 1978; Vieira, 1982; Matta, 2003 Branch & Silva, 1983; Amorozo & Gèly, 1988; MEB 1993; Revilla, 2002;

Chicória Eryngium foetidum Corrêa, 1926; Lorenzi & Matos, 2002; Rodrigues, 2006 Amorozo & Gèly, 1988; Vieira, 1992; Berg, 1993; Ming, 1997; Revilla, 2002; Rodrigues, 2006

Cipó-pucá Cissus sicyoides Branch & Silva, 1983; Ducke & Martinez, 1994; Revilla, 2002

Almeida, 1993; MEB 1993; Ducke & Martinez, 1994; Gupta, 1995; Revilla, 2002

Copaíba Copaifera sp. Corrêa, 1926; Branch & Silva, 1983; Revilla, 2002; Matta, 2003; PDR, 2007;

Corrêa, 1926; Cid, 1978; Berg & Silva, 1986; Tenório et al., 1991; Vieira, 1992; MEB, 1993; Revilla, 2002; Lorenzi & Matos, 2002; Oliveira, 2004; PDR, 2007; De La Cruz, 2008

Cumaru Dipteryx odorata Cruz, 1982; Rodrigues, 1989; Vieira, 1992; Ducke & Martinez, 1994; Oliveira et al. 2011a

Figueiredo, 1979; Branch & Silva, 1983; Vieira, 1992; Ducke & Martinez, 1994; Di Stasi & Hiruma-Lima, 2002; Matta, 2003; Oliveira, 2004; PDR, 2007; Pinto & Barbosa, 2009

Diabinho Bryophyllum calycinum

Rodrigues, 2006 Corrêa, 1926; Almeida, 1993; Ming, et al., 1997; Rodrigues, 2006; Berg, 1993; Vieira, 1992; Lorenzi & Matos, 2002; Oliveira, 2004; Albuquerque et al., 2007; De La Cruz, 2008; Pinto & Barbosa, 2009

Esturaque ------------------------------ Nadembega et al., 2011

Gergelim Sesamum indicum Cid, 1978; Cruz, 1982; Almeida, 1993; Martins, 1989; Revilla, 2002;

Amorozo & Gèly, 1988; MEB, 1993; Di Stasi & Hiruma-Lima, 2002




Goiaba Psidium guajava Gupta, 1995 Gupta, 1995; Pasa et al., 2008

Guaribinha Polypodium decumanum

------------------------------ Figueiredo, 1979; Branch & Silva, 1983; Amorozo & Gèly, 1988; Schultes & Raffauf, 1990; Berg, 1993; Ming, 1997; Lorenzi & Matos, 2002; Oliveira, 2004

Hortelã-grande Plectranthus amboinicus

Tenório et al., 1991; Revilla, 2002; Vieira, 1992; Berg, 1993; Longuefosse & Nossin, 1996; Ming, 1997; Lorenzi & Matos, 2002; Oliveira, 2004; IEPA, 2005; Ramos et al, 2005; Albuquerque et al., 2007; Monteles & Pinheiro, 2007; Mosca & Loiola, 2009

Japana Eupatorium triplinerve

Cid, 1978; Corrêa, 1926; Rodrigues, 1989; Di Stasi & Hiruma-Lima, 2002; Matta, 2003

Figueiredo, 1979; Vieira, 1992; Berg, 1993; Longuefosse & Nossin, 1996; Pinto & Barbosa, 2009

Jaramacaru Cereus sp. Cruz, 1982; Rodrigues, 1989. Corrêa, 1926; Cid, 1978; Figueiredo, 1979; Branch & Silva, 1983; Berg & Silva, 1986; Amorozo & Gèly, 1988; Rodrigues, 1989; Martins, 1989; Vieira, 1992; Berg, 1993; Lorenzi & Matos, 2002; Revilla, 2002; Agra et al., 2007; Pinto & Barbosa, 2009

Jucá Caesalpinia ferrea Branch & Silva, 1983; De La Cruz, 2008; Pinto & Barbosa, 2009

Cid, 1978; Figueiredo, 1979; Cruz, 1982; Amorozo & Gèly, 1988; Tenório et al., 1991; Vieira, 1992; Almeida, 1993; Di Stasi & Hiruma-Lima, 2002; Revilla, 2002; Matta, 2003; Oliveira, 2004; Ramos et al, 2005; Albuquerque et al., 2007

Jutaí/Jatobá Hymenaea spp. Corrêa, 1926; Figueiredo, 1979; Cruz, 1982; Branch & Silva, 1983; Berg & Silva, 1986; Rodrigues, 1989; Tenório et al., 1991; Vieira, 1992; Berg, 1993; Almeida, 1993; Lorenzi & Matos, 2002; Revilla, 2002; Di Stasi & Hiruma-Lima, 2002; Oliveira, 2004; Rodrigues, 2006; Matos, 2007; De La Cruz, 2008; Pinto & Barbosa, 2009; Oliveira et al. 2011a;

Corrêa, 1926; Cid, 1978; Cruz, 1982; Branch & Silva, 1983; Berg, 1984; Berg & Silva, 1986; Berg & Silva, 1988; Amorozo & Gèly, 1988; Vieira, 1992; Berg, 1993; Ducke & Martinez, 1994; Gupta, 1995; Ming, 1997; Lorenzi & Matos, 2002; Revilla, 2002; Di Stasi & Hiruma-Lima, 2002; Matta, 2003; Oliveira, 2004; Ramos et al, 2005; Franco & Barros, 2006; Maciel & Guarim-Neto, 2006; Rodrigues, 2006; Matos, 2007; Albuquerque et al., 2007; De La Cruz, 2008; Pasa et al., 2008; Pinto & Barbosa, 2009

Limão Citrus limon Corrêa, 1926; Cruz, 1982; Branch & Silva, 1983; Martins, 1989; Teixeira et al., 1991; Rodrigues, 2006; PDR, 2007

Cruz, 1982; Branch & Silva, 1983; Teixeira et al., 1991; Vieira, 1992; Berg, 1993; Schardong & Cervi, 2000; Oliveira, 2004; Monteles & Pinheiro, 2007; PDR, 2007; Pasa et al., 2008; Pinto & Barbosa, 2009

Língua-de-vaca Orthopappus angustifolius

Cruz, 1982 Amorozo & Gèly, 1988; Martins, 1989; Cruz, 1982; Vieira, 1992; Oliveira, 2004

Manaiara Campsiandra comosa

Corrêa, 1926; Oliveira et al. 2011a -----------------------------------------------

Mangarataia Zingiber officinale

Corrêa, 1926; Cid, 1978; Cruz, 1982; Branch & Silva, 1983; Martins, 1989; Teixeira et al., 1991; Di Stasi & Hiruma-Lima, 2002; Rodrigues, 2006; Oliveira et al. 2011a

Corrêa, 1926; Figueiredo, 1979; Martins, 1989; Teixeira et al., 1991; Vieira, 1992; Ducke & Martinez, 1994; Schardong & Cervi, 2000; Revilla, 2002; Di Stasi & Hiruma-Lima, 2002; Oliveira, 2004; De La Cruz, 2008; Pinto & Barbosa, 2009

Mangueira Mangifera indica Branch & Silva, 1983 Cruz, 1982; Branch & Silva, 1983; Guarim-Neto, 1987; Amorozo & Gèly, 1988; Vieira, 1992; MEB, 1993; Berg, 1993; Longuefosse & Nossin, 1996; Schardong & Cervi, 2000; Oliveira, 2004; Rodrigues, 2006; Maciel & Guarim-Neto, 2006; Monteles & Pinheiro, 2007; Pasa et al., 2008; Mosca & Loiola, 2009; Nadembega et al., 2011

Mastruz Chenopodium ambrosioides

Branch & Silva, 1983; Berg & Silva, 1988; Tenório et al., 1991; Berg, 1993; Matta, 2003; Rodrigues, 2006; Matos, 2007; Oliveira et al. 2011a

Branch & Silva, 1983; Rodrigues, 1989; Tenório et al., 1991; Vieira, 1992; Almeida, 1993; MEB, 1993; Berg, 1993; Ducke & Martinez, 1994; Lorenzi & Matos, 2002; Revilla, 2002; Maciel & Cardoso, 2003; Matos et al., 2004; Ramos et al, 2005; Franco & Barros, 2006; Rodrigues, 2006; Matos, 2007; Albuquerque et al., 2007; De La Cruz, 2008; Pinto & Barbosa, 2009; Mosca & Loiola, 2009



Acknowledgements

This work was supported by CNPq and FAPERJ. Authors wish to thanks Lucia Andrade from Comissão Pró-Índio de São Paulo for providing original files of the map of the quilombola region, upon which a new one was built by Paula S. de O. Barbosa. Carlos Bêta and Mira Carvalho, directors of Unidade Avançada José Veríssimo, of the Universidade Federal Fluminense, located in Oriximiná, contributed with infrastructure used for this project. We are especially thankful to the quilombolas who provided housing for the researchers involved in this ethnoknowledge study.

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*Correspondence

Danilo R. OliveiraNúcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de JaneiroCentro de Ciências da Saúde, Bloco H, Ilha do Fundão, 21941-590 Rio de Janeiro-RJ, [email protected].: +55 21 2562 6413

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Article


695X2011005000126

Received 7 Dec 2010Accepted 22 Apr 2011

Available online 8 Jul 2011


21(5): 807-813, Sep./Oct. 2011Cranberry in children: prevention of recurrent urinary tract infections and review of the literature

Angelica Dessì,*,1 Alessandra Atzei,2 Vassilios Fanos1

1Department of Paediatrics, Neonatal Intensive Care Unit, Neonatal Pathology, Puericultura Institute and Neonatal Section, University of Cagliari, Italy,2Department of Paediatrics, Neonatal Intensive Care Unit, Neonatal Pathology, Puericultura Institute and Neonatal Section, Azienda Ospedaliero Universitaria, University of Cagliari, Italy.

Abstract: Urinary tract infections (UTI) are common in childhood. In 30-50% of children with UTI the infections occur recurrently, especially in those with vesicoureteral reflux (VUR), neurogenic bladder (NB), previous cystitis or pyelonephritis and malformative uropathies. To reduce the likelihood of UTI, antibiotic prophylaxis has been regarded as the therapeutic standard for many years. However, the disadvantage of long-term antibiotic therapy is the potential for development of collateral effects and resistant organisms in the host. Such reasons have induced scientists to search for alternative modalities of UTI prevention and have contributed to determining the increasing desire for “naturalness” of the population and preventing excessive medication. The use of cranberry fulfils these needs by potentially replacing or enhancing traditional procedures. The purpose of this study was to assess the effectiveness of cranberry in preventing UTI in pediatric populations. We searched Pubmed, the Cochrane Central Register of Controlled Trials and Internet. Cranberry in patients with previous UTI was evaluated in three studies, cranberry in patients with VUR in three studies and four studies analyzed the efficacy of cranberry in children with NB. In seven of nine studies cranberry had a significant effect in preventing UTI.

Keywords:children

cranberryneurogenic bladder

pediatricsUTI

VUR

Introduction

Urinary tract infections (UTI) are the most frequent bacterial infections in pediatrics after infections of the respiratory tract: at the age of seven, 8% of females and 2% of males present with at least one episode of UTI. Only in the first three to six months is the incidence in males higher, after which the pathology appears more often in females. Children who have had a UTI run a 10 to 30% risk of recurrence and in 15% of cases may develop high blood pressure (Foxman et al., 2000). A study carried out in all of Denmark showed that in one thousand patients in pediatric age followed for one year by an area physician there were nineteen episodes of UTI (Kwock et al., 2006). A 2006 study (Wald, 2006) showed that UTI represents the most frequent and serious bacterial infection in the United States since some bacterial infections (S. pneumoniae and H. influenzae) have been weakened through vaccinations. From the aetiological standpoint the most common bacterium in discussion is E. coli (almost 80% of cases) and more rarely other enterobacteria,

especially Proteus spp. (mostly in males), Klebsiella spp., Enterococcus spp., Citrobacter spp., Providencia spp., Morganella spp., Serratia spp. and Salmonella spp., which, similarly to the strains of Pseudomonas spp. with low virulence, may be involved in complicated infections. In the first months of life, UTI, especially relapsing (infections caused by the same germ) and recurrent (UTI caused by different microorganisms), represent an important clinical element for further investigations and diagnoses of possible urinary tract malformations. Indeed, in children there are factors that favor relapses and/or recurrences of UTI such as malformative uropathies, especially obstructive; vesicoureteral reflux (VUR), previous repeated episodes of cystitis and/or pyelonephritis (three or more episodes per year) even without urinary tract anomalies and the frequently catheterized neurogenic bladder (NB) (Mangiarotti et al., 2000). Although the literature supplies no univocal indications, in the pediatric age it is common practice to use antibiotic prophylaxis in the treatment of relapsing and recurrent UTI, especially in patients with

Cranberry in children: prevention of recurrent urinary tract infections and review of the literatureAngelica Dessì et al.


VUR (Fanos & Cataldi, 2004). However, the many side effects of antibiotics, the increasing number of antibiotic-resistant bacteria and the economic impact of the therapy have promoted research on alternative measures to prevent UTI. An important contribution to this research has come from the ever-increasing desire for "natural remedies" among the population as concerns the excessive (or perceived as such) use of drugs in special periods of life, such as pregnancy and infancy (Fanos et al., 2006a). The use of cranberry (Vaccinium macrocarpon Ainton, Ericaceae) juice represents one of the possible solutions that may potentially go hand in hand with traditional procedures or, in some cases, even replace them.

What do we know about the cranberry?

Historical notes

The interest in cranberry juice goes back to the times of the American colonies. The traditional use of the cranberry is associated with Thanksgiving Day starting from 1961 when the pilgrims served it with turkey and lobster (Kemper, 1999). The common name cranberry comes from the colonial name of crane berry, which later became contracted. The withered flower resembles the neck and head of the sandhill crane, which the colonists often saw feeding on this fruit. It was appreciated by the North American natives owing to its beneficial effects both in the diet (as a sweetener or in the drying of meat and fish) and as a remedy in disinfecting wounds, reducing pain caused by indigestion and in oral and dental hygiene. The Penobscot tribe in Maine used the cranberry for treating kidney stones and other urinary problems (Duthie et al., 2005). In East Europe it was used as an antipyretic and anticancer remedy and, being rich in vitamin C, it was loaded on ships to prevent scurvy. Used as a folk remedy for UTI before the introduction of antibiotics, it continues to be widely used for this purpose (Kemper, 1999); in 1997 it was one of the ten most sold herbal remedies in the United States (Reid & Bruce, 2003). The part used for this purpose is the mature berry, which is extremely bitter so that many preparations are sweetened to improve their taste.

Main components and how it acts

The main components of the cranberry are flavonoids (among which the anthocyanins), catechins, triterpenoids, organic acids (butyric, malic, glucuronic, citric and quinic), vitamins (A and C), carbohydrates (especially fructose), mineral salts and tannins such as the proanthocyanidins (PAC) (characteristic of type

A). This plant has recently been monographed in the ESCOP (ESCOP, 2009) where we find a liquid preparation in compliance with the United States Pharmacopoeia with no less than 2.4% of dextrose, 0.7% of fructose, 0.9% of quinic acid, 0.9% of citric acid and 0.7% of malic acid. The recommended preparation is the concentrated juice extract titrated in proanthocyanidin of type A at 1.2 to 1.4% (or polyphenols at 15%). For a long time the antiseptic and antibacterial action of the cranberry was attributed to its capacity to acidify the urine owing to its vitamin C content and its capacity to metabolize benzoic acid into hippuric acid with antibacterial properties. More recent pharmacological studies have also revealed that cranberry juice is capable of inhibiting the adhesion of type 1 (activity attributed to fructose) and type P (activity attributed to the proanthocyanidins) fimbriated E. coli to the epithelial cells of the bladder wall (Zafriri et al.,1989; Ahuja et al., 1998; Howell et al., 2010). In fact, the pathogenicity of these bacteria is connected to the presence of fimbriae at the extremities of which there are proteinic structures called adhesins which make it possible for the pathogens to adhere to the membrane of uroepthelial cells. The type A proanthocyanidins appear to be those mainly responsible for the antiseptic and antibacterial action. In a study by Howell & Foxman. (2002) the antiadhesive activity of the cranberry against type B uropathogen fimbriated E. coli was studied in vitro by examining PAC A, which has been shown to inhibit adhesion (Howell & Foxman, 2002). PAC, by acting as receptor analogues and adhering to the extremities of the fimbriae, competitively inhibit the adhesion of E. coli, thus causing an elongated deformation of the bacterium cell bodies, a reduction in the length and density of the P-fimbriae and finally an inhibition of their synthesis with the total disappearance of the adhesins. The antiadhesive activities of the cranberry help to prevent UTI also indirectly by selecting at the intestinal level less adhesive uropathogenic bacteria. A study in 2005 showed in subjects who regularly drank cranberry juice an increase in urinary salicylicates, thus suggesting a possible local anti-inflammatory effect and a further probable mechanism of action (Duthie et al., 2005). The research demonstrated that besides the urinary antiseptic action, the cranberry possesses an antioxidant and radical scavenger (being rich in polyphenols) activity and an antibacterial action against many germs (recently it has been demonstrated as acting against H. pylori and S. mutans) (Gotteland et al., 2008; Koo et al., 2010).

Clinical studies in the adult There are numerous studies in the literature concerning the use of the cranberry in the adult which provide data that confirm the therapeutic action of this plant in the prevention of UTI.

Cranberry in children: prevention of recurrent urinary tract infections and review of the literature

Angelica Dessì et al.


A double-blind randomized placebo-controlled cross-over trial on twenty healthy volunteers showed a significant dose-dependent reduction of bacterial adhesiveness following the administration of cranberry (Di Martino, 2006). The Cochrane group has recently demonstrated a significant reduction in UTI in women following the regular assumption of cranberry juice (Jepson & Craig, 2008). The study included consultation of databases, the involvement of companies producing products based on the cranberry and the search for lists of bibliographic references of systematic revisions and important studies. All controlled and randomized studies on cranberry-based products used in the prevention of UTI were examined and studies on patients of both sexes and different ages were included. The authors concluded that there is definite evidence that cranberry juice can be effective in reducing the number of symptomatic urinary tract infections in females after twelve months of treatment. However, certainties are lacking as concerns its pharmaceutical preparation and the most appropriate dosages, and finally there are no clinical studies in support of the fact that the cranberry is just as effective in the pediatric age. The most recent study has demonstrated that besides E. coli, the cranberry can significantly reduce the adherence of other common pathogens of the urinary tract, including P. mirabilis, S. aureus, K. pneumoniae, Enterobacter and P. aeruginosa (Magariños et al., 2008).

Dosage and side effects

The daily dose recommended in ESCOP Monographs (ESCOP, 2009) is in adults 300 to 750 mLl/day of a liquid preparation containing 25 to 100% juice in two to three administrations or 200 to 500 mg/day of dry extract or concentrated juice in two daily administrations. As concerns children between the ages of two and eighteen, 15 mL/kg of juice or equivalent doses are recommended. No limits on use in the pediatric age are reported (some studies included breastfeeding infants a few months after birth). The cranberry is normally well tolerated. In a study by Kontiokari et al. (2005) in 341 children cranberry juice appeared to have beneficial effects on urinary health and this was not compromised by other unexpected antimicrobic effects (Kontiokari et al., 2005). A significant increase in the urinary concentration of oxalates (controversial finding) was reported and this suggests caution in patients predisposed for the formation of urinary oxalate stones (Gettman et al., 2005). Against this, the cranberry has a protective effect on the formation of urate stones. A single case of autoimmune thrombocytopenic purpura

was reported in the case of an elderly patient (Davies et al., 2001) and a probable interaction between cranberry and warfarin in anticoagulant therapy (Suvarna et al., 2003) has also been described. The product obviously cannot be administered to patients allergic to cranberry. As concerns the use of cranberry in pregnancy, in Italy this was expressly prohibited by a decree law in 2002 (Official Gazette no.167 of 18 July 2002), concerning the use of products containing biflavonoids. It is to be pointed out that this is an isolated provision, but one that has recently been confirmed by the Supplement to the ESCOP Monographs 2009 (ESCOP, 2009) which states that in pregnancy the administration must be under the control of a physician whenever the dose greatly exceeds the amount found in foodstuffs. The recommendation is extended to the period of breastfeeding. On this subject, the data in a study by Wing et al. (2008) in California on 188 women below the sixteenth week of pregnancy demonstrated that there may be a protective effect in the assumption of cranberry against asymptomatic bacteriuria and UTI in pregnancy (Wing et al., 2008). As concerns costs, these were analyzed in a controlled clinical study that evaluated the effects of different cranberry-based preparations on UTI in women. One hundred and fifty sexually active women between the ages of 21 and 72 were enrolled; they assumed per os 150 mg of dry cranberry extract or 250 mL of cranberry juice or a placebo for one year. The assessment was made on the basis of the number of cystitis episodes and the consumption of antibiotics during the year of the study. At the end of the study it was found that both the dry extract and the juice reduced the number of infections by 20 and 18% respectively. The yearly cost of the therapy was 624 USD for the dry extract and 1400 USD for the juice. The consumption of antibiotics was significantly lower in the two verum groups compared to the placebo group. The study indicates that both the dry extract and the juice of cranberry are effective in UTI, although the dry extract is preferable from the economic standpoint (Stothers, 2002).

The cranberry in the child

Materials and methods

Studies on the use of cranberry in the pediatric age are few; however, the literature shows an ever-increasing interest in its use in this delicate age. This is supported by results obtained in adults, by the documented scarcity of side effects, by the ease in administration and the fair and verified compliance by the patients treated. The purpose of this research was to evaluate the efficacy of cranberry juice in the prevention of UTI



in pediatrics through a review of the entire literature. We searched through Pubmed, Embase, the Cochrane Central Register of Controlled Trials (Central Cochrane Library) and Internet and examined the proceedings of international congresses and the review bibliography. Information was collected on methods, researchers, patients, operations, drugs administered, side effects, compliance with therapy and results. The study, considering the few works now available, included all written publications which were analyzed by type of study (retrospective study, randomised controlled trials etc.) (Table 1).


Recent studies affirm that the use of antibiotic

prophylaxis in children with relapses of UTI is not only ineffective in preventing infection and long-term kidney damage, but is also associated with important side effects. A very recent meta-analysis involving five studies revealed no or little benefit with prophylaxis in the prevention of UTI recurrence (Mattoo, 2010). Also to be considered is the result of a study by Smolkin et al. (2007): one out of two couples does not perform the long-term (e.g. one year) antibiotic prophylaxis prescribed by specialists and 50% of parents cannot explain why they perform antibiotic prophylaxis even though this has been explained to them (Smolkin et al., 2007). It is also of interest to note that in a Unites States study, about 30% of parents administered cranberry to their children to prevent UTI, but only 25% of them spoke to their physician about this (Super et al., 2005).

Table 1. Summary of clinical trials on cranberry products for UTI prophylaxis in children.

Study Year of study Patients Mean age

(months) Type of study Intervention Outcome

Bircan et al. 201050 (16M-34F) with NB

(32 overactive detrusor/18 hypocompliant bladder)

84.9±51.4 Retrospective study

Cranberry 1 capsule/day for 25.94±24.12

months

A significant reduction in UTI (4.36/year)

in overall group

Goj et al. 2010 63 (24M-39F) with VUR 20 Retrospective study

Cranberry concentrate 0.5 mL/kg daily; 24

month trial

A significant reduction in UTI (4.7%) in patients with VUR,

including high grade VUR and associated urinary tract

malformations

Maringhini et al. 2010 79 (23M-56F) with

previous UTI 62.4 Retrospective study

Cranberry concentrate 0.5 mL/kg daily in

two doses for a mean of 6.4 months

UTI average rate during treatment was 0.03/month

compared with 0.4/month in the preceding period

Ferrara et al. 2009 84F with previous UTI 90 Randomized

controlled trial

Cranberry juice (C.J.) 50 mL daily vs

Lactobacillus (L.) GG drink on five days a

month/vs controls; six month trial

A significant reduction in UTI: 18.5% for C.J. vs 42.3%

for L.GG vs 48.1% for controls

Nishizaki et al. 2009 31 (18M-13F) with VUR

32.5±19.6 (C.J.)

18.2±22.9 (Cefaclor)

Randomized controlled trial

Cranberry juice (C.J.) 100 mL daily vs

Cefaclor 5-10 mg/kg/day; for 17.2±7.90

(C.J.) and 10.2±3.29 (Cefaclor) months

No significant difference in the risk of having recurrent UTI between the cranberry

and cefaclor groups (p> 0.05)

Fanos et al. 2006b 20 (4M-16F) with previous UTI 85.2±52.3 Retrospective

studyCranberry 1 capsule/

day for 2.8±10 months 0.36% UTI/patient/year

Erculiani et al. 2004 47 (25M-22F) with NB and/

or VURBetween

24 and 168Retrospective

studyCranberry 2 capsule/day for 11.2 months

Cranberry appears to have beneficial effects in patients

with low grade VUR

Schlager et al. 1999 15 children with NB Between

24 and 216

Double-blind placebo-

controlled cross-over

study

300 mL cranberry concentrate vs

placebo, each for three months

No benefit in preventing UTI or bacteriuria

Foda et al. 1995 40 children with NB (21 finished) -

Randomized single-blind cross-over

study

Cranberry cocktail 15 mL/kg/day vs water, each for six months

No benefit in preventing UTI or bacteriuria




Congress of the International Pediatric Nephrology Association. They deal with the cranberry treatment of three types of different patients: with VUR (Goj et al., 2010), NB (Bircan et al., 2010) and previous UTI (Maringhini et al., 2010). All three of these works report significantly positive results with the use of cranberry in UTI prevention. In the site of the United States National Health Institute (ClinicalTrials.gov) we found four studies (three completed and one recruiting) concerning cranberry in children, but no results have been published to date (Uhari, 2009; Godbolt, 2010; Anderson, 2006; Wing, 2006). Some of these authors are studying the interaction between the antibiotic and the cranberry in acute UTI. In all works examined, the cranberry was well tolerated and there was good compliance both by patients and parents. We must indicate the critical points in this review: the scarcity of data, the heterogeneity of case histories as concerns the population studied, the types of products, their dosage and the duration of treatment.

Conclusions

Recurrent and relapsing UTI are quite common. Their frequent finding in given age groups, such as infancy, indicate the need to find a therapy that is the least invasive possible and one without side effects. The data in pediatric literature, although few and not definitive, provide encouraging results. The cranberry may be a possible valid alternative to traditional antibiotic prophylaxis, which today is under strong attack. Together with the good tolerability of cranberry there is also good patient and parent compliance as concerns the use of this natural element with proven special characteristics. The potential uses of the cranberry are in cases of: a) documented relapsing and recurrent cystitis once organic causes (e.g. VUR) have been excluded, b) breastfeeding children of three to six months with grade 1 and 2 reflux, normal ultrasonography and with careful, informed and cooperating parents, c) association with the antibiotic in the treatment of acute UTI (in the near future). The results of the studies analyzed may make it possible for pediatricians to take the cranberry into consideration in cases of this kind since its use does not lead to a risk of antimicrobial resistance and to date no side effects have been associated with it in pediatric patients.In any case, further studies are propitious so as to confirm the efficacy and tolerability in the pediatric age.

References

Ahuja S, Kaack B, Roberts J 1998. Loss of fimbrial adhesion with the addition of Vaccinum macrocarpon to the

They explained this by saying that since the product was natural it was by definition without side effects and also because they thought physicians knew very little about the subject. Recent literature offers works with promising results on the use of cranberry in the prevention of UTI in the pediatric age; despite the difficulty of performing a comparative evaluation of the different studies owing to the heterogeneity of the populations, the use of different preparations containing cranberry (concentrated juice, juice, cocktails, capsules and so on) at different dosages, but in any case the data appear encouraging. Nine studies were included in this review. Some studies were not included in the meta-analyses due to methodological issues or lack of available data. Cranberry in patients with previous UTI was evaluated in three studies, cranberry in patients with VUR in three studies and four studies analyzed the efficacy of cranberry in children with NB (one study assessed both VUR and NB). While Foda (Foda et al., 1995) and Schalager (Schalager et al., 1999) found no effect on the reduction of bacteriuria in children affected by NB treated with cranberry, a subsequent study by Erculiani et al. (2004) on a group of 47 children with the same pathology found favorable results (Erculiani et al., 2004) as in all other works written to date (Table 1). In a preliminary study by one of the authors (Fanos et al., 2006b), twenty children were placed in prophylaxis with a special extract of cranberry having a high content of phenols with vitamin C for the purpose of evaluating the possible use of cranberry in the prevention of UTI in children. During the prophylaxis only three episodes of UTI were found in different patients, for which the cranberry therapy was suspended. In the remaining seventeen patients the treatment was well tolerated and no side effects were reported. Various studies have compared the use of cranberry juice in the prophylaxis of UTI with the administration of a placebo, Lactobacillus GG and antibiotic prophylaxis. In a recent work by Ferrara et al. (2009) 84 girls aged from 3 to 14 years with UTI were studied: they were divided into three groups which received three different treatments: G1 (cranberry juice 50 mL/day), G2 (Lactobacillus GG 100 mL/5 days per month) and G3 (controls). The first group presented five episodes of UTI (18.5%) against 11 (42.3%) and 18 (48.1%) episodes in groups G2 and G3 respectively (Ferrara et al., 2009). Only one work (Nishizaki et al., 2009) compared the effect of cranberry with that of an antibiotic (Cefaclor) in UTI prophylaxis. This study shows that the effect and efficacy of the two treatments are comparable and without the presence of side effects caused by cranberry juice (Nishizaki et al., 2009). The latest works in 2010 were published in abstract form in the proceedings of the Fifteenth



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Super EA, Kemper KJ, Woods C, Nagaraj S 2005. Cranberry use among pediatric nephrology patients. Ambul Pediatr 5: 249-252.

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prophylaxis. Pediatrics 117: 919-922.Wing DA 2006. Cranberry juice for preventing bacteria in

urine during pregnancy. http://clinicaltrials.gov/ct2/show?term=cranberry+children&rank=4, accessed in November 2010.

Wing DA, Rumney PJ, Preslicka CW, Chung JH 2008. Daily cranberry juice for the prevention of asymptomatic bacteriuria in pregnancy: a randomized, controlled pilot study. J Urol 180: 1367-1372.

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*Correspondence

Angelica DessìNICU, Puericultura Institute and Neonatal Section, University of CagliariVia Ospedale 119, 09124 Cagliari, [email protected]/fax: +390706093495

814

Article


Received 21 Dec 2010Accepted 29 Mar 2011Available online 12 Aug 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 814-817, Sep./Oct. 2011 Ethnopharmacology in Dublin: surveys on the

medicinal plants use profi le

Francis Cunningham, Fabio S. Menezes*

School of Pharmacy and Pharmaceutical Scieces, Panoz Institute, Trinity College Dublin, Ireland.

Abstract: This study aimed to examine the use of natural products as medicines in the greater Dublin area, Ireland. A field study was carried out on eighteen natural health stores; store assistants were interviewed and asked questions from a previously prepared questionnaire. Results were evaluated and a picture could be drawn about the use of natural products in Dublin. Questions asked included best sellers, new products on the market, common prescribers, ailments for which product is requested. Results indicated that as much as 60% of customers requests specific products were advice from staff. Nutritionists accounted for approximately one third of the medical profession that prescribed products for sale in health stores. The survey also examined common medicinal products requested which were not available without prescription. St. John’s Wort and Gingko biloba were the most requested. Undoubtly, there is a revival to searching for natural alternatives for the treatment of diseases with no medical advice.

Keywords:Dublinethnopharmacologymedicinal plantsnatural products

Introduction

Ethnopharmacology concerns the intersection of medical ethnography and biological studies of therapeutic action i.e., a transdisciplinary exploration that spans the biological and social sciences. (Etkin & Elisabetsky, 2005). The main goal of ethnopharmacology has been to discover novel compounds, derived from plants and animals used in indigenous medical systems, which can be employed in the development of new pharmaceuticals (Waldstein, 2006). Ethnopharmacologic exploration, involving both field visits, as well as experimental research has lead in past to highly valuable information about medicinal plants used in different cultures and many were developed into drugs (Bruhn & Holmstedt, 1981; Farnsworth et al., 1985). In years past enormous ethnopharmacological research was carried out by in the early days of medicinal plant research 250 years ago. Ethnopharmacology was initiated by the missionaries in the colonies interested in the use of pharmacologically active plants, like the Jesuits in 16th century Latin America (Anagnostou, 2005). Throughout that time, anthropologists worked together with chemists and biochemists often leading to fruitful collaborations. Examples of such partnerships are the isolation of caffeine by Johann Wolfgang von Goethe and Friedlieb Ferdinand Runge, and the elucidation of

the pharmacological principles in foxglove (Digitalis spp.). Typically, ethnopharmacological discoveries started with field observations and ended in new pharmacological insights. With the advancement of synthetic approaches in recent years, there has been a general lack of classical ethnopharmacological approaches in medicinal plant research both as a source of lead compounds for new drugs as well as in published research (Etkin, 2001; Cordell & Colvard, 2005). Another problem facing the future of ethnopharmacology is the highly competitive nature of drug research in the medical field. Thus, the success of drug discovery depends on the stringent criteria that help to avoid development of false positive candidates (Gertsch, 2009). As stated by Moerman (2008) “the low-hanging fruits have already been picked”, the miracle molecule that can cure cancer and so forth will not be found easily. Thus the aims of this study were to find out if naturally derived medicinal products are still used in Dublin city and if so, what uses they were intended for. Evaluating the main requests and uses of natural products is vital to filling a niche that is not fully filled by modern and more synthetically produced medicines. There are no records of any such interviews in the areas investigated, and thus the interviews carried out in health shops in the Dublin area were the first of their kind.

Ethnopharmacology in Dublin: surveys on the medicinal plants use profileFrancis Cunningham and Fabio S. Menezes



Fieldwork

A total of eighteen health shops in the Dublin area were interviewed to gather information on the different types of natural medicinal products supplied. A total of 34 trained staff members were interviewed with the prepared survey. The survey contained twelve questions, and asked in general to at least one person that worked in each health shop. To increase the number of answers, and therefore the accuracy of results two members of staff from each store were generally interviewed to remove any possible bias.

Research site

Dublin is the largest city and capital of Ireland (Figure 1). It is located near the midpoint of Ireland's east coast, at the mouth of the River Liffey. There are over 1,6 million residents in the county of Dublin. The use of Health shops in the Dublin area has increased in recent years as residents are becoming tired with the, perceived lack healthcare provided by the state. Statistics provided in 2007 by the central statistics office showed the following about Ireland health system; 47.6% of Ireland's population were covered by private health insurance, and 31.9% of the population were covered by Medical Cards, and there were 53 publicly-funded acute hospitals, with a total of 12,094 in-patient beds available and 1,253 day beds available.

Figure 1. Map of Dublin City and its neighbourhoods (numbers are the postcode areas in the inner city) and the areas where shops were visited to gather information on medicinal plants/natural products use (stars).

The map provided in Figure 1 shows the greater Dublin area divided into sections based on postcode. A sample of stores from a broad area in Dublin was undertaken. Areas in the South and South east of the

county are generally considered more affluent from the map that can be seen as areas 6, 14 and the constituencies of Rathdown/Dun Laoghaire. Areas such as Dublin 24, 1, and 7 would be viewed upon as having lower income families. Whilst carrying out the survey these observations were validated by staff members. General cold and flu preparations were very common requests in the poorer areas, whilst a wider choice of medicines was requested in the more affluent areas. Examples included anti-ageing products, vitamin supplements, and a wide range of health food organically grown and harvested. Many of the shops, although natural health stores, had very different functions to the community. While some focused purely on the medicinal side others focused more on a dietary approach thus giving a wider range of answers to the questions asked. To tailor for this the questions were adapted, if required, to allow the maximum amount of information to be gathered. During the month of October, the selected health stores were visited on the list provided. The following stores were selected: 1. Country Cellar, 8 Patrick St. Dun Laoghaire; 2. General Health Food Store, GPO Arcade Dublin 1; 3. Health Matters, Ashleaf S.C. Crumlin Dublin 12; 4. Health Matters, 8 Grafton St. Dublin 2; 5. Hopsack Health Store, The Swan Centre Rathmines; 6. La Sante, Marine Mall Dun Laoghaire; 7. Nourish, GPO Arcade Dublin 1; 8. Nourish, Liffey St. Dublin 1; 9. Nourish, 16 Wicklow St. Dublin 1; 10. Organic Supermarket, 2c Main Street, Blackrock; 11. The Health Store, The Square Tallaght Dublin 24; 12. The Health Store, Nutgrove Office Park Dublin 24.; 13. Vegeplanet, 19a Main Street Blackrock Market; 14. Down To Earth, Great Georges Street South Dublin; 15. Full life health food store, Dun Laoghaire shopping centre; 16. Nature Store, 324A NCR Phibsboro Dublin 7; 17. Nourish, Omni S.C Santry Dublin 9; 18. Nourish, Nutgrove S.C. Rathfarnham Dublin 16.

Questionnaire

1. What is the main natural product sold in your shop?2. What is the main complaint presented by customers that require the use of Natural Medicines from your shop?3. What are the most commonly sold new releases at the moment?4. What are the main side effects that you know about if any associated with Natural products that were investigated?5. Is there any particular plant that you don’t sell for which people request?6. If yes what is the plant used for?7. How many people come in on a daily basis to buy natural products without any previous advice?



8. How many people come in on a daily basis to buy natural products knowing exactly what they want or a detailed knowledge on the subject?9. What are the main “prescribers” for Natural products? Acupuncturists, healers or reiki, shiatsu, homeopathy, chiropractic, osteopathy, reflexology, naturopathy or anthroposophy. Kinesiologists were later added to the list due to the volume of answers it was included in.10. Do you have any product which the main claim is to protect against oxidative disorders or oxidative stress or excess free radicals or could have anti ageing properties?11. Do you only sell manufactured products or do you sell any medicinal plants just dried or fresh with no manufacturing?12. If so what is the best seller?


The main results show Echinacea as the main “natural remedy” sold in Dublin. Other herbal remedies that were in the top ten sellers included green/white tea, flaxseed and elderberry (Figure 2).

0

2

4

6

8

10

12

14

16

18

20

Echinacea Fish Oil ManukaHoney

Edelberry Green/whitetea

Flaxseed

Figure 2. Main products sold in health shops in Dublin (Y = number of staff answers).

Regarding the plants not available, Ginkgo biloba, St. John’s Wort (Hypericum perforatum), Cannabis (for arthritis) and Tribulus were the most requested (Figure 3). Ginkgo biloba is a medicinal plant used for the treatment of several conditions related to oxidative stress that is just available in Ireland under prescription. The same applies to St. John´s Wort, Hypericum perforatum, a plant used for the treatment of depression. Cannabis sativa, a drug of abuse, is constantly asked in shops for the treatment of arthritic pains. Tribulus terrestris, a plant used as aphrodisiac due to the contents of saponins is stated to possess androgenic activities and this is probably the reason why it is looked for. Around 60% of the population that enter health shops to buy these natural products have at least moderate knowledge on the relevant plant and how to use such medicinal product. The main prescribers

of natural products, including plants, are nutritionists and kinesiologists, followed by a third category that encompasses all the other complementary and alternative medicine prescribers (Figure 4).

0

5

10

15

20

25

30

Ginkgo biloba St. John's Wort Cannabis Tribulus

Figure 3. Plants not available in health shops that are constantly asked about (Y = number of staff answers).

0

5

10

15

20

25

30

35

Acupu

nturis

ts

Healer

s (Reik

i)

Shiatzu

prac

tition

ers

Homeo

pathy

pres

cribe

rs

Chirop

ractic

s

Osteop

athy p

ractiti

oners

Reflex

ology

prac

tition

ers

Naturop

athy p

ractiti

oners

Kinesio

logist

s

Nutritio

nists

Figure 4. Main prescribers of Natural Products and medicinal plants (Y = number of staff answers).

Natural products or medicinal plants available in Ireland must first pass stringent safety tests before they are available to the public in an OTC manner (Over the counter, i.e. no formal prescription needed) manner. There is no recognition of phytotherapy or medicinal plants use by any Irish Medical Board. What is interesting though is that the use of plant derived remedies is still a vital area in medicinal product invention. Approximately 60% of the anti-tumoral and anti-infective agents, either commercially available or in late stages of clinical trials are of natural origin (Silva et al., 2005). However at present there is no single prescribed medication existing (with an active principle isolated or inspired by plant metabolism) on the world market that is off plant origin and as a result this puts a great emphasis on health stores to fill this niche. It is also interesting to see the main reasons why people enter in a health shop to buy medicines. There are usually a broad variety of ailments they are seeking treatment for; however it is always mentioned, among the diseases themselves, the fact that people are seeking other



approaches for one main reason, dissatisfaction with other medicinal approaches and current medicine (Figure 5).

0

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8

10

12

14

16

Tiredn

ess/F

atigu

e

Cold an

d Flu

Natural

App

roach

Food A

lterna

tive

Dissati

sfacti

on

Migrain

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Fat Burn

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id

Figure 5. Main reasons of Natural Products/Medicinal Plants use in Dublin (Y = number of staff answers).

Echinacea and general multi-vitamin were the dominant products selling in the health stores visited. When the survey was carried out, in early October, the swine flu epidemic had just become publicized; along with the impending winter sales of medicines with the ability to boost immune function, were among the best sellers, hence its position as the number one seller. The second best seller, multi-vitamins (not shown in Figure 2), was number 1 for the rest of the year commonly, as stated by staff when the interview was conducted. Staff claimed this reason was due to customer request for a supplement that could re-energize and reduce fatigue. From the survey it becomes apparent that there is an almost equal amount of people that enter stores with a clear view on what they want, to those who enter and seek advice. There were also two clear products for which patients had researched privately and requested in these stores, which could not be legally sold. The two products were Gingko biloba and St. John’s Wort, the former available in the United Kingdom and the later being made prescription only in 2000. Nutritionists were named by all health stores as the main prescribers of their medicines. Kinesiologists, which had been previously been excluded from the list was the second highest profession prescribing natural medicinal products. All others professions appeared with approximately the same ratio of prescriptions. All stores stated that prescribing was generally from a mixture of all the professions present on the list. The main reason for potential patients visiting health stores at the time of visiting was for herbal products for the prophylaxis of cold and flu’s. Mentioned previously personnel in the stores visited stressed the point that many people had become worried about contracting such diseases and the public outbreak of the H1N1 virus had caused people to become more fearful of their health. The general pattern of the oncoming winter season and outbreak of new diseases annually ensures that preparations with immune boosting

function become the largest seller at that period. The second largest reason for the rise in popularity of Health Stores was a more natural approach to health. Many of the store assistants interviewed said that people purchasing medicines from their store were becoming weary of general practitioners and pharmacies and believed natural remedies would provide a safer option.

Acknowledgment

The authors would like to thank the staff from the Pharmacognosy area in the School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, especially Mr. Joe O´Reilly.

References

Anagnostou S 2005. Jesuits in Spanish America: contributions to the exploration of the American materia medica. Pharmacy in Histor, 47: 3-17.

Bruhn JG, Holmstedt B 1981. Ethnopharmacology: objectives, principles and perspectives. In: Beal JL, Reinhard E (Eds.), Natural Products as Medicinal Agents. Hippocrates-Verlag, Stuttgart, p. 405-430.

Cordell GA, Colvard MD 2005 Some thoughts on the future of ethnopharmacology. J Ethnopharmacol 100: 5-14.

Etkin NL 2001. Perspectives in ethnopharmacology: forging a closer link between bioscience and traditional empirical knowledge. J Ethnopharmacol 76: 177-182.

Etkin NL, Elisabetsky E 2005. Seeking a transdisciplinary and culturally germane science: the future of ethnopharmacology. J Ethnopharmacol 100: 23-26.

Farnsworth NR, Akerele O, Bingel AS, Soejarto DD, Guo Z 1985. Medicinal plants in therapy. B World Health Organ 63: 965-981.

Gertsch J 2009. How scientific is the science in ethnopharmacology? Historical perspectives and epistemological problems. J Ethnopharmacol 122: 177-183.

Moerman DE 2008. Personal Communication at the 10th

International Congress of Ethnopharmacoloy 16-19th

September, Sao Paulo, Brazil.Silva CG, Herdero RS, Mathias CJ, Panek AD, Silveira CS,

Rodrigues VP, Renno MN, Falcao DQ, Cerqueira DM, Minto ABM, Nogueira FLP, Quaresma CH, Silva JFM, Menezes FS, Eleutherio ECA 2005. Evaluation of antioxidant activity of Brazilian plants. Pharmacol Res 52: 229-233.

Waldstein A 2006. Mexican migrant ethnopharmacology: Pharmacopoeia, classification of medicines and explanations of efficacy. J Ethnopharmacol 108: 299-310.

*Correspondence

Fabio S. MenezesSchool of Pharmacy and Pharmaceutical Scieces, Panoz Institute, Trinity College Dublin, [email protected]. +353 1 8964154Fax: +353 1 8962810

818

Article


Received 21 Dec 2010Accepted 9 Jun 2011Available online 29 Jul 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 818-823, Sep./Oct. 2011 HPTLC fingerprint: a modern approach for

the analytical determination of Botanicals

Marcello Nicoletti

Department Enviromental Biology, University Sapienza, Italy.

Abstract: Availability of rapid and reliable methods for detection of quality in plant raw materials and botanicals is urgently needed. The recent HPTLC instrumentation allows to obtain fingerprints useful to ascertain identity and composition. The results of direct application of HPTLC devices in selected cases, using the fingerprint approach, are here reported, considered and compared with other methods. HPTLC is proposed as an useful tool for analytical validation of the novel forms of natural products.

Keywords:botanicalsfingerprintHPTLCnatural products

Introduction

Recently, the market of natural substances dramatically changed after the appearance of several new products characterized by elements of novelty as well as confusion, including a plethora of imaginative denominations, i.e. nutraceuticals, food supplements, botanicals, dietary supplements, medical devices, multifunctional foods, herbal drug preparations and others. As a matter of fact, these are modern forms of natural products utilizations, inheriting natural origin from the tradition like herbal medicines and herbalist products and offering solutions for the treatment of increasing minor pathologies or health maintenance. However, they urgently need adequate analytical controls, since their nowadays success could continue and be insured only by quality assessments, first by the exact determination of the composition. So far limited but significant cases of adulteration, substitution or changes, with the aim of improving efficacy, could invert the positive phenomenon. Dangers clearly also come from the utilization method: registered products are prescription drugs, that must be used under medical supervision, whereas herbal products are self administered and generally regarded as being harmless because of their natural origin. Therefore, the availability of analytical tools specially tailored to face the complexity of natural products mixtures is

crucial. Controls should be based on simple, viable, comprehensible and low cost methods. From the beginning, started with the first application by Michael Tsweet (Pelick et al., 1966), the future of the chromatography was marked: suited for separation and identification of plant constituents. Separation allowed the identification of thousands and thousands of natural substances, generating the construction of an amazing catalogue of more or less complicated structures, a flippant evidence of the endless chemical fantasy of plant metabolism and the necessary basis for any further phytochemical advancement, also in the analytical frontiers. The central position must be conceived to the planar chromatography, in particular TLC, the daily tool of any phytochemical student. Nowadays, TLC remains the immediate, simplest, reliable analytical friend in the laboratory of organic chemistry to check the presence and the identity of known marker compounds, to follow the improvement of a synthesis, to test column fractions separation trend, and others. However, the current challenge of complex mixtures, as those present in the botanicals, needs more effective proper analytical tools (Kaiser, 1988).


Preparation of the samples before HPTLC was simply performed by filtration on cotton. All chemical

HPTLC fingerprint: a modern approach for the analytical determination of Botanicals

Marcello Nicoletti


standards were of analytical grade. Arbutin (AR, purity ≥96%) and 3,4-dihydroxybenzoic acid (IS, purity ≥97%), hydroquinone (purity 99.0%), were purchased from Sigma-Aldrich (Milan, Italy). Rutin (≥98.5%) and gallic acid were procured from Prodotti Gianni (Milan, Italy); other flavonoid standards were obtained in other researches and their purity checked by NMR analysis. HPLC grade solvents were purchased from Sigma-Aldrich (Milan, Italy), Merck (Darmstadt, Germany) and Carlo Erba (Milan, Italy). HPLC-grade water was prepared with a Milli-Q gradient (Millipore, Vimodrone, Italy) water purification system. Formic acid was purchased by BDH prolabo (Poole, England). Analysed products were obtained from the market. Market materials were procured from different Italian companies of raw plant material or were obtained from the market. The origins of Arctostaphylos spp. raw materials were from Macedonia, Canada, Mexico, Albania and Siberia. Neem products from India and Italian different importer companies. Filtered solutions were applied to HPTLC 60 silica gel glass-backed layers (Merck, Darmstadt, Germany). For this purpose, a Camag Linomat IV sample applicator (Muttenz, Switzerland) with nitrogen flow was used. The operating conditions were as follows: syringe delivery speed, 10 s μL-1; injection volume, 10 μL; band width, 6 mm; space between bands, 9 mm; start position, 9 mm; distance from bottom, 8 mm. The HPTLC plates were developed in a horizontal chamber (Camag 20x10) after saturation with the same mobile phase. The optimised chamber saturation time for the mobile phase was 20 min. at room temperature. The length of the chromatogram run was 80 mm. The developed layers were dried in an oven at 100-105 °C for 15 min and then detected. Initially, the separated components were visually detected. The layers were allowed to dry in air for 30 min and then analysed under the proper detection way. For the densitometric analysis a Camag TLC scanner 3 linked to winCATS software was set at 350 nm, after multi-wavelength scanning between 250 and 400 nm in the absorption mode had first been tried. The sources of radiation were deuterium and tungsten lamps. The slit dimension was kept at 6.00 × 0.45 mm and the scanning speed used was 20 mm s-1.


HPTLC vs. TLC

HPTLC arises from the need for major separation capacity, has it is obtained by the use of precoated plates with smaller particles (5 vs. 15 µm), in order to obtain the efficacy needed for plant mixtures, but problems for TLC have also other origins. Planar

chromatography is mainly based on critical manual steps: it is an open system, dependent on environmental factors (temperature, light, fumes, humidity), that can influence the resulting data; also using the best rigor, despites all the extracautions of the same operator, results can not be fully controlled and analyses are not totally reproducible. In other words, data are scientifically not totally reliable and confident. TLC still remains a craftsman’s performance. HPTLC, High Performance Thin Layer Chromatography - the TLC of the 21th Century - is the last evolution of planar chromatography, whose mission is change the weakness into strengths. In modern HPTLC, the plate is the central tool of a complex automatic instrumentation system developed to control analysis conditions, to optimize reproducible results and to allow a complete comparison between different laboratories. The operator is the director of a tentative to reproduce Nature’s symphony tricking with sophisticated machines. In this regard, HPTLC is much more complicated than TLC, but complex problems need complex solutions. Being a multistep process, HPTLC performance requires separated devices for each step of the sequence: sample application, chromatogram development, derivatisation, visualization and documentation. The full power of HPTLC comes from the proper use, compatible and complementary, of each device in an integrated system (Reich & Schibli, 2007). Frequent goal of HPTLC is the fingerprint, as authentic maker of the biological complexity. A fingerprint is the individual chromatographic track representing, as near as possible, a mixture of organic substances. By the fingerprint approach, it is possible to obtain a proper identification of the plant material, but also determine and accept the limits of the biological changes. Variations in HPTLC tracks of the same species are mainly quantitative, not qualitative.

HPTLC vs. HPLC

Although HPLC remains the current best analytical tool, other instrumental possibilities have been proposed. Nowadays, HPTLC is ready to become one of the best methods for control of quality, purity, stability and identity, in one word validation of the composition of complex botanical products. The final coherent step of an evolution of analytical applications in natural products determination. Besides the achieved great increasing in efficacy, due to the use of minor size silica gel that means larger surface, it is possible to perform high quality HPTLC analytical determinations, due to a novel dedicated machinery, in order to finally achieve the necessary reliability, repeatability and flexibility. Substantially, the advantages of HPTLC were the rapidity and the

HPTLC fingerprint: a modern approach for the analytical determination of BotanicalsMarcello Nicoletti


possibility of analysing many samples at the same time under the same chromatographic conditions. Therefore, the consumption of the mobile phase is reduced and the results are rapid because HPTLC only needs chamber saturation and running time is only a few minutes. The mobile phase is completely removed from the plate during detection so that interferences may be eliminated, costs are limited, and all of the sample components may be seen together as part of the chromatogram. The same HPTLC plate can be visualised with and without derivatisation using different light sources. Reference materials can be chromatographed side by side on the same plate, that means exactly in the same conditions. Among the advantage of the HPTLC is important to report the sensitivity: the limit of detection are very low in accordance with the tradition of TLC, and, using the proper standard, quotient of each area can be defined and used for quantitative analysis. The main feature is in flexibility and complexity: HPTLC is able to generate a chromatographic fingerprint in the form of an unique sequence of peaks corresponding to the analyzed sample in its fullness. The aim is obtain resulting fingerprints that can be compared with respect to the number, sequence, position (Rf) and colour of the separated zones, also in reference to a data bank, using a computerized digitalic data bank. Furthermore, densitometry allows a quantitative analysis. Analogous and complementary data can be obtained using the NMR fingerprint of the total extract or parts due to simple treatment. The fingerprint approach is very useful in the analysis of complex mixtures. Usually, operator set HPLC analysis on a single standard or a class of substances, therefore other compounds are invisible or not detected. Therefore, botanicals can be easily and simply compared using their fingerprint and presence of adulterants as well detected. Main default concerns the still high cost of instrumentation and main advantage consists into the clear and simple evidence of the understandable result typical of TLC, avoiding the limits derived by the use of too specialized analyses. The successful use of HPTLC performed in our laboratory is here reported in cases concerning: a) adulteration, the use of other constituents besides those reported in label composition b) substitution, the utilization of botanical species not reported in pharmacopoeias and other regulatory references instead of the official ones c) comparison, between extracts of the same species differently obtained.

HPTLC vs. NMR

NMR has been proposed as a possible tool in natural products fingerprint (Krishnan et al., 2005; Piccin et al., 2009). However, also using spectrometer

of high resolution, the possibility of overlapping remains very high. The difficulty is enhanced in the case of comparison of species taxonomically near and in the absence of typical markers. Therefore, sensitivity is in contrast with the appearance of signals of the same constituent in several parts of the spectrum, whose unity must be reconstructed. On the contrary, the NMR study remains crucial in the identification of single substances.

Adulterations

Herbal products spiked with synthetic drugs are dangerous to consumers and noxious for future correct developing of the use of natural products. In 2010 HPTLC was used in our laboratories to evidence several adulterations in food supplements commercialized in Italy: a) presence of synthetic substances: nimesulide in a herbal remedy marketed for relieving pain (Mobile phase: toluene: ethylacetate 2:8 v/v; Detection: UV 254 nm); b) presence of hemisynthetic products: bromexine in a product commercialized for antiallergic properties, containing Adathoda vasica among other plant extracts; bromexine is obtained by chemical transformation of vasicine, the main alkaloid present in A. vasica (Mobile phase: toluene: ethylacetate: diethylamine 5:4:1 v/v/v; Detection: Dragendorff Reagent); c) presence of natural substances: several terpenoid alkaloids of reserpine type probably from a Rauwolfia sp. were detected (Mobile phase: toluene: ethylacetate: diethylamine 5:4:1 v/v/v; Detection: Dragendorff Reagent) in a herbal product sold for hypotensive effects, whose composition reported plants without any alkaloid relevant presence. As well known, most problems come from the products that can be obtained by web site selling, including herbal dietary supplements marketed as natural products for the enhancement of sexual function. In particular, HPTLC plates clearly showed an anomalous relevant constituent in a herbal product marketed from China as a potent aphrodisiac, named Sensual Tea or Jinshenkang, heavily present in internet sites as able to rapidly solve any sexual problem of females and males without any collateral effect (www.thesensualtea.com.mx; www.okokchina.com/p/Herbal-Medicine/Herbal-Jinshenkang-better-than-Viagra). The product examined, also marketed in Italy, came from Spain, where later the product was removed from the market. HPTLC showed a great spot very strong at UV lamp at 366 nm, as evident clue of the adulteration (Figure 1). Direct HPTLC comparison with sildenafil and vardenalafil - the PDE-5 inhibitors approved as anti-impotence drugs for the treatment of erectile dysfunction, excluded the identity with these substances. Also, densitometry analysis confirmed the differences,


Marcello Nicoletti


showing different UV absorption. A simple extraction with ethylacetate completely removed the substance from the product and the NMR spectrum of the extract resulted in an almost pure compound identified as thiosildenafil, by analysis of spectral data (Balayssac et al., 2009). The analysed Jinsenkahn product came from 2009 market, but the analysis of a 2008 product revealed the presence of sildenafil (1): therefore, thiosildenafil (2) has the same structure of sildenafil with a S instead of O, and it was artfully inserted to bypass the controls, too specific and specialised, as really happened. Both HPLC and HPTLC spectrometric analyses allowed to ascertain a quantity of the adulterant compound slightly superior to that contained in a tablet of Viagra. Thiosildenafil has been detected in another Chinese similar supplement, but using complicated 2D and 3D DOSY 1H NMR spectroscopy experiments (Balayssac, 2009).

Figure 1. The HPTLC plate of Jinshenkang. From left to right, the three first spots are the product in different concentrations (1, total extract; 2, 1:2 total extract/ethyl acetate; 3, 1:5 total extract/ethyl acetate) and the last two vardenafil and sildenafil, respectively. Mobile phase: dichlomethane: methanol (9:1 v/v). Detection: UV 366 nm.

NN

S

O

N

HNN

N

H3C CH3 CH3

CH3R

1 R = O2 R = S

OO

Substitution

Regulatory documents on medicinal plants, including Pharmacopoeias, are resistant to modifications and not able to follow the changes of the market. In the global market, several new species are already

used from several years and are still waiting a legal recognition. Meanwhile, these species are used and confused with old ones. The use of HPTLC fingerprint allows to obtain an easy identification of the different species, certifying in some cases also the geographic origin, as reported in the HPTLC volume of the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission, 2009). In particular, we applied the fingerprint method to the study of Arctostaphylos uva-ursi and A. pungens. The second species is nowadays increasingly used instead of the first one, traditionally employed as medicinal plant to treat urinary infections. A. pungens is collected in good quantity in Mexico, whereas A. uva-ursi is even more difficult to find and costly The activity is attributed to arbutine, a glucosidic parachinone metabolically converted into the corresponding aglucone, hydrochinone (WHO, 1996; Frohne, 1970). However, HPTLC analysis clearly showed a different composition for the two species: arbutine is practically absent in A. pungens, which is very rich in flavonoids. Also, NMR confirmed this absence, whereas HPLC analysis was able to verify a very low presence of arbutine, below the 0.5% (Nicoletti et al., 2010a). However, recent pharmacological studies showed a potent activity of flavonoids against bacterial infection of the urinary tract, validating the use of A. pungens. Other similar studies were obtained for products containing Equisetum sp. (Gallo, 2011) (Mobile phase: ethyl acetate:formic acid:water 82:9:9, v/v/v; Detection: NPR). According to the European Pharmacopoeia only E. arvense should be used, but other species, i.e. E. maximum, resulted present in the marketed products. Also in this case pharmacological data are lacking about the effects of other Equisetum species.

Comparison

HPTLC is a potent tool for easy comparison of similar products. Lots of the same product can be compared in the same plate and quantities assessed by densitometric inspection. In particular, we were able to examine the composition of several products marketed by the name of neem cake. Neem tree, Azadirachta indica A. Juss, features a long traditional utilization in agriculture, for the body care (cosmetics) and medicine (Mordue & Blackwell, 1993) but currently, there is a huge amount of products obtained from this multipurpose tree, due to the anti-fungal, anti-bacterial and insecticide properties. The most commercially important product is the neem oil extracted from the seeds, also known as margosa oil. Neem, identified by WHO/UNEP1989 as an environmentally powerful natural pesticide, is considered to be one of the most promising trees of the 21st century for its great potential

HPTLC fingerprint: a modern approach for the analytical determination of BotanicalsMarcello Nicoletti


middle of the first two tracks were identified as linear fatty acids after separation and NMR analysis, whereas spots on the top were identified as methyl esters of fatty acids. The fingerprint approach is extremely effective in the comparison of populations of the same species or lots of the same extract. It was used to verify the different production of flavonoids and hypericine in Hypericum perforatum plants collected in different parts of Italy. As evident in Figure 3, the contents resulted very different and this is in contrast with the current regulation requiring a fixed relationship between the constituents.

Conclusion

The utilization of HPTLC, owing to the high automatization recently obtained, can be considered as an useful tool in the analysis of complex mixtures of natural products, such as those introduced nowadays in the market. Still some aspects remain to be improved, like the cost of instruments, and the sensibility, still not comparable with HPLC. Also in this field, like in the other analytic approaches, the use of hyphenated techniques, as already obtained in GC/MS. Hyphenated HPTLC/MS instruments are already available, and HPTLC/NMR are under study, to solve in one step the problem of separation and identification. Adoption of HPTLC by USP (USP, 2007), Ph. Eur., AHP and PhPRC (Chinese Pharmacopoeia, 2009) constitute a clear recognition of the importance of this technique as the method of choice for handling complex analytical task involving herbal drugs and botanicals.

in pest management, environment protection and medicine. Neem cake is the raw material derived by cold pressing neem kernels from handpicked and cleaned neem fruits and seeds. Neem cake is commercially important for its use as fertilize material, as well as potential insecticide (Nicoletti et al., 2010b). By the HPTLC neem cakes resulted different depending by the extraction process used, the different origins and the method of commercialization (Neem America Incorporated, 2010). In particular, the content of fatty constituents appeared highly variable, as evident in the plates (Figure 2). Therefore, a different utilization of neem cakes should be inferred according to the compositions.

Figure 2. Different compositions of neem cakes from the market. Mobile phase: toluene: ethylacetate (8:2, v/v). Detection: anisaldehyde reagent (sulphuric acid (10 mL) added to methanol (170 mL) and acetic acid (20 mL) and to further anisaldehyde (1 mL). The main spots evident in the

Figure 3. Comparison between different populations of Hypericum perforatum. On the left: tracks of Hypericum perforatum collected in different Italian regions at the same vegetative stage including a commercial extract (tract 3); on the right: the flavonoids standards, track 10 hyperoside (not visible in these conditions), 11 rutin (yellow spot at low Rf), 12 quercetin (green spot), 13 isoquercetin (yellow spot), 15 luteolin (white spot) and 16 apigenin; hypericine was track 9, evident in red, and chlorogenic acid in track 14 was used as stardard. Mobile phase: formic acid: water: ethyl acetate 10:5:85, v/v/v). Detection: the layers were treated with a solution containing the Natural Product Reagent (diphenylborinic acid aminoethylester (1 g ) in ethyl acetate (200 mL)), dried in the open air and then dipped into Macrogol reagent (polyethylene glycol 400 (1 g) in dichloromethane (20 mL)).


Marcello Nicoletti


Acknowledgements

The author would like to thank F.E.I. (Federazione Erboristi Italiani) for providing most of the market analyzed products.

References

Balayssac S, Trefi S, Gilard V, Malet-Martino M, Martino R, Delsuc M-A 2009. 2D and 3D DOSY 1H NMR, a useful tool for analysis of complex mixtures: Application to herbal drugs or dietary supplements for erectile dysfunction. J Pharmaceut Biomed 50: 602-612.

Chinese Pharmacopoeia Commission 2009. TLC Atlas of Chinese Crude Drugs in Pharmacopoeia of the People’s Republic of China. People’s Medical Publishing House.

Frohne D 1970. The urinary disinfectant effect of extract from leaves uva ursi. Planta Med 18: 1-25.

Gallo FR, Multari G, Federici E, Palazzino G, Giambenedetti M, Petitto V, Nicoletti M 2011. Chemical fingerprinting of Equisetum arvense L. using HPLC and HPTLC densitometry Nat Prod Rep, in press.

Kaiser RE 1988. Scope & limitation of modern planar chromatography. Part 1: Sampling. JPC-J Planar Chromat 1: 182-199.

Krishnan P, Kruger NJ, Ratcliffe 2005. Metabolite fingerprinting and profiling in plants using NMR. J Exp Bot 56: 255-265.

Mordue AJ, Blackwell A 1993. Azadirachtin: an update. J Insect Physiol 39: 903-924.

Nicoletti M, Petitto V, Federici E, Gallo FR, Multari G, Palazzino G 2010a. La politica dello struzzo conviene? Erboristeria Domani 350: 69-73.

Nicoletti M, Serafini M, Aliboni A, D’Andrea A, Mariani S 2010b. Toxic effects of neem cake extracts on Aedes albopictus larvae. Parasitol Res 107: 89-85.

Neem America Incorporated 2010. Preparation of neem products and their uses. Manufactures of Premium Neem Products. Available in ww.neemamerica.com/research06.asp.

Pelick N, Bolliger H, Mangold H 1966.The history of thin-layer chromatography. In: Giddings JC, Keller RA (eds). Advances in chromatography, vol. 3. New York, Marcel Dekker. p. 85-118.

Piccin A, Serafini M, Nicoletti M 2009. Phytochemical profile of Irix tenax. Nat Prod Comm 4: 1643-1644.

Reich E, Schibli A 2007. High-performance thin-layer chromatography for analysis of medicinal plants. NY: Thieme Medical Publishers Inc.

The United States Pharmacopoeia, 31st Edition 2007. The National Formulary, 26th edition. Rockville: The United States Pharmacopeia Convention.

World Health Organization 1996. IPCS International Programme on Chemical Safety. Health and Safety Guide No. 101. Hydroquinone health and safety guide. United Nations Environment Programme. International Labour Organisation. Geneva (available in www.inchem.org/documents/hgs).

*Correspondence

Marcello NicolettiDepartment Enviromental Biology, University SapienzaP.le A. Moro, 5 I-00185 Rome, [email protected]. +39 6 4991 2195Fax: +39 6 4991 2195

824

Article


Received 20 Dec 2010Accepted 19 Jun 2011Available online 26 Aug 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 824-828, Sep./Oct. 2011 Quinolizidine alkaloid composition in different

organs of Lupinus aschenbornii

Edith Montes Hernández,1 María L. Corona Rangel,1 Aidee Encarnación Corona,1 Jorge A. Cantor del Angel,1 Jesús Arnoldo Sánchez López,1 Frank Sporer,2 Michael Wink,2 Kalina Bermúdez Torres*,1

1Centro de Desarrollo de Productos Bióticos. Instituto Politécnico Nacional, México,2Institute of Pharmacy & Molecular Biotechnology, Heidelberg University, Germany.

Abstract: Lupinus aschenbornii S. Schauer, Fabaceae, grows in the Central Highlands of Mexico, at altitudes between 2800 to 4300 m above sea level. The alkaloid patterns in organs of L. aschenbornii were analyzed by Gas-Liquid Chromatography-Mass Spectrometry (GLC-MS). Quinolizidine alkaloids (QA) were identified according to their mass fragmentation patterns, in combination with their Kovats retention indeces. Total QA content in organs differed substantially: seed contained 3.3 mg/g dry weight, flowers 2.8 mg/g DW, leaves 1.9 mg/g DW, stems 1.5 mg/g DW, and pods 1.4 mg/g DW. Roots do not accumulate QA and their profiles differed considerably: while seed stored N-formylangustifoline (17%), 17-oxolupanine (16%), multiflorine (11%) and an unidentified alkaloid (n.i.) 2869 (11%) as main QA, sparteine was absent. In flowers, sparteine reached 73%, in leaves up to 80%, in stems up to 32% and in pods up to 96%. Other QA present were lupanine (32% in stem, 9% in flower and 7% in seed); N-formylangustifoline (9% in stem and 4% in flower); multiflorine (6% in stem and 3% in flower). Differences in QA profile might be a strategy of lupins to avoid adaptation of possible predators because the different QA have different pharmacological properties.

Keywords:mexican Lupinusphylogenyquinolizidine alkaloid profiles

Introduction

The genus Lupinus, Fabaceae, is widely distributed in the New World. About 230-350 species have been reported of which 200-320 occur in North and South America (Lewis et al., 2005). Mexico is a secondary diversifi cation center; in the Mexican National Herbarium collection, our group found more than 100 species, however Sousa & Delgado (1993) recognized only 65. Species delimitations in the genus Lupinus are poorly defi ned and their taxonomy needs a thorough revision. Lupinus aschenbornii S. Schauer grows in the Central Highlands of Central America as part of the subalpine vegetation at altitudes between 2800 m and 4300 m above the sea level (Sousa & Delgado, 1993; Stanley & Steyermark, 1946). This species has several common names such as “frijolillo” and “cantuez”. The plant is not known to be used by any local communities for food or medicine. Many Lupinus species are characterized by the presence of quinolizidine alkaloids (QA) as part of a defense strategy against herbivores and microorganisms (Wink, 1992; 1983). QA profi les are rather constant within

species but substantial variation is found in the alkaloid patterns of different organs (Wink, 1993b). More than 170 alkaloids of the quinolizidine group have been identifi ed in different Lupinus species (Wink, 1993b; Wink, 1988). The main structural types of QA belong to the groups lupinine, sparteine/lupanine/multifl orine, α-pyridone, matrine, Ormosia, piperidine and dipiperidine alkaloids (Wink, 1993a; Kinghorn & Balandrin, 1984). Tetracyclic QA such as sparteine and lupanine are the major alkaloids present in almost all Lupinus species (Wink et al., 1995; Wink, 1993b). QA are constitutive metabolites; however their presence and abundance can vary during development and depends on plant organ (Zamora Natera et al., 2009; Cortes Sánchez et al., 2005; Wink, 1992). Some authors have shown that the QA profi les of North American species present a high degree of intraspecifi c variation (Wink & Carey, 1994). Bermúdez Torres et al. (1999) compared QA profi les of seeds and leaves of L. aschenbornii. They found that main QA of these organs are almost similar; both organs accumulate13α-hydroxylupanine (56 and 54.9%, respectively) and an unidentifi ed compound with

Quinolizidine alkaloid composition in different organs of Lupinus aschenborniiEdith Montes Hernández et al.


a retention index RI 2849 (19.7 and 17.9%, respectively); however angustifoline was detected only in seeds (18.9%). As minor QA, a high diversity of QA-esters (less than 10%) were found in seeds. Leaves contain tetrahydrorhombifoline (8.07%), 13α-tigloyloxilupanine (3.97%), lupanine (3.65%), and albine (1,4%). On the other side, Bermúdez Torres et al. (2009) analyzed leaves of L. aschenbornii in order to evaluate its activity against Spodoptera frugiperda, they found sparteine as main QA (85%) and lupanine, multiflorine and an unknown compound RI 1940 (9.3; 3.3; 2.1%, respectively) as minor QA. These results show that QA profiles differ in dependence of organ and probably origin. The aim of the present work was to evaluate QA composition in different organs (stems, leaves, flower, roots, pods and seeds) of L. aschenbornii plants in the same physiological state by Gas-liquid Chromatography-Mass Spectrometry (GLC-MS). For L. aschenbornii, this is the first report of QA profile for the other organs than leaves.


Plant material

Ten plants of Lupinus aschenbornii S. Schauer, Fabaceae, were randomly collected in September 2009 from a single population of L. aschenbornii growing in La Joya, Iztaccihuatl Volcano, Estado de México, Mexico, 3800 m above sea level. All organs (root, leaf, stem and flower), except seeds and pods, were collected during the flowering stage. Seeds and pods were collected in October 2009. Plant material from each organ was ground and stored at room temperature in paper bags until use. Voucher specimens were deposited at the National Herbarium MEXU (Voucher No. 1297270, 1297311 and 1297317).

Alkaloid extraction

Alkaloids were extracted as described in Wink et al. (1995). Each organ (0.5 g) was homogenized separately in 20 mL 1M HCl. The homogenate was adjusted to pH 12 with a 3 M aqueous solution of NH4OH. Alkaloids were extracted by solid phase extraction using Extrelut-columns® and dichloromethane as eluent.

Alkaloid identification by GLC-MS

The alkaloid extracts were analyzed by GLC-MS on a HP5890 instrument. The separation conditions were: column type: OV1; 30 m; 0.25 mm i.d.; 0.25 µm film thickness; Split ratio 1:30; carrier gas: He; flow 1 mL min-1; 120 °C; 2 min isothermal; 120-310 °C at a rate of 4 °C min-1; then 4 min isothermal; the data system was started at 138 °C oven temperature. The capillary column was directly coupled to a quadrupole mass spectrometer

(SSQ 7000, Thermo-Finnigan, Bremen, Germany). The injector temperature was 250 °C. Helium carrier gas flow rate was 2 mL/min. All the mass spectra were recorded with the following conditions: filament emission current, 100 mA; electron energy, 70 eV; ion source, 175 °C. The components were identified by their retention times, retention indices relative to C9-C28 n-alkanes, computer matching with the Wiley Registry of Mass Spectral Data 8th edition, NIST Mass Spectral Library (December 2005) and the MS library at IPMB for alkaloids. Lupanine and sparteine were used as external standards for quantification.


GLC-MS separations of alkaloid extracts from seed, leaves, flowers and stems of L. aschenbornii are shown in Figure 1. Total Ion Chromatograms of pods and roots are not shown as for pods only two QA (sparteine and n.i. 2484) and for roots no QA were detected. It is evident that the presence and abundance of some individual QA varies in an organ-specific fashion. The diversity of QA is highest in seeds (11 QA) and in this organ relative abundances of individual QA are mostly equal, while QA diversity in leaves is poor, and sparteine is the predominant alkaloid. These results contrast with the observations of Planchuelo-Ravelo et al. (1993) and Wink (1992) that QA diversity is generally higher in green organs where QA are being synthesized. As seeds are the reproductions organ of plants, they have to be especially protected against predators to allow species survival; a higher diversity and concentration of defense compounds affords a wide range of protection. As shown in Table 1, total QA content in organs differs substantially; seeds contain 3.3 mg total QA/g DW, flowers 2.8 mg/g DW, leaves 1.9 mg/g DW, stems 1.5 mg/g DW, and pods 1.4 mg/g DW. These results confirm the assumption that QA are accumulated in the reproductive organs, and might play a role as part of the defense strategy against predators, but also contribute to N metabolism in these organs (Wink & Witte, 1985). While in seeds N-formylangustifoline, a tricyclic molecule is the main QA, the other evaluated organs contain mainly tetracyclic QA such as sparteine, lupanine and multiflorine. Seeds of L. aschenbornii are characterized by the presence of several QA esters (Bermúdez Torres, 2009; Bermúdez Torres, 1999), in this study we found N-formylangustifoline to be the main QA, at 17%, and some other esters to be minor QA. However, European lupin species produce esters of 13-hydroxylupanine and hydroxymultiflorine mainly in shoots and leaves, but not in seeds (Wink et al., 1995). L. aschenbornii accumulates esters mostly in seeds. The results of the present study show that L. aschenbornii contains a similar QA pattern to other



American species which are all closely related (Käss & Wink, 1997). It is remarkable that there was no sparteine in seeds, a possible explanation is that during transport to this organ QA are transformed to QA esters and, finally, they will be accumulated in this way.

Table 1. Alkaloid composition in organs of L. aschenbornii.

KI AlkaloidAlkaloid composition (%)

Seed Stem Flower Leaf Pod Root

1853 sparteine 0 32 73 80 96 0

1858 n.i. 1858 0 0 0 10 0 0

1864 n.i. 1864 0 0 0 5 0 0

2039 tetrahydrorombifoline 3 0 0 0 0 0

2046 angustifoline 2 0 0 5 0 0

2056 n.i. 2056 5 0 0 0 0 0

2060 lupanine 7 19 9 0 0 0

2085 multiflorine 11 6 3 0 0 0

2095 N-formylangustifoline 17 9 4 0 0 0

2097 n.i. 2097 5 0 0 0 0 0

2476 n.i. 2476 0 7 4 0 0 0

2484 n.i. 2484 0 0 0 0 4 0

2494 17-oxolupanine 16 6 0 0 0 0

2497 n.i. 2497 0 0 3 0 0 0

2869 n.i. 2869 11 0 0 0 0 0

Sparteine and lupanine are the most widely distributed QA in the genus Lupinus (Wink et al., 1995). The major alkaloid in stem, flower, leaf and pod was sparteine (32, 73, 80 and 96%, respectively). Wink et al. (1995) evaluated 56 species of the Old World, and the Americas; they found that almost half of the North American species contain sparteine as main QA. In relation to Mexican Lupinus species, Bermúdez Torres et al. (2009) reported sparteine as main QA in leaves of L. aschenbornii and L. montanus. Lupanine, another tetracyclic QA, was present in stem (19%), flower (9%) and seed (7%) of L. aschenbornii. Multiflorine is one of the characteristic QA of seed (11%), stem (6%) and flower (3%) of L. aschenbornii Schauer. Whereas sparteine, lupanine and their derivatives are very common in lupin species, multiflorine and their derivatives have a restricted distribution (Wink et al., 1995). They were found in Old World species such as L. albus, L. cosentinii, L. micranthus and L. varius and in South American species such as L. albescens and L. aureonitens (Planchuelo-Ravelo & Wink, 1993; Planchuelo-Ravelo et al., 1993). Traces of this alkaloid were also found in some North American species (Wink et al., 1995). Table 2 presents the mass spectral characteristics of individual QA (alkaloid name, Kovat’s retention index, molecular ion M+, significant fragment ions and their relative abundance). Fifteen QA were detected in the extracts and seven were identified unequivocally by

Figure 1. Chromatographic profile of seeds (A), stems (B), flowers (C), and leaves (D).

KI: Kovat’s Index; n.i.: not identified.



comparing their retention index and informative mass spectra with those in the data bases. Eight QA could not be identified; some of them are main QA (Table 1), and according to their mass spectra some are QA esters. With these results it is obvious that profiles differ between organs, however we are currently not able to explain such diversity in the alkaloid composition between organs of L. aschenbornii, although some possibilities merit consideration and discussion: i) Structural changes of QA usually imply differences in their pharmacological and toxicological properties (Schmeller et al., 1994). The difference in QA profiles in the organs evaluated might be a measure to reduce predator adaptation. “One predator, one organ” might be a defense strategy that would confer Lupinus the possibility to survive and reproduce in an adverse environment. This means that each predator would be adapted to specific QA profiles and their intrinsic pharmacological properties, only being able to feed on the respective organ; ii) Other possible explanations for the variation of QA profiles in organs could be an uneven transport of QA through the plant. The leaf is the organ of QA biosynthesis; a higher QA diversity should be expected in this organ. However, in the population studied, leaves had fewer QA than other organs, maybe because they are transported to various organs necessary for the survival of the plant (Wink, 1985). Flowers and seeds are the reproductive organs, so a survival strategy for the species might be to accumulate higher amounts of QA, on the one hand as defense against predators but also as a N source for seedlings to provide amino acids and proteins (Wink & Witte, 1985). Stem (and specially the phloem) is the organ for the translocation of QA; therefore we should find here QA in a form that can be transported.

QA have been suggested to be of use as chemotaxonomical markers. However the results of this work show that QA profiles vary dramatically between organs, and as shown in other studies, also between developing stages (Wink, 1992). More importantly, QA profiles do not match lupin phylogeny and it has been assumed that a patchy distribution results from differential gene regulation (Wink et al., 2010). Another question which should be discussed, is the observation that published QA profiles of L. aschenbornii (Bermúdez Torres et al., 2009; Bermúdez Torres et al., 1999), differ considerably: How can this phenomenon be explained? i. The first but least likely explanation could be that plants were collected in different physiological stages. As shown by other authors (Wink 1992; Zamora-Natera et al., 2009) QA levels might vary during plant development. ii. Alkaloid profiles are ultimately the result of chemical warfare between the plant and herbivores (Adler & Kittelson, 2004), and the presence and abundance of QA-inducing predators may be influenced by ambient factors such as the season. iii. L. aschenbornii like all North American Lupinus species is phylogenetically a young species (Käss & Wink, 1997), and its traits are still evolving. Another possibility should be that species limits are not well defined for L. aschenbornii and that the presently recognized taxon is in reality a species complex. Or hybridization with a population of L. montanus growing very close to it, may have influenced the QA of L. aschenbornii. To try to respond all these questions, we plan to pursue the chemical and molecular characterization of individuals of this population, to evaluate inter population variability of QA profiles and genetic variation.

Table 2. Quinolizidine alkaloids identified in L. aschenbornii by GLC-MS.Alkaloid KI M+ Characteristics ions (abundance %)

Angustifoline 2046 234 193(100) 112(85) 150(15) 55(20) 94(11)

Sparteine 1853 234 137(100) 98(90) 234(44) 193(25) 84(10)

n.i. 1858 1858 254 135(100) 149(75) 107(58) 121(52) 191(25)

n.i. 1865 1865 282 135(100) 121(53) 107(39) 149(35) 220(25)

Tetrahydrorombifoline 2039 248 207(100) 58(80) 112(15) 108(10) 248(1)

n.i. 2056 2056 394 272(100) 137(31) 273(28) 127(25) 82(23)

Lupanine 2060 248 136(100) 149(60) 248(40) 150(34) 219(8)

Multiflorine 2085 246 134(100) 246(65) 148(20) 110(15) 217(5)

N-Formylangustifoline 2095 262 112(100) 193(98) 221(41) 262(2) 150(19)

n.i. 2097 2097 366 272(100) 207(23) 273(18) 136(14) 57(15)

n.i. 2476 2476 294 245(100) 112(83) 263(43) 55(36) 149(35)

n.i. 2484 2484 284 246(100) 134(56) 55(44) 112(30) 148(23)

17-Oxolupanine 2494 262 150(100) 262(40) 110(30) 234(10) 55(20)

n.i. 2493 2493 282 245(100) 112(83) 263(50) 55(35) 149(34)

n.i. 2869 2869 282 245(100) 112(80) 203(53) 149(35) 55(33)n.i.: not identified.



Guatemala. Feldiana Botany, vol. 24, part. V., p. 287-290.

Sousa SM, Delgado SA 1993. Mexican Leguminosae: phytogeography, endemism, and origins. In Ramamoorthy T, Bye R, Lot A, Fa J (eds.) Biological diversity of Mexico: origins and distribution. New York: Oxford Univ. Press, p. 459-511.

Wink M, Botschen F, Gosmann C, Schäfer H, Waterman PG 2010. Chemotaxonomy seen from a phylogenetic perspective and evolution of secondary metabolism. In Wink M (ed.) Biochemistry of plant secondary metabolism. Blackwell: Annual Plant Reviews, vol. 40, 2 ed, p. 364-433.

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Wink M 1983. Wounding-induced increase of quinolizidine alkaloids accumulation in lupin leaves. Z Naturforsch 38c: 905-909.

Wink M, Witte L 1985. Quinolizidine alkaloids as nitrogen source for lupin seedlings and cell suspension cultures. Z Naturforsch 40c: 767-775.

Wink M 1985. Metabolism of quinolizidine alkaloids in plants and cell suspension cultures: Induction and degradation. In Neumann KH, Barz W, Reinhard E (eds.) Primary and secondary metabolism of plants and cell cultures. Berlin: Springer Verlag, p. 107-116.

Wink M 1988. Plant breeding: importance of plant secondary metabolites for protection against pathogens and herbivores. Theor Appl Genetics 75: 225-233.

Wink M 1992. The role of quinolizidine alkaloids in plant insect interactions. In Bernays EA (ed.) Insect-plants interactions. Boca Raton: CRC Press, p. 133-169.

Wink M 1993a. Quinolizidine alkaloids. In Waterman PG (ed.) Methods in plant biochemistry. London: Academic Press, p. 197-239.

Wink M 1993b. Allelochemical properties and the raison d'être of alkaloids. In Cordell G (ed.) The Alkaloids. Academic press, vol. 43, p. 1-118.

Zamora-Natera F, García-López P, Ruiz-López M, Rodríguez-Macías R, Salcedo-Pérez E 2009. Composición y concentración de alcaloides en Lupinus exaltatus Zucc. durante su crecimiento y desarrollo. Interciencia 34: 672-676.

*Correspondence

Kalina Bermúdez TorresCentro de Desarrollo de Productos Bióticos. Instituto Politécnico NacionalApartado Postal 24, C.P. 62731, Yautepec-Morelos, Mé[email protected].: +55 57 29 60 00 Ext. 82520 Fax: +55 57 29 60 00 Ext. 85252

Acknowledgments

This work was funded by the Secretaría de Investigación y Posgrado del Instituto Politécnico Nacional (SIP 20090435 and 20100688) and by CONACYT (100808). E. Montes Hernández and J. Cantor del Angel are indebted to Instituto Politécnico Nacional (PIFI-IPN) and CONACYT for the fellowship awarded. A. Encarnación Corona thanks CONACYT for support. K. Bermúdez Torres thanks EDI and SIBE-IPN for support. Astrid Backhaus provided valuable help at the IPMB (Heidelberg). We thank Peter Winterton and Luc Legal for valuable help on reviewing the manuscript.

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Bermúdez-Torres K, Robledo-Quintos NR, Martínez-Herrera J, Te, Wink M 1999. Patrón de acumulación de alcaloides en hojas y semillas de Lupinus aschenbornii crecidos en México. Rev Latinoamericana Quim 27: 101-105.

Cortés-Sánchez M, Altares P, Pedrosa MM, Burbano C, Cuadrado C, Goyoaga C, Muzquiz M, Jiménez-Martínez C, Dávila-Ortiz G 2005. Alkaloid variation during germination in different lupin species. Food Chem 90: 347-335.

Käss E, Wink M 1997. Molecular Phylogeny and phylogeography of the genus Lupinus (family Leguminosae) inferred from nucleotide sequences of the RbcL Gene and ITS 1+2 sequences of rDNA. Plant Syst Evol 208: 139-167.

Kinghorn AD, Balandrin MF 1984. Quinolizidine alkaloids of the Leguminosae: structural types, analysis, chemotaxonomy, and biological activities. In Pelletier WS (ed) Alkaloids: chemical and biological perspectives. New York: Wiley, p. 105-148.

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Article


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Available online 5 Aug 2011


21(5): 829-834, Sep./Oct. 2011Effect of the microfi ltration process on antioxidant activity and lipid peroxidation protection capacity of blackberry juice

Gabriela Azofeifa,1 Silvia Quesada,*,1 Ana-Mercedes Pérez 2

1Departamento de Bioquímica, Escuela de Medicina, Universidad de Costa Rica,2Centro Nacional de Tecnología de Alimentos, Universidad de Costa Rica.

Abstract: Phytochemicals are highly concentrated in berries, especially polyphenols as anthocyanins and ellagitannins. These compounds have been associated with antioxidant capacity, lipid peroxidation protection, anti-inflammatory activity, anti-carcinogenic activity, obesity prevention and others. Blackberries are commonly grown and consumed as juice in Latin-American countries. However, blackberry juice is easily fermented and different industrial techniques are being applied to enable the juice to be stored for longer periods. One important issue required for these techniques is to preserve the health-promoting capacities of blackberries. This study compared the antioxidant activity and the lipid peroxidation protector effect between a fresh blackberry juice (FJ) and a microfiltrated blackberry juice (MJ). Chemical analysis of both juices show less polyphenols concentration in the MJ. Despite this difference, values for biological activities, such as protection of lipid peroxidation, was not significantly different between FJ and MJ. These results suggest that the compounds responsible for the antioxidant activity are maintained even after microfiltration and the free radical scavenging capacity of these compounds could protect the initiation of lipid peroxidation. Microfiltration could be used as an industrial technique to produce blackberry juice that maintains biological activities of polyphenols.

Keywords:antioxidant capacity

blackberry juicelipid peroxidation

microfiltrationRubus adenotrichus

Introduction

Several studies have suggested a potential health benefi t of fl avonoids (Beattie et al., 2005; He & Giusti, 2010). Particularly, there has been documented an inverse association between high consumption of fl avonoids and the incidence of cardiovascular diseases (Mazza, 2007). Phytochemicals, such as fl avonoids, can act as free radical scavengers or can up regulate endogenous antioxidant defenses. Some fl avonoids suppress reactive oxygen species formation, by chelation of transition metal ions or by the suppression of superoxide-driven reactions that generate active oxygen species (Pietta, 2000). Compared with most fruit, berries have high concentrations of a type of fl avonoids known as anthocyanins (Beattie et al., 2005). Anthocyanins have been associated also with antioxidant activities, lipid peroxidation protection, anti-infl ammatory activity, anticarcinogenic activity, obesity prevention and others (He & Giusti, 2010). Blackberries are promoted as rich sources of polyphenols with antioxidant activities (Jiao & Wang, 2000; Acosta-Montoya et al., 2010). Blackberry juice consumption demonstrates the capacity to increase the

plasma antioxidant capacity in humans (Hassimotto et al., 2008). Moreover, purifi ed anthocyanin fractions of blackberry demonstrated anti-infl ammatory activity by inhibition of COX-1 and COX-2 enzymes (Rossi et al., 2003; Cuevas-Rodriguez et al., 2010; Bowen-Forbes et al., 2010). Major phenolic compounds in the blackberry variety Rubus adenotrichus Schltdl., Rosaceae, are anthocyanins cyanidin 3-glucoside and cyanidin 3-(6’-malonyl) glucoside and ellagitannins lambertianin C and sanguiin H-6 (Mertz et al., 2007). Both types of molecules were associated as main contributors to the antioxidant capacity in another berry Rubus idaeus L., Rosaceae, (Mullen et al., 2002). Juice processing conditions cause differences in antioxidants content (Gil et al., 2000). Industrial processing with the addition of sugar and pectin; thermal treatments; and long term storage, can have marked effects on the phenolic content of berries, sometimes causing the increase of some compounds and the decrease of others (Zafrilla et al., 2001; Gancel et al., 2010). According to Mullen et al. (2002), in raspberries these effects are more important in ellagitannins than in anthocyanins; however there is no effect in the antioxidant

Effect of the microfiltration process on antioxidant activity and lipid peroxidation protection capacity of blackberry juiceGabriela Azofeifa et al.


capacity of the fruit. Gil et al. (2000) described that pomegranate hand press juices and commercial juices had the same anthocyanin pigments, but quantitative differences. Pomegranate anthocyanins are partially degraded or transformed into other products due to freezing procedures prior to juice extraction. Some publications have studied changes in blackberries anthocyanins properties due to processing, storage and heat treatments. Cisse et al. (2009) explained that anthocyanins of Rubus adenotrichus are less sensitive to heat when compared to anthocyanins from other fruits and other varieties of blackberry. Hager et al. (2008) reported a decrease in the scavenging activity against peroxyl radicals and superoxide radicals (ORAC and PCL) after thermally processed blackberry products, but not after storage time. Microfiltration is an application with special importance in fruit-juices. Efforts have been done to produce practical models that can be used for industrial scale and to test parameters that influence the performance of microfiltration techniques (Vaillant et al., 2008). However, it is also important to evaluate the possible effects of the microfiltration technique in beneficial roles documented for fruits-juices rich in polyphenols. The aim of this study was to evaluate the effect of microfiltration of blackberry juice on the antioxidant capacity and the lipid peroxidation protection effect.


Sample collection

Samples were prepared with full-ripe blackberries (Rubus adenotrichus Schltdl., Rosaceae, cv. ‘vino’) harvested in different farms of the province of Cartago, Costa Rica (altitude 1864 m-2517, latitude 09º 39' 57.1''N - 09º44'40.3''N, longitude 83º53'32.1''W - 84º00'06.3''W). To prepare the fresh juice (FJ), blackberries were blended and filtrated in gauze to reduce suspended solids. The microfiltrated juice (MJ) was prepared according to Vaillant et al. (2008). Blackberries were pressed and the juice treated with a commercial enzymatic preparation (Klerzyme® 150 from DSM Food Specialties, Heerlen, Netherlands) for 1 h at 35 °C with constant agitation. The microfiltration was perform in a tubular ceramic membrane (Membralox® 1 P19-40, Pall Exekia, Bazet, France) with an average pore size diameter 0.2 μm. Both samples were stored at -30 °C until analysis.

Analytical methods

Moisture content

The moisture content was determined by the weight difference of the samples after freeze-dried. Each

sample was analyzed in duplicate.

Total polyphenol content

Total phenolic content was determined with Folin-Ciocalteu assay modified by George et al. (2005). Results were expressed as milligrams of gallic acid equivalent (GAE) per gram of dry matter (DM). Each sample was analyzed in four replicates.

Anthocyanin content

Total anthocyanins content was determined by pH differential method (Lee et al., 2005b). Briefly, juice (pH 1.0 and 4.5) absorbance was measured at 510 nm and corrected with the absorbance at 700 nm. The calculation of total anthocyanin content was performed using the molecular weight (449.2 g.mol-1) and molar extinction coefficient of the cyanidin 3-glucoside (26900 L.mol-1.cm-1). Results were expressed as mg cyanidin 3-glucoside per gram of dry matter (DM). Each sample was analyzed in four replicates.

Antioxidant capacity

DPPH radical- scavenging activity

The radical-scavenging activity (RSA) of the fresh juice and the microfiltrated juice were evaluated by assessing their direct DPPH-scavenging activity (Hyun-Jin et al., 2004). DPPH 0.25 mM was prepared in methanol and 0.5 mL of this solution incubated with 1 mL of sample dilutions. Mixtures were incubated at room temperature in the dark for 30 min and the absorbance of DPPH was measured at 517 nm. Methanol (0.5 mL) plus juice dilution (1.0 mL) were used as sample blank. Percentage of radical scavenging activity of the juice was calculated according the formula: %RSA = [1-(Abs sample/Abs control)]* 100. RSA percentage was plotted against the sample concentration and a linear regression curve was established in order to calculate the IC50, which means that amount of juice necessary to reach the 50% radical scavenging activity. Results were expressed as μg DM/mL. Each sample was analyzed in triplicate.

Oxygen radical absorbance capacity (ORAC)

ORAC assay was performed according to Ou et al. (2001). Fluorescein was used as fluorescent probe and the oxidation was induced with AAPH (2,2-azobis-2-methyl-propanimidamide-dihydrochloride). Assays were performed in spectrofluorimeter equipment (Biotek Instruments). ORAC values were expressed as Trolox equivalents (TE/g DM).

Effect of the microfiltration process on antioxidant activity and lipid peroxidation protection capacity of blackberry juice

Gabriela Azofeifa et al.


Protection to lipid peroxidation in liposomes

Liposomes were prepared according to Pérez et al. (2003). Briefly, 25 mg of commercial lecithin was dissolved in 2.15 mL of chloroform and then 350 μL of methanol was added. This mix was dried using a nitrogen atmosphere and finally the lecithin was resuspended in 4.5 mL of warm phosphate buffered saline (PBS) and sonicated for 1 h at 4 °C to form liposomes. To test the juice capacity to protect lipid peroxidation, an oxidative stress was induced with AAPH (2,2-azobis-2-methyl-propanimidamide-dihydrochloride). Fifty microliters of juice dilutions were mixed with 0.45 mL of liposomes and 0.2 mL of AAPH in a final concentration of 10 mM. These solutions were incubated in the dark for 2 h at 37 °C. To determine malondialdehyde (MDA) concentration, 0.2 mL of TCA 5% (trichloroacetic acid) and 1mL TBA 0.75% (thiobarbituric acid) were added and samples were heated at 100 °C for 30 min. After cooling, 0.25 mL SDS 3% was added and centrifuged at 4000 rpm for 15 min. The absorbance of the supernatant was measured at 532 nm. MDA concentrations were plotted against the sample concentration and a linear regression curve was established, results were expressed as the amount of juice that inhibits 50% of lipid peroxidation (IC50).

Protection to lipid peroxidation in liver homogenates

Sprague-Dawley rats (220±20 g) were anesthetized and sacrificed by decapitation according to the Institutional Committee for Care and Handling of Experimental Animals of Universidad de Costa Rica (CICUA # 19-06). Liver tissue of each rat was obtained and homogenized in phosphate buffered saline (PBS) using an Ultraturrax T-25 equipment (Ika-Labortechnik) to obtain a tissue suspension at 20%. The suspension was centrifuged at 9000 x g during 15 min to reduce suspended solids. Seventy-five microliters of different concentrations of juices were added to 0.75 mL of liver-supernatant and incubated for 30 min at 37 °C. Subsequently, an oxidative

stress was induced with TBHP (tert-butyl hydroperoxide) in the final concentration of 1.7 mM and incubated for 1 h at 37 °C. Finally, thiobarbituric acid reactive substances (TBARS) were measured as the end product of lipid peroxidation. TBARS was assayed according to Uchiyama & Mihara (1978). Briefly, 0.25 mL of liver homogenate, 0.25 mL of 35% TCA and 0.25 mL of Tris-HCl buffer (50 mM, pH 7.4) were mixed and incubated 10 min at room temperature. Then, 0.5 mL of 0.75% TBA was added and heated at 100 °C for 45 min. After cooling, 0.5 mL of 70% TCA was added, mixed and centrifuged at 4000 rpm for 15 min. The absorbance of the supernatant was measured at 532 nm. Concentration of TBARS was assessed using the molar absorption coefficient for malondialdehyde (MDA) of 1.56 x 105 cm-1-M-1 and results were expressed as nmol MDA/g liver tissue. MDA concentrations were plotted against the sample concentration and a linear regression curve was established to calculate the IC50. The assay was performed using liver tissue from five rats. To establish basal levels, MDA levels without TBPH were assessed in each rat. Due to the color of blackberries, sample blanks were prepared in each experiment. Each juice sample was tested in triplicate.


To compare FJ and MJ results, statistical analysis was undertaken using ANOVA and Tukey’s test. A p value <0.05 was accepted as statistically significant.

Results

Analytical methods

Table 1 shows the moisture content, total polyphenols (TP) and anthocyanins content of the fresh juice and the microfiltrated juice. The amount of total polyphenols and anthocyanins for both samples are significantly different (p<0.001)

Table 1. Total polyphenols and anthocyanins content in two blackberry samples.Moisture content

(% FW-1)TP

mg GAE g-1 DMAnthocyanins

mg cyanidin 3-glucoside g-1 DMFresh Juice (FJ) 92.0±0.1a 45.0±0.5a 11.5±0.1a

Microfiltrated Juice (MJ) 90.1±0.1a 24.9±1.0b 5.6±0.3b

DM, dry matter, TP, total polyphenols, GAE, Gallic acid equivalents. Each value is mean±SE of four replicate experiments. Means in columns followed with different letters differed significantly (p<0.001).

Table 2. Radical-scavenging activity of two blackberry samples.DPPH IC50 (μg DM mL-1) ORAC (μmol TE g-1 DM )

Fresh Juice (FJ) 101.2±4.8a 547±10a

Microfiltrated Juice (MJ) 94.08±4.2a 417±11b

Each value is mean±SE of three replicate experiments. DM, dry matter. Means in columns followed with different letters differed significantly (p<0.05).



Antioxidant capacity

The DPPH and ORAC assay were utilized to evaluate the antioxidant capacity of both juices. Table 2 shows the amount of each juice necessary to reach the 50% radical scavenging activity (DPPH), and for ORAC the μmol of trolox equivalents for each sample. The comparison of values for DPPH do not show significant differences (p>0.05) but for ORAC the values are significantly different (p<0.05).

Protection to lipid peroxidation

To evaluate protection to lipid peroxidation, lecithin liposomes were incubated with both samples of blackberry juices and exposed to an oxidative stress induced with AAPH. Both juices decrease levels of lipid peroxidation measured as MDA concentration. The IC50 indicates the amount of juice necessary to decrease 50% of MDA concentration of liposomes treated with AAPH. The IC50 was 155.7±13.1 μg DM/mL for MJ and 134.7±11.0 μg DM/mL for FJ. These IC50 values do not show significant differences (p>0.05). Figure 1 shows the inhibitory effect of both blackberry juices against lipid peroxidation induced by TBHP in a liver homogenates of rats. Results indicate a hepatoprotective effect against oxidative stress. FJ and MJ decrease levels of lipid peroxidation in a dose-dependent manner. The amount of FJ and MJ necessary to decrease 50% of MDA concentration of the control liver tissue treated with TBHP are 462±72 μg DM/mL and 429±118 μg DM/mL, respectively. Samples do not show significant differences (p>0.05).

Discussion

Microfiltration technique to produce juices involves mechanical and enzymatic treatments, transmembrane pressure and elimination of some compounds due to retained particles (Vaillant et al., 2008;

Riedl et al., 1998). All these factors affect polyphenols composition of the final juice. As it is shown in this investigation, the microfiltration process decreases the concentrations of total polyphenols and anthocyanins when it is compared to the fresh juice. Gil et al. (2000) emphasize that in the industrial process to produce juice some phenolic compounds are extracted from the fruit, but depending on the technique other ones could increase. The antioxidant activity measured by ORAC was significantly different between FJ and MJ but is not significantly different when is measured by DPPH. According to Niki (2010) variations among antioxidant activity measured by DPPH and ORAC are expected because DPPH is based in the reaction with a stable free radical while ORAC is an assessment by a competition method. This distinction causes a different relative contribution of the compounds responsible for the antioxidant activity in each technique. The results show that the capacity to protect lipid peroxidation is not affected by the differences in the total polyphenol composition of FJ and MJ. This was confirmed both in the liver model and in the liposomes model. It is well known that free radicals are involved to initiate lipid peroxidation (Lee et al., 2005a). Therefore, the high antioxidant capacity shown by FJ and MJ permits an effective radical scavenging and therefore protects to lipid peroxidation. Lee et al. (2005a), suggested that the ability of different compounds to scavenge ROS in liver tissue is particularly important because it’s main role in metabolism of exogenous chemicals that usually generate ROS and induce oxidative stress. In this study, despite ORAC differences for FJ and MJ, the capacity to protect against lipid peroxidation is similar for both samples, even when the lipid peroxidation is induced with AAPH or TBHP. This is in agreement with Niki (2010) who suggests that the capacity of free radical scavenging by antioxidants does not necessarily correlate with the capacity of inhibition of lipid peroxidation. Specifically, this author states that ORAC method does not show the capacity of antioxidants to inhibit lipid peroxidation.

0

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0 56 127 286 643

[MD

A] (

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ver)

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*

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0 5 112 252 566

[MD

A] (

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ver)

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**

Figure 1. Protective effect of fresh juice (A) and microfiltrated juice (B) on TBHP induced oxidative stress in liver homogenates model. Each value is mean±SE (three independent experiments).*p<0.05, **p<0.01 compared with homogenates treated with TBHP.

Effect of the microfiltration process on antioxidant activity and lipid peroxidation protection capacity of blackberry juice

Gabriela Azofeifa et al.


from blood orange, blackberry, and roselle using the Arrhenius, Eyring, and Ball models. J Agric Food Chem 57: 6285-6291.

Cuevas-Rodriguez EO, Díaz VP, Yousef GG, García PA, López J, Paredes O, Gonzalez E, Lila MA 2010. Inhibition of pro-inflammatory responses and antioxidant capacity of Mexican blackberry (Rubus spp.) extracts. J Agric Food Chem 58: 9542-9548.

Gancel AL, Feneuil A, Acosta O, Pérez AM, Vaillant F 2010. Impact of industrial processing and storage on major polyphenols and the antioxidant capacity of tropical highland blackberry (Rubus adenotrichus). Food Res Int (In Press) doi:10.1016/j.foodres.2010.06.013.

George S, Brat P, Alter P, Amiot MJ 2005. Rapid determination of polyphenols and vitamin C in plant-derived products. J Agric Food Chem 53:1370-1373.

Gil MI, Tomás FA, Hess B, Holcroft DM, Kader AA 2000. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J Agric Food Chem 48: 4581-4589.

Hager TJ, Howard LR, Prior RL 2008. Processing and storage effects on monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed blackberry products. J Agric Food Chem 56: 689-695.

Hassimotto NMA, Da Silva Pinto M, Lajolo FM 2008. Antioxidant status in humans after consumption of blackberry (Rubus fruticosus L.) juices with and without defatted milk. J Agric Food Chem 56:11727-11733.

He J, Giusti M 2010. Anthocyanins: natural colorants with health-promoting properties. Food Sci Technol 1: 163-187.

Hyun-Jin K, Feng C, Changqoing W, Xi W, Hau YC, Zhengyu J 2004. Evaluation of antioxidant activity of Australian tea tree (Malaleuca alternifolia) oil and its components. J Agric Food Chem 52: 2849-2854.

Jiao H, Wang SY 2000. Correlation of antioxidant capacities to oxygen radical scavenging enzyme activities in blackberry. J Agric Food Chem 48: 5672-5676.

Lee H, Lee HS, Won NH, Kim KH, Jun W, Lee KW 2005a. Antioxidant effects of aqueous extract of Terminalia chebula in vivo and in vitro. Biol Pharm Bull 28: 1639-1644.

Lee J, Durst W, Wrolstad RE 2005b. Determination of total monomeric anthocyanins pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. J AOAC Int 88: 1269-1278.

Mazza GJ 2007. Anthocyanins and heart health. Ann Ist Super Sanita 43: 369-374.

Mertz C, Cheynier V, Günata Z, Brat P 2007. Analysis of phenolic compounds in two blackberry species (Rubus glaucus and Rubus adenotrichus) by high-performance liquid chromatography with diode array detection and electrospray ion trap mass spectrometry. J Agric Food Chem 55: 8616-8624.

Mullen W, McGinn J, Lean MEJ, MacLean MR, Gardner P, Duthie GG, Yokota T, Crozier A 2002. Ellagitannins, flavonoids, and other phenolics in red raspberries and their contribution to antioxidant capacity and vasorelaxation properties. J Agric Food Chem 50: 5191-5196.

Niki E 2010. Assessment of antioxidant capacity in vitro and in vivo. Free Radic Biol Med 49: 503-515.

This study concludes that differences in the amounts of polyphenols and anthocyanins after the microfiltration could alter the antioxidant capacity but do not alter the capacity to protect against lipid peroxidation. Mullen et al. (2002) evaluated changes in polyphenols quantity due to industrial process with similar results to our study. Mullen’s study in raspberries evaluates the effect of short-term treatments, such as storage and freezing. For raspberries, significant differences in total phenolics were observed and changes between free and conjugated forms of anthocyanins were evident. Mullen concluded that antioxidant capacity is not due to a single anthocyanin, instead it is a combined contribution of different anthocyanins. Blackberry juice has a very short shelf-life and that is why microfiltration process coupled to freezing storage is important to maintain the juice available for longer periods. Several other industrial processes are available, but some of them such as pasteurization present an important reduction of the health-promoting properties of processed-juice (Gancel et al., 2010). Microfiltration technique coupled to UHT packaging provides a sterile juice thanks to the pore used in the membranes. This study suggests that this procedure could permit the preservation of the capacity to inhibit lipid peroxidation in the microfiltrated juice. However, further research is needed to characterize the compounds, the interactions of polyphenols after the microfiltration process, and how these affect biological activities.

Acknowledgment

Financial support for this work was funded by Vicerrectoría de Investigación, Universidad de Costa Rica (Project No. 422-A7-028), Producing Added-Value from Under-Utilized Crops project (PAVUC-FP6-INCO project- DEV-2, contract 015279). Authors thank Marielos Torres (CITA, UCR) and AL Torres (Departamento de Bioquímica, Escuela de Medicina, UCR) for their technical assistance.

References

Acosta-Montoya O, Vaillant F, Cozzano S, Mertz C, Perez AM, Castro MV 2010. Phenolic content and antioxidant capacity of tropical highland blackberry (Rubus adenotrichus Schltdl.) during three edible maturity stages. Food Chem 119: 1497-1501.

Beattie J, Crozier A, Duthie GG 2005. Potential health benefits of berries. Curr Nutr Food Sci 1: 71-86.

Bowen-Forbes CS, Yanjun Z, Muraleedharan GN 2010. Anthocyanin content, antioxidant, anti-inflammatory and anticancer properties of blackberry and raspberry fruits. J Food Compos Anal 23: 554-560.

Cisse M, Vaillant F, Acosta O, Dhuique-Mayer C, Dornier M 2009. Thermal degradation kinetics of anthocyanins



Ou B, Hampsch-Woodill M, Prior RL 2001. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J Agric Food Chem 49: 4619-4626.

Pérez RM, Vargas R, Martínez FJ, García EV, Hernández B 2003. Antioxidant activity of alkaloids from Bocconia arborea. A study on six testing methods. Ars Pharm 44: 5-21.

Pietta PG 2000. Flavonoids as antioxidants. J Nat Prod 63: 1035-1042.

Riedl K, Girard B, Lencki RW 1998. Influence of membrane structure on fouling layer morphology during apple juice clarification. J Membrane Sci 139: 155-166.

Rossi A, Serraino I, Dugo P, Di Paola R, Mondello L, Genovese T, Morabito D, Dugo G, Sautebin L, Caputi AP, Cuzzocrea S 2003. Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation. Free Radic Res 37: 891-900.

Uchiyama M, Mihara M 1978. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal

Biochem 86: 271-278.Vaillant F, Pérez AM, Acosta O, Dornier, M 2008. Turbidity of

pulpy fruit juice: a key factor for predicting cross-flow microfiltration performance. J Membrane Sci 325: 404-412.

Zafrilla P, Ferreres F, Tomás-Barberán FA 2001. Effect of processing and storage on the antioxidant ellagic acid derivatives and flavonoids of red raspberry (Rubus idaeus) jams. J Agric Food Chem 49: 3651-3655.

*Correspondence

Quesada SilviaDepartamento de Bioquímica, Escuela de Medicina, Universidad de Costa Rica, San Pedro, 2060, San José, Costa [email protected]. +506 2511 4450 Fax: +506 2511 5338

835

Article


695X2011005000076

Received 22 Dec 2010Accepted 20 Jan 2011



21(5): 835-840, Sep./Oct. 2011The activity of flavones and oleanolic acid from Lippia lacunosa against susceptible and resistant Mycobacterium tuberculosis strains

Aline Castellar,1 Tatiane S. Coelho,2 Pedro E. A. Silva,2 Daniela F. Ramos,2 Maria Cristina S. Lourenço,3 Celso L. S. Lage,1 Lisieux S. Julião,1 Ymira G. Barbosa,4 Suzana G. Leitão*,4

1Programa de Biotecnologia Vegetal, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Brazil,2Laboratório de Micobactérias, Fundação Universidade Federal do Rio Grande, Brazil,3Laboratório de Bacteriologia e Bioensaios em Micobactérias, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Brazil,4Departamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Brazil.

Abstract: Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world’s number one killer among infectious diseases. The search for new natural products that can act as drugs against TB has received increased attention during the last years. In this work we describe the isolation and identification of the active antimycobacterial principles of the dichloromethane extract from Lippia lacunosa Mart. & Schauer, Verbenaceae. Compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis (susceptible and rifampicin resistant strain) using a redox bioassay. From the dichloromethane extract of L. lacunosa leaves, seven methoxy-flavones named cirsimaritin (1), eupatilin (2), eupatorin (3), salvigenin (4), 3′-O-methyl-eupatorin (5), 3′,7-dimethoxy-5,6,4′- trihydroxyflavone (6), and 7′-O-methylapigenin (7), and one triterpene, named oleanolic acid (8), were isolated. All compounds were found to display antimycobacterial activity against susceptible strain, with MIC ranging from 25 to 200 µg/mL. None of them was active against rifampicin resistant strain. This is the first report in the antimycobacterial activity of 6-substituted flavones, as well as the first report of the occurrence of these substances in L. lacunosa.

Keywords:Lippia lacunosa

Verbenaceaeflavonoids

antimycobacterial activity Mycobacterium tuberculosis

methoxyflavonesoleanolic acid.

Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world’s number one killer among infectious diseases (Lin et al., 2002). Each year, about 400,000 people develop TB with isoniazid- and rifampicin-resistant strains (MDR) (Silva et al., 2008). The emergence of MDR strains with human immunodeficiency virus (HIV) co-infection, as well as the decline of socioeconomic conditions in several places around the world, has amplified the difficulty in controlling this disease (Lin et al., 2002; Lechner et al., 2008). The search for new drugs against TB has received increased attention during the last couple of years. Several candidates are being investigated and an interesting array of compounds constitutes the current portfolio of promising drugs. These drugs should be

active against both susceptible and resistant strains (Silva & Ainsa, 2007). In the last couple of years, several reports and review articles have appeared in the literature about medicinal plants and natural products with antimycobacterium activity (Okunade et al., 2004; Copp, 2003; Newton et al., 2002; Cantrell et al., 2001). Over 350 natural products, mainly from plant species, have been assessed for their antimycobacterial activities (Newton et al., 2002). A number have demonstrated significant in vitro activity and active plant-derived compounds belonging to various chemical classes have been isolated (Newton et al., 2002). The genus Lippia (Verbenaceae) includes approximately 200 species and is found in Central and South America, as well as in some areas of Tropical Africa (Salimena-Pires, 1991). One of the main diversity centers of this genus is located in the

The activity of flavones and oleanolic acid from Lippia lacunosa against suscep-tible and resistant Mycobacterium tuberculosis strains Aline Castellar et al.


central region of Brazil, mainly in “cerrado” biome, at the “Cadeia do Espinhaço” Mountains, in the State of Minas Gerais (Leitão et al., 2008). Some species, such as Lippia alba (Mill.) N. E. Brown, Lippia origanoides H. B. K., Lippia sidoides Cham., and Lippia graveolens Kunth, are widely used for different purposes such as medicinal and culinary use (Pascual et al., 2001). The interest in this genus is based on the presence of flavonoids which is a class of low molecular weight phenolic compounds that are widely distributed in the plant kingdom. They exhibit different biological functions that allow interactions between plants and their environment: they are involved in the plant pathogen, plant-plant interactions and plant-insect interactions (Treutter, 2005). Recent studies have also indicated that flavonoids can act as cellular modulators by interaction with enzymes, receptors and transcription factors of intracellular signaling. These interactions could explain their potential anticancer and neurodegeneration-inhibiting activities (Williams et al., 2004). During the course of our search for Brazilian plant extracts that are active against Mycobacterium tuberculosis, the dichloromethane extract from the leaves of Lippia lacunosa Mart. & Schauer showed moderate anti-mycobacterial activity (minimal inhibitory concentration, MIC of 25 µg/mL) (Leitão et al., 2006). In this study, we report the fractionation of this extract, which resulted in the isolation of the seven flavonoids and one triterpene, besides the evaluation of their antimycobacterial activity against Mycobacterium tuberculosis by the same method.


Plant material and morphological analysis

Fresh leaves of Lippia lacunosa Mart. & Schauer, Verbenaceae, cultivated from clones originally from Diamantina, MG, Brazil (Viccini et al., 2004), were collected in October 2006, at the campus of the Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil, (22o46’48.6”S, 43o22’24.5”W). The plant was authenticated by Dr. Fatima Regina Gonçalves Salimena, and a voucher of specimen is deposited at the Herbarium of the Departamento de Botânica, Universidade Federal de Juiz de Fora (CESJ 41.691).

Plant extracts

Leaves (800 g) of L. lacunosa were air-dried, powdered, and extracted by maceration exhaustively with ethanol at room temperature. After filtration and concentration under reduced pressure, the aqueous residue was sequentially extracted with organic solvents

of increasing polarities, to afford the new extracts: n-hexane (LLFH, 12.2 g), dichloromethane (LLFD, 6.1 g), ethyl acetate (LLFA, 15.4 g) and n-butanol (LLFB, 46.5 g) extracts, in this order, as well as the remaining water-soluble extract (LLFW).

Isolation and identification of substances from the extracts

Part of the dichloromethane extract (LLFD, 3.0 g) was chromatographed on silica gel, and eluted with dichloromethane containing increasing amounts of ethyl acetate. Then it was eluted with ethyl acetate containing increasing amounts of methanol, affording thirty fractions (I-XXX). Fractions V and VI (375 mg), eluted with dichloromethane-ethyl acetate (9:1), were pooled (monitored by TLC) and re-chromatographed on Sephadex LH-20 (methanol), affording oleanolic acid (8, 66 mg), (Mahato & Kundu, 1994), and a mixture of flavonoids. The flavonoid mixture was further separated by preparative layer chromatography on silica gel plates using dichloromethane-ethyl acetate (9:1) as the solvent system. Using this procedure, salvigenin (4, 1.5 mg) (Skaltsa & Shammas, 1988); 3’-O-methyl-eupatorin (5, 2 mg) (Valant-Vetshera & Wollemweber, 1988), and cirsimaritin (1, 9 mg) (Valant-Vetshera & Wollemweber, 1988) were isolated. Fraction XII (637 mg), eluted with dichloromethane-ethyl acetate (8:2), was re-run on silica gel CC, affording 27 fractions. Fraction XII-8, eluted with dichloromethane-ethyl acetate (9:1), afforded additional 3’-O-methyl eupatorin, (5, 1.2 mg); fraction XII-15 yielded eupatilin (2, 2.3 mg) (Valant-Vetshera & Wollemweber, 1988), and fraction XII-20 afforded eupatorin (3, 1.8 mg) (Valant-Vetshera & Wollemweber, 1988). Fractions XIII-XV (466 mg), eluted with dichloromethane-ethyl acetate (7:3) on the original column, were re-submitted to purification on silica gel CC and eluted with dichloromethane-ethyl acetate (9:1 to 8:2), to afford 35 fractions. From fraction XIII-XV-23, 3’,7-dimethoxy-5,6,4’-trihydroxyflavone (6, 10.7 mg) (Christensen & Lam, 1991) was isolated, and from fraction XIII-XV-30, 7’-O-methylapigenin (7, 12 mg, also known as genkwanin) (Wollemweber, 1986) was isolated.

O

OOH

R1

R2

R4

R3

1 R1 = R2 = OCH3; R3 = H; R4 = OH2 R1 = OH; R2 = R3 = R4 = OCH33 R1 = R2 = R4 = OCH3; R3 = OH4 R1 = R2 = R4 = OCH3; R3 = H5 R1 = R2 = R3 = R4 = OCH36 R1 = R3 = OCH3; R2 = R4 = OH7 R1 = OCH3; R2 = R3 = H; R4 = OH

HHOH

CO2HH

8

The activity of flavones and oleanolic acid from Lippia lacunosa against suscep-tible and resistant Mycobacterium tuberculosis strains

Aline Castellar et al.


oleanolic acid, dissolved in DMSO. The minimal inhibitory concentration (MIC) was carried out by two fold serial dilutions of the drugs, starting from 200 µg/mL.


In a previous study from our group (Leitão et al., 2006) the dichloromethane extract from leaves of L. lacunosa showed antimycobacterial activity (MIC 25 µg/mL) against M. tuberculosis H37Rv. In this work, seven methoxyflavones (1-7) and one triterpene (8) were isolated from this extract and evaluated against M. tuberculosis H37Rv and rifampicin resistant strains (Table 1). These compounds were identified by the combination of 1D and 2D NMR techniques, as well as UV data of flavonoids. All tested substances showed activity against M. tuberculosis H37Rv. The most active compound was 3’-O-methyl-eupatorin (5, MIC 25 µg/mL), followed by cirsimaritin (1), eupatilin (2) and eupatorin (3, MIC 50 µg/mL). Salvigenin (4), and 3’,7–dimethoxy-5,6,4’-trihydroxyflavone (6) were the least active substances tested, affording MIC of 100 and 200 µg/mL, respectively. Even though there are many reports on the antimycobacterial activity of the triterpene oleanolic acid (MIC of 50-69 µg/mL) in the literature (Jiménez-Arellanes, 2007; Copp, 2003; Cantrell et al., 2001; Caldwell et al., 2000; Koysomboon et al., 2006; Tanachatchairatana et al., 2008) it was re-evaluated against H37Rv as well as against the rifampicin resistant strain. Our results for H37Rv are compatible with those previously reported. However, oleanolic acid was inactive against this rifampicin-resistant strain at the maximum assayed concentration of 200 µg/mL. Due to the isolation of very small amounts of the flavonoids 2-7, they were not assayed against the rifampicin-resistant strain. The antimycobacterial activity of flavonoids has already been reported by many authors. Lin and coworkers (2002) have described the antimycobacterial activity of 142 synthetic chalcones, flavonoids (including some methoxyflavones) and chalcones-like compounds against M. tuberculosis H37Rv, and concluded that flavones showed decreased activity with respect to the corresponding chalcones. Another paper on antimycobacterial activity of flavonoids describes the results of the susceptibility of M. tuberculosis H37Ra, an avirulent strain, against fourteen flavonoids, including rotenoids and prenylated isoflavones from Derris indica (Murillo et al., 2003). In this paper, the authors regret that even with the concomitant isolation of fourteen different compounds from the same class (flavonoids), the careful inspection of the comparison of their molecular structures and their activities did

The identity of the isolated compounds was confirmed by 1D and 2D-NMR techniques (1H, HMBC and HSQC) on a Bruker Avance DRX400 (Karlsruhe, Germany) at 25 °C, operating at 400.13 MHz for 1H and 100.61 MHz for 13C. NMR spectra were recorded in CDCl3 (flavonoids) or deuterated pyridine (oleanolic acid), using TMS as an internal standard. The data obtained was in accordance with that found in the literature. Additionally, UV data of the flavonoids in methanol, followed by the use of AcONa as a shift reagent was used for identification purposes (Mabry et al., 1970).

Tested material

Cirsimaritin (1), eupatilin (2), eupatorin (3), salvigenin (4), 3’-O-methyl-eupatorin (5), 3’,7-dimethoxy-5,6,4’-trihydroxyflavone (6), 7’-O-methylapigenin (genkwanin) (7) and oleanolic acid (8), obtained as described above.

Bacterial strains

M. tuberculosis strain H37Rv (ATCC-27294) was used for all experiments in both of the microbiology laboratories. Additional assays with a rifampicin-resistant strain (ATCC-35838, His-526-Tir) were performed at the FURG laboratories. The isolates were maintained in Ogawa-Kudoh medium for ca. fourteen days. The bacterial suspensions were prepared in sterile water containing 3-mm glass beads. The suspensions were homogenized by vortex agitation and turbidity was adjusted in agreement with tube one of the McFarland scale (3.2 x 106 colony-forming units/mL). The inoculums were prepared by diluting the bacterial suspension 1:25 in Middlebrook 7H9 OADC medium (4.7 g Middlebrook 7H9 base; Difco, Becton Dickinson) enriched with 10% (v/v) oleic acid-dextrose-albumin-catalase (BBL).

In vitro evaluation of anti-tuberculosis activity and Minimum Inhibitory Concentration (MIC)

Samples were screened against Mycobaterium tuberculosis strain H37Rv (ATCC-27294) using microtiter assays containing a redox indicator (Franzblau et al., 1998; Palomino et al., 2002). The final concentration of the substances and fractions was either 100 or 200 µg/mL. Media plus bacteria, with and without rifampicin, were used as controls. MIC determination was carried out as described in literature (Franzblau et al., 1998; Palomino et al., 2002). In brief, redox assays were performed in 96-well microplates using resazurin, Alamar Blue or MTT as the indicator of cellular viability, and the extracts, flavonoids or



not reveal any useful or consistent trends. Three other publications on antimycobacterial flavonoids report the activity against M. tuberculosis H37Rv of methoxyflavones obtained from Haplopappus sonorensis (Yenjai et al., 2004), Kaempferia parviflora (Gu et al., 2004), and Valeriana laxiflora (Haermers et al., 1990). Again, no conclusion on structure-activity could be drawn. High lipophilicity of substances has been reported as an important feature for antimycobacterial activity (Silva et al., 2007). Due to the fact that the cell wall of mycobacteria contain lipophilic substances such as mycolic acid, more lipophilic substances are likely to penetrate more easily into the cell (Palomino et al., 2002). It has been demonstrated for a series of terpenes that the activity improves with the lipophilicity of a given substance when compared to their more polar analogues (Cantrell et al., 2001). To date, no flavonoid glycosyl derivative has been shown to be active against M. tuberculosis, and compounds such as quercetin have demonstrated only weak activity. Thus, if polarity is considered as an important factor of the antimycobacterial activity of flavonoids, it would be expected that in a series of methoxyflavones, that the higher the degree of methyl substitution the higher the activity of the derivatives. Analysis of the MIC of methoxyflavones 1-7 in Table 1, shows, as expected, that the tetramethoxy flavone derivative 5 (3’-O-methyl-eupatorin) is the one displaying the best activity (MIC 25 µg/mL). However, this hypothesis does not hold

when we compare the MIC of the dimethoxy flavone 6 (200 µg/mL) with that of monomethoxy flavone 7 (50 µg/mL). Furthermore, a critical analysis of the structures of the trimethyl derivatives 2 and 3, as well as the dimethyl derivatives 1 and 6 does not reveal any structure-activity relationship. Recently, the mechanisms of resistance of mycobacteria have garnered a lot of attention. Besides the classic resistance mechanisms, other mechanisms related to intrinsic and acquired resistance, such as efflux pump mechanisms, have been described. Drugs that can decrease resistance by inhibition of the efflux pump have been an important goal in the research of new drugs. For instance, INH resistance has been associated with the activation or induction of an energy-dependent efflux pump (Lechner et al., 2008). In this work, two of the isolated compounds, cirsimaritin (1), and oleanolic acid (8), were obtained in large enough quantities to be assayed against the rifampicin–resistant Mycobacterium tuberculosis strain 35838. One interesting feature about oleanolic acid (8), is its inhibitory activity on the multidrug resistance protein ABCC1 (MRP1) (Braga et al., 2007). Furthermore, a number of flavonoids have been described as MRP1 inhibitors (Felipe et al., 2008) to date. However, even at 200 µg/mL, compounds 1 and 8 failed to show any activity against this rifampicin-resistant strain. The antimycobacterial activity of oleanolic acid against a rifampicin-resistant strain has already been reported in literature (Jimenez-Arellanes, 2007) with a MIC of

Table 1. Minimal inhibitory concentration (MIC) of flavonoids (1-7) and of oleanolic acid (8), isolated from the dichloromethane extract from leaves of Lippia lacunosa, against Mycobacterium tuberculosis strains.

Substances

O

OOH

2

346

7

8

2'

3'

4'

5'

6' MIC (μg/mL)

Flavonoids 6 7 3’ 4’ H37Rv 35838cirsimaritin (1) OCH3 OCH3 - OH 50 Inactive at 200 µg/mLeupatilin (2) OCH3 OH OCH3 OCH3 50 *eupatorin (3) OCH3 OCH3 OH OCH3 50 *salvigenin (4) OCH3 OCH3 - OCH3 100 *3’-O-methyl-eupatorin (5) OCH3 OCH3 OCH3 OCH3 25 *3’,7– dimethoxy-5,6,4’-trihydroxyflavone (6) OH OCH3 OCH3 OH 200 *7’-O–methyl-apigenin (7) - OCH3 - OH 50 *TriterpeneOleanolic acid (8) 50 Inactive at 200 µg/mLRifampicin 1 -

*not tested.

The activity of flavones and oleanolic acid from Lippia lacunosa against suscep-tible and resistant Mycobacterium tuberculosis strains

Aline Castellar et al.


50 μg/mL. However, as we don’t know the molecular basis of rifampicin resistance of the strain assayed by Jimenez-Arellanes (2007) it is very difficult to establish some comparative analysis between these different results, since we can’t infer that they are genetically identical. From the dichloromethane extract of Lippia lacunosa seven flavones, including six 6-substituted flavones, were isolated. Our results of the antimycobacterial activity of 6-substituted flavones from Lippia lacunosa show the importance of investigating the phytochemistry area to discover new drugs against TB. This is the first report concerning the non-volatile chemistry of this plant, as well as the first report of the antimycobacterial activity of 6-substituted methoxyflavones.

Acknowledgements

The authors wish to thank CNPq (Edital MCT-CNPq/MS-SCTIE-DECIT 25/2006, Process no. 410475/2006-8) and FAPERJ (E-26/111.614/2008) for financial support. We are also indebted to Centro Nacional de Ressonância Magnética Nuclear Jiri Jones, UFRJ, Rio de Janeiro, for the use of NMR equipment, and to Prof. Lyderson Viccini and Prof. Fatima G. Salimena, from Universidade Federal de Juiz de Fora, for supplying us with plant material. Some of us are indebted to CAPES and CNPq for fellowships, CDTS/Plataforma de Bioensaios II, FIOCRUZ, and for UNICEF/UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR). Collaborative work was performed under the auspices of the Iberoamerican Program for Science and Technology (CYTED), Project X.11:PIBATUB.

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*Correspondence

Suzana G. LeitãoDepartamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro Bloco A, 2º andar, sala 4, 21.941-590 Rio de Janeiro-RJ, [email protected].: +55 21 2562 6413Fax: +55 21 2562 6425

841

Article


695X2011005000123

Received 21 Dec 2010Accepted 10 Mar 2011

Available online 8 Jul 2011


21(5): 841-849, Sep./Oct. 2011Enantiomeric distribution of key volatile components in Citrus essential oils

Ivana Bonaccorsi,*,1 Danilo Sciarrone,1 Antonella Cotroneo,1 Luigi Mondello,1,2 Paola Dugo,1,2 Giovanni Dugo1

1Dipartimento Farmaco-chimico, Università di Messina, Italy,2Università Campus Bio-Medico, Rome, Italy.

Abstract: Citrus as many other plants present characteristic distribution of some enantiomers, thus it is often possible to use this parameter for identifi cation, characterization, genuineness, and pharmacological activity assessment. In particular, it is possible to reveal adulteration of different nature, such as addition of synthetic compounds, or natural components of different botanical origin, with drastic changes in the biological and olfactory properties. This study is focused on the evaluation of the enantiomeric excesses of numerous samples of different Citrus species: C. deliciosa Ten., C. limon (L.) Burm., C. bergamia, C. aurantifolia (Christm.) Swing., C. latifolia Tan., C. sinensis (L.) Osbeck, and C. aurantium L. The enantiomeric distribution is determined by direct esGC and, depending on the complexity of the essential oil, by MDGC with a chiral column in the second dimension. The research is focused on the determination of fourteen chiral components which present specifi c distribution in the essential oils investigated. Particular attention is given to the trend of the enantiomeric distribution during the productive season, so to identify useful parameters for quality assessment also in consideration of the wide range of variability often reported in literature. The components investigated were the following: α-thujene, α-pinene, camphene, β-pinene, sabinene, α-phellandrene, β-phellandrene, limonene, linalool, camphor, citronellal, linalyl acetate, terpinen-4-ol, α-terpineol. The use of MDGC allowed the separation of the enantiomers of camphor and citronellal, otherwise not separated by conventional esGC; however for the separation of the enantiomers of α-pinene it was preferable to use conventional esGC. The MDGC system allowed to determine the enantiomeric distribution of camphene, α- and β-phellandrene in lime essential oil for the fi rst time. The results are discussed in function of seasonal variation and, when possible, in function of the extraction technology, with particular regards to lime oils.

Keywords:Citrus essential oilsenantiomeric purity;

monoterpenesesGC

MDGC

Introduction

Citrus essential oils are widely used in flavor, fragrances, food, soft drink and liqueurs, they are included in cosmetics, perfumes, detergents, body-care products, and in a large number of pharmaceutical preparations (gynecological, ophthalmic, surgical, dentistry etc.). These essential oils are appreciated mostly for their fragrances but also for their solvent properties and their well-known antiseptic properties. Citrus as many other plants present highly characteristic distribution of some enantiomers, thus it is often possible to use this parameter for identification, characterization, genuineness and pharmacological activity assessments. In particular, by the enantiomeric distribution of key components it is possible to reveal adulteration of different nature, such as addition of synthetic compounds, or natural components of different botanical origin, which may drastically

change the biological and olfactory properties. In literature numerous articles on the biological activities exerted by citrus essential oils are available. Most of these were recently revised by Bisignano & Saija, 2011. Since essential oils are chemically complex matrices (containing tens of different compounds) their biological activity is difficult to explain. Very little is known on the effectiveness of single compounds. Many reports on the activities of essential oils often apply the terms “synergism”, “antagonism”, “additivity” to overcome the need of identifying the single components activities; others simply cite the main components present in the whole oil without further discussion. Few are the references available on the different biological properties exerted by pure compounds (mainly limonene and linalool) naturally present in citrus oils (Ruberto & Baratta, 2000; Heuberger et al., 2001; Peana et al., 2002, 2006; Frum & Viljoen, 2006; Kamatou & Viljoen, 2008; Ozek et al., 2010).

Enantiomeric distribution of key volatile components in Citrus essential oilsIvana Bonaccorsi et al.


It is widely known that enantiomers of the same molecule possess different properties, they can work as growth regulators, support plant reproduction, defend the plants from external attacks. Within a vegetable individual enantiomers generally show quantitative differences, although sometimes they can be present as racemic mixtures, and exert different activity based on their stereochemical configuration. This study may provide useful information to stimulate researches focused on the evaluation of biological activity exerted by citrus oils by testing the properties of single enantiomers in function of their chiral purity in different citrus species. The composition of citrus oils was deeply investigated in the last decades by modern analytical techniques. Most of the literature on the volatile fraction was recently revised by Dugo et al., 2011; the non-volatile fraction was revised by Dugo & Russo, 2011; the chiral compounds were revised by Mondello et al., 2011 and the carotenoid fraction by Dugo & Giuffrida, 2011. Recently few articles were published by our research group on the characterization of citrus oils by means of enantioselective separation (Dugo et al., 2010; Dugo et al., 2011a,b; Bonaccorsi et al., 2009. Successful enantioselective-GC (esGC) is performed by cyclodextrin derivatives (CDD) to evaluate essential oils through the enantiomeric distribution of their optically active components. New CDD greatly enlarged the number of separable racemates and it is possible to evaluate the essential oils through several of its optically active components in a single GC run. The recovery of pure reference is not always possible and often the identification of the correct enantiomer is based on literature information. In natural matrices the separation of enantiomers of components present at different concentrations is quite complex. Direct enantioselective GC (esGC) separation is often compromised by the presence of very large peaks (e.g. limonene in sweet orange) which overload the capillary column, and by numerous peak overlaps that occur between the enantiomers and other components, therefore different analytical approaches should be evaluated. In particular, to avoid this inconvenient and to reduce peak overlapping a good choice is the approach by multidimensional gas chromatography (MDGC). Using MDGC each peak eluting from the first dimension of the system (usually a conventional apolar column), well separated from other components, is transferred, partially or totally, to the second dimension (chiral column). It is possible to transfer numerous peaks from the first to the second dimension during the same analysis. In this study the enantiomeric distribution is determined by direct esGC or, depending on the complexity of the essential oil, by MDGC.

The determination of the chiral components with a specific distribution in each of the essential oils are investigated. Particular attention was paid to the trend of the enantiomeric distribution during the productive season, useful to determine parameters for quality assessment considering of the wide range of variability often reported in literature for specific components. The components investigated were: α-thujene, α-pinene, camphene, β-pinene, sabinene, α-phellandrene, β-phellandrene, limonene, linalool, camphor, citronellal, linalyl acetate, terpinen-4-ol, α-terpineol. The use of MDGC allows the separation of the enantiomers of camphor and citronellal, otherwise not separated by conventional esGC; however for the separation of the enantiomers of α-pinene it is preferable to use conventional esGC. The MDGC system allowed to determine the enantiomeric distribution of camphene, α- and β-phellandrene in lime essential for the first time.


Samples

The analyses were carried out on of 251 samples of different Citrus species: C. deliciosa Ten., C. limon (L.) Burm., C. bergamia, C. aurantifolia (Christm.) Swing., C. latifolia Tan., C. sinensis (L.) Osbeck, and C. aurantium L. were analyzed by esGC or by MDGC to determine the enantiomeric purity of selected compounds. All the samples were surely genuine with the exception of one commercial sample of Italian bitter orange oil. All the samples were obtained by cold-extraction by the most appropriate machines as described below. With the exception of Key lime oil Type A none of the extraction techniques used allow the contact of the oil with the acid juice of the fruits. The 124 samples of Italian mandarin, were cold-extracted by screw press and by Brown International Corp. (BOE) machines from fruits collected during an entire productive season; The 92 samples of Italian lemon oil were cold-pressed by FMC (FMC Corporation, Citrus Machinery Divison) and BOE machines from fruits produced during an entire productive season; the eight samples of Italian bergamot oil were cold-pressed by Pelatrice machines from fruits collected during an entire productive season; the three samples of Key lime type A were produced by cold-extraction, using screw press on the crushed fruits and centrifuge to separate the oil from the emulsion; the three of Type B were produced by cold-extraction (Raspatrice and Speciale) in Mexico; the two samples of Mexican



Persian lime were cold pressed by FMC; the three genuine samples of bitter orange oil, one Italian and two from Egypt were obtained by Pelatrice machines; The seventeen samples of Italian sweet orange oil, were produced in Sicily by FMC extractors collected during an entire productive season.

Analysis

EsGC: Shimadzu GC2010 gas chromatograph equipped with a Flame Ionization Detector (FID), a split/splitless injector and an AOC-20i series autoinjector. Capillary chiral column was a Megadex DETTBS-β-(diethyl-tert-butyl-silyl β-cyclodextrin) 25 m x 0.25 mm I.D. x 0.25µm df (Mega, Legnano, Italy). Temperature program: 50-200 °C at 2 °C/min. Inlet pressure 96.6 kPa (220 °C), split mode 1:20 (gas carrier He); injected volume, 1.0 µL (1:10 in n-hexane); linear velocity, 35 cm/s (constant). Data handling was made by means of GCsolution software Multidimensional enantio-GC (MDGC) analyses were carried out on a MDGC system consisted of two GC2010 (defined as GC1 and GC2) gas chromatographs, equipped with a Deans type switch transfer device, an MS-QP2010 quadrupole mass spectrometer, and an AOC-20i autosampler (Shimadzu, Japan). GC1 was equipped with a split/splitless injector and a flame ionization detector (FID1). The MDGC switching element, located inside the oven, was connected to an advanced pressure control (APC) system which supplied carrier gas (He) at constant pressure. GC1: column, SLB-5MS (silphenylene polymer, virtually equivalent in polarity to 5% diphenyl, 95% methylpolysiloxane) 30 m x 0.25 mm i.d. x 0.25 µm df (Supelco, Milan, Italy). The operational conditions were as follows: constant inlet pressure 220 kPa (300 °C), split mode 1:20 (gas carrier He), injected volume 1.5 µL (1:10 in n-hexane), initial linear velocity 30 cm/s. Temperature program: 50 to 280 °C at 3 °C/min. The FID (300 °C) was connected, via a stainless steel retention gap, to the transfer system; sampling rate: 80 s. APC constant pressure: 130 kPa. GC2 was equipped with a split/splitless injector and a flame ionization detector (both not used in the present research). Transfer line between the two GC systems was kept at 180 °C. GC2: column: Megadex DETTBS-β-(diethyl-tert-butyl-silyl β-cyclodextrin) 25 m x 0.25 mm i.d. x 0.25 µm df (Mega, Legnano, Italy). Temperature program: 40 °C (20 min) to 100 °C at 1 °C/min, to 160 °C at 3 °C/min. Detector MS, Ion source: 200 °C, Interface temp.: 220 °C, Interval scan: 40-400 m/z, Scan speed: 2000 a.m.u. per second (5 Hz).

Identification and quantitative analysis

Peak identification was based on the results obtained from previous studies carried out in the authors’ laboratory by injecting standard references and by GC-MS identification. The methods (esGC and MDGC) were tested for repeatability by triplicates with RSD values ranging from 3 to 9% (highest value refer to (+)-α-thujene) (Sciarrone et al., 2010).


The use of esGC and MDGC allowed the determination of the enantiomeric distribution of monoterpene hydrocarbons (α-thujene, α-pinene, camphene, β-pinene, sabinene, α-phellandrene, β-phellandrene, limonene) monoterpene alcohols (linalool, terpinen-4-ol, α-terpineol), linalyl acetate, camphor and citronellal in 251 samples of essential oils of seven different citrus species. The choice of using both techniques was dictated by the complementarity of the results. In fact by direct enantioselective chromatography it is not possible to separate citronellal and camphor isomers in all the essential oils analyzed depending on the complexity of the oil. The determination of the exact value of β-phellandrene can be compromized by the coelution of the (+)-isomer with α-terpinene. In this case MDGC is mandatory in order to obtain reliable results. On the other hand due to the chromatographic conditions applied in MDGC the separation of the enantiomers of α-pinene is compromized. These enantiomers can be well separated by direct esGC. With the exception of β-phellandrene, camphor and citronellal, which were all determined by MDGC, and for the α-pinene enantiomers, separated by esGC, the enantiomeric distribution determined by the two chromatographic techniques were in excellent agreement. Table 1 reports the ranges determined in lemon, bergamot, mandarin, limes and sweet orange oils of the enantiomeric distribution and the total percentages in the whole volatile fraction of the fourteen components investigated. Table 2 reports the enantiomeric distribution and the total percentages in the whole volatile fraction, determined in the samples of bitter orange oil. For the quantitative composition of the oils please refer to the previously published results reported in the review by Dugo et al., 1999. Among the seven Citrus species analyzed it is possible to identify key components with characteristic enantiomeric distribution, useful for their characterization. In the graphs reported in Figure 1-4 it is possible to see the most significant seasonal behavior of the enantiomeric excesses for lemon, bergamot, mandarin and sweet orange oils. Figure 5 compares the enentiomeric excess in Key lime types A and B and Persian lime. With regards to bitter orange oil it was not possible



Table 1. Enantiomeric distribution (ranges) determined by esGC and MDGC, and relative peak area % determined by GC on the whole volatile fraction. Number of samples are indicated in brackets.

Lemon Bergamot Mandarin Key Lime Persian Lime Sweet Orange

Italy Italy Italy Mexico Mexico Italy

(92) (8) (124) (3) (3) (2) (17)

Type A Type B

1 (+)-α-thujene 0.7-1.5 0.9-1.4 0.5-0.8 0.9-1.1 0-1.2 0.5-0.7 10.2-62.0

2 (-)- 99.3-98.5 99.1-98.6 99.5-99.2 99.1-98.9 100-98.8 99.5-99.3 89.8-38.0

% 0.27-0.54 0.15-0.49 tr-1.06 0.27-0.43 0.46-0.6 tr-0.03

3 (+)-α-pinene 25.5-31.5 30.2-32.6 21.8-22.5 22.3-23.3 28.6-30.0 91.1-99.4

4 (-)- 74.5-68.5 69.8-67.4 78.2-77.5 77.7-76.7 71.4-70.0 8.9-0.6

% 0.88-4.40 0.46-1.84 1.55-5.24 1.91-2.70 1.78-2.25 0.36-1.40

5 (-)-camphene 86.2-92.4 87.9-89.3 44.7-48.4 92.8-92.9 91.8-94.6 88.4-89.3

6 (+)- 13.8-7.6 12.1-10.7 55.3-51.6 7.2-7.1 8.2-5.4 11.6-10.7

% 0-0.13 0-0.05 tr-0.02 0.07-0.12 0.04-0.11

7 (+)-β-pinene 4.3-6.8 8.5-9.6 87.7-98.7 3.5-4.5 3.7-4.5 9.4-10.3 10.6-70.2

8 (-)- 95.7-93.2 91.5-90.4 12.2-1.3 96.5-95.5 96.3-95.5 90.6-89.7 99.4-29.8

% 8.57-17.79 2.97-10.70 1.0-2.44 18.25-25.45 10.01-12.20 tr-0.11

9 (+)-sabinene 12.4-15.0 16.1-17.2 71.3-81.5 15.2-15.4 15.2-15.4 18.6-19.3 89.8-97.5

10 (-)- 87.6-85.0 83.9-82.8 28.7-18.5 84.8-84.6 84.8-84.6 81.4-80.7 10.2-2.5

% 1.13-2.79 0.51-1.69 0.10-0.59 2.31-3.28 0.91-2.07 0.24-0.80

11 (-)-α-phellandrene 46.9-52.6 49.5-54.0 44.3-55.0 50.9-53.7 52.1-58.4 55.2-55.8

12 (+)- 53.1-47.4 50.5-46.0 55.7-45.0 49.1-46.3 47.9-41.6 44.8-44.2

% tr-0.13 0.01-0.18 0.03-0.11 0.02-0.05 tr-0.05

13 (-)-β-phellandrene 31.1-53.9 27.6-33.7 0.4-2.7 63.9-65.6 68.5-73.7 54.5-55.1 0.4-1.4

14 (+) 68.9-46.1 72.4-66.3 99.6-97.3 36.1-34.4 31.5-26.3 45.5-44.9 99.6-98.6

% tr-0.48 0.02-0.21 tr tr tr 0.40-1.40

15 (-)-limonene 1.4-1.6 1.7-1.9 1.6-2.2 2.8-2.9 2.8-3.0 2.7 0.5

16 (+)- 98.6-98.4 98.3-98.1 98.4-97.8 97.2-97.1 97.2-97.0 97.3 99.5

% 59.57-71.82 23.5-54.85 65.30-77.82 47.85-51.14 51.47-59.81 91.15-96.10

17 (+)-camphor 25.3-42.1 17.0-36.5

18 (-)- 74.7-57.9 83.0-63.5

% 0-0.01 tr-0.02

19 (-)-linalool 52.0-74.6 99.5-99.6 13.2-20.4 68.7-73.7 65.2-66.1 63.5-63.9 7.8-17.9

20 (+)- 48.0-25.4 0.5-0.4 86.8-79.6 31.3-26.3 34.8-33.9 36.5-36.1 92.2-82.1

% 0.05-0.46 1.58-36.14 0.02-0.31 0.15-0.24 0.11-0.24 0.17-0.8

21 (-)-citronellal 84.6-94.8 3.9-8.9 77.0 72.8-81.0 78.7-80.5 37.4-52.6

22 (+)- 15.4-5.2 96.1-91.1 33.0 27.2-19.0 21.3-19.5 62.6-47.4

% tr-0.05 tr-0.04 tr-0.11

23 (-)-linalyl acetate 99.7-99.8

24 (+)- 0.3-0.2

% 11.80-41.36

25 (+)-terpinen-4-ol 12.1-26.7 15.0-21.7 9.5-18.3 28.8-29.2 20.8-29.1 19.3-20.4

26 (-)- 87.9-73.3 85.0-78.3 90.5-81.7 71.2-70.8 79.2-70.9 80.7-79.6

% 0.01-0.10 tr-0.29 tr-0.08 0.04-0.70 0.04-0.17

27 (-)-α-terpineol 66.4-82.7 21.2-54.2 67.7-76.5 84.1-89.6 79.5-83.8 77.4-78.0 5.1-15.7

28 (+)- 33.6-17.3 78.8-45.8 32.3-23.5 15.9-10.4 20.5-16.2 22.6-22.0 94.9-84.3

% 0.05-0.84 0.03-0.13 0.04-0.46 0.21-0.80 0.16-0.37 0.02-0.15



Bergamot

In the eight samples analyzed the enantiomeric distribution of all components is almost constant during the season of production with the exception of α-terpineol. The distribution of the enantiomers is characterized by the prevalence, during the entire productive season of the levorotatory enantiomers of: α-thujene, α-pinene, β-pinene, sabinene, camphor, linalool, terpinen-4-ol, linalyl acetate; an almost racemic distribution is noticed for α-phellandrene, with a very small excess of the (-)-isomer (3.95-7.67) in seven of the eight samples; the dextrorotatory isomer of limonene and of β-phellandrene are prevalent. Very characteristic is the inversion of the excess of α-terpineol from levorotatory to the dextrorotatory during the season shown in Figure 2. From a comparison with previously published data by Dugo et al. (1999) the values of the enantiomeric excess of (-)-β-pinene, (-)-sabinene, (-)-terpinen-4-ol and (+)- α-terpineol are slightly different. These discrepancies could be addressed to the different period of production of the samples analyzed. This is particularly true for α-terpineol, due to its great seasonal variation.

Figure 2. Seasonal variation of the enantiomeric ratios determined in Bergamot oil.

to evaluate particular behaviors due to the limited number of samples available. Therefore for this citrus oil it is simply reported the enantiomeric distribution determined for each sample. Lemon

The chiral distribution of the components analyzed in the 92 samples of lemon are characterized by the prevalence, during the entire productive season of the levorotatory enantiomers of: α-thujene, α-pinene, camphene, β-pinene, sabinene, linalool, citronellal, terpinen-4-ol and α-terpineol; the dextrorotatory enantiomer of limonene is always the most abundant; characteristic is the inversion of the excess of α- and β-phellandrene from levorotatory to the dextrorotatory during the season as shown in Figure 1. The range of the enantiomeric ratios determined in the 92 samples of lemon oils for linalool and α-terpineol are wider than those previously reported by Mondello et al. 1996. This is due to the larger number of samples here analyzed and to seasonal variation. However the values reported by Mondello et al. 1996 are within the ranges here determined.

Figure 1. Seasonal variation of the enantiomeric ratios determined in Lemon oil.



Mandarin

The graphs in Figure 3 show the enantiomeric excess variation of 124 samples of mandarin oil collected during the entire productive season. The enantiomeric distribution is characterized by the prevalence of the following dextrorotatory isomers: camphene, β-pinene, sabinene, β-phellandrene, limonene, linalool, citronellal. For α-thujene, camphor, terpinene-4-ol and α-terpineol exceeds the levorotatory isomer. The enantiomeric excess of (-)-α-phellandrene inverts during the season, starting with a small negative value in September. However the variation during the season is very small, with a slight excess of the (-)-enantiomer from December until the end of the season. The range of the enantiomeric ratios determined in the 124 samples of mandarin oils for linalool and α-terpineol are wider than those previously reported by Mondello et al. (1996). This is due to the larger number of samples here analyzed and to seasonal variation. This set of samples include results obtained for mandarin oils produced in September and in March, never considered in previous seasonal evaluations. However the values reported by Mondello et al. (1996) are within the ranges here determined.

Figure 3. Seasonal variation of the enantiomeric ratios determined in Mandarin oil.

Sweet orange

In this essential oil the enantiomeric distribution is quite variable. However, in all the samples analyzed is noticed the prevalence of all dextrorotatory enantiomers with the exception of α-thujene and β-pinene which show prevalence of the levorotatory isomer and for citronellal which shows an inversion. This great variability could be explained by the numerous different cvs. used to extract the essential oil which differently contribute to the industrial production during the season. To put some light on this aspect it may be necessary to manually extract the oils from fruits of selected cultivars during the season. Figure 5 shows the enantiomeric excess only of the enantiomers that show a regular trend during the season.

Figure 4. Seasonal variation of the enantiomeric ratios determined in Sweet orange oil.

Key and Persian limes

The enantiomeric distribution of these oils is characterized by the excess of the levorotatory isomer for all the components determined with the exception of limonene which, as in all citrus peel oils, shows a large excess of the dextrorotatory isomer. Between Key and Persian limes little differences can be noticed between the enantiomeric distributions of the components analyzed. The most evident is the lower enantiomeric excess of (-)-β-phellandrene in Persian lime. The enantiomeric excess of the monoterpene hydrocarbons are very similar between the two Key limes, with the exception, for small differences in distribution of α- and β- phellandrenes. The alcohols show some differences as well: in Key lime type A the excess of (-)-linalool and (-)-α-terpineol is slightly higher than in Key lime type B and in Persian; an opposite trend is observed for (-)-terpinen-4-ol, which is lower in Key lime type A.



Bitter orange In the samples analyzed, when determined, it is noticed the prevalence of the dextrorotatory enantiomers of α-pinene, camphene, β-phellandrene, limonene and α-terpineol. For α-thujene, β-pinene, α-phellandrene, linalool and linalyl acetate the levorotatory enantiomer is the most abundant. Sabinene shows an almost racemic distribution in the commercial sample and a prevalence of the dextrorotatory enantiomer in all the other samples. Linalool shows in two of the four samples analyzed values of the enationtiomeric excess of the levorotatory isomer lower than that reported by Mosandl & Juckelka, (1997) for genuine bitter orange oil. This results highlight the necessity of the acquisition of deeper knowledge on bitter orange essential oil, in consideration of the high commercial value of this niche product, hitherto not exhaustively studied.

Enantiomeric Excess

0,00

20,00

40,00

60,00

80,00

100,00

120,00

(-)-a-

Thujen

e

(-)-a-

Pinene

(-)-C

amph

ene

(-)-be

ta Pine

ne

(-)-S

abine

ne

(-)-a-

Phella

ndren

e

(-)-b-

Phella

ndren

e

(+)-Li

monen

e

(-)-C

itrone

llal

(-)-Li

naloo

l

(-)-T

erpine

n-4-ol

(-)-a-

Terpine

ol

EE

Type A Type B Persian

Figure 5. Graphical comparison of the enantiomeric excesses in Key type A and B, and Persian limes.

Based on the enantiomeric distribution of the components analyzed, the oils analyzed could be divided into two groups. The first group, characterized by the prevalence of the same enantiomers or by values close to racemates (α- and β-phellandrene). In this group fall lemon, bergamot and all the limes. Exception to this common behavior is given by the enantiomeric excess of α-terpineol in bergamot oil which presents an opposite value at the beginning of the season. The esGC chromatograms of these citrus oils are reported in Figure 6. The second group, composed by mandarin, sweet orange and bitter orange is characterized by similarities (mainly between mandarin and sweet orange) of the enantiomeric distribution of numerous components. Bitter orange could fall into the first group if are considered the enantiomeric distributions of linalool and linalyl acetate, but it well fits into the second group for the distribution of other components. The ratio of the α-terpineol enantiomers are similar for sweet and bitter orange oils, while the values determined in mandarin are similar to those obtained for the citrus oils of the first group. The esGC chromatograms of these citrus oils are reported in Figure 7. In conclusion the enantiomeric distributions determined for the fourteen components in the essential oils of seven different citrus specie was useful to determine: genuineness parameters; seasonal variationand to some extent the extraction technique. Characteristic are the enantiomeric ratios of linalool and linalyl acetate in bergamot, which are constant during the entire season and can be used to determine possible addition of synthetic or natural compounds obtained from sources different from bergamot; the enantiomeric ratios of linalool in sweet orange and bitter orange which allow to determine contaminations or fraudulent addition of the less valuable sweet orange to the more valuable bitter orange oil; the enantiomeric ratios of limonene provide a useful tool to reveal the addition in the more valuable citrus oils of products containing

Figure 6. Overlay of the esGC chromatograms of the following cold-pressed essential oils: A. Bergamot; B. Key lime Type B; C. Lemon.

Figure 7. Overlay of the esGC chromatograms of the following cold-pressed essential oils: D. Bitter orange; E. Sweet orange; F. Mandarin.



(-)-limonene, added to correct the optical rotation of oils reconstituted from sweet orange terpenes. Lime oils present slight differences (Figure 6) between the two different species (Key vs. Persian). In Persian lime the enantiomeric excess of (-)-α-pinene, (-)-camphene, (-)-β-pinene, (-)-sabinene, (-)-β-phellandrene, (-)-linalool and (-)-α-terpineol are always lower.

Table 2. Enantiomeric distribution determined in bitter orange oils by esGC and MDGC, and relative peak area % determined by GC on the whole volatile fraction.

Italy Egypt Egypt

commercial laboratory Red Green

1 (+)-α-thujene 10.3 30.8 23.6

2 (-)- 89.7 69.2 76.4

% 0-0.02

3 (+)-α-pinene 89.6 97.4 97.4 96.9

4 (-)- 10.4 2.6 2.6 3.1

% 0.29-0.89

5 (-)-camphene 41.6 37.1 35.8 45.0

6 (+)- 58.4 62.9 64.2 55.0

% tr-0.01

7 (+)-β-pinene 2.3 4.3 7.9 6.1

8 (-)- 97.7 95.7 92.1 93.9

% 0.10-0.18

9 (+)-sabinene 49.4 72.5 80.6 75.4

10 (-)- 50.6 27.5 19.4 24.6

% 0.09-0.45

11 (-)-α-phellandrene 74.9 99.9 100

12 (+)- 25.1 0.1 0

% tr-0.08

13 (-)-β-phellandrene 5.7 1.9 1.1 0.6

14 (+) 94.3 98.1 98.9 99.4

% tr

15 (-)-limonene 0.5 0.5 0.5 0.5

16 (+)- 99.5 99.5 99.5 99.5

% 91.54-96.52

19 (-)-linalool 89.8 60.7 61.1 80.2

20 (+)- 10.2 39.3 38.9 19.8

% 0.06-0.37

23 (-)-linalyl acetate 99.4 97.8

24 (+)- 0.6 2.2

% 0.18-1.17

25 (+)-terpinen-4-ol 71.5

26 (-)- 28.5

% tr-0.03

27 (-)-α-terpineol 6.6 23.5 29.8 17.4

28 (+)- 93.4 76.5 70.2 82.6

% 0.03-2.94

Key lime Type A and B are obtained from the same fruits, but in Type A the oil is in contact with the juice and then separated by centrifuge. Between the two Key lime oils the enantiomeric purities are very similar, with the exception of those relative to the phellandrenes, and to the alcohols. The two hydrocarbons present a slight tendency to racemization in Type A. The enantiomeric excess of (-)-linalool and (-)-α-terpineol, strangely increased in Type A. This is probably due to the formation of these components during the contact of the oil with the juice/pulp phase which occurs during the extraction of Key lime type A. A similar phenomenon was observed for (+)-α-terpinenol in bergamot oils recovered by screw press from solid residues (Mondello et al. 1998). In this case these authors assumed that the stereoselective formation of this alcohol could be due to presence of selected microorganisms responsible of this phenomenon, or to the enzymatic hydrolysis of the glycosidically bonded enantiomer. Terpinen-4-ol seams to exhibit a slight tendency to racemization, as predictable when acid catalyzed reactions occur. Differences between the enantiomeric excess in Italian and Egyptian bitter orange oils are neglectable. In this case the geographic origin could be characterized by different analytical approach, such as the determination of isotopic ratios by IRMS.

References

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Bonaccorsi I, Dugo P, Trozzi A, Cotroneo A, Dugo G 2009. Characterization of mandarin (Citrus deliciosa Ten.) essential oil. Determination of volatiles, non-volatiles, physico-chemical indices and enantiomeric ratios. Nat Prod Commun 4: 1595-1600.

Dugo G, Cotroneo A, Bonaccorsi I, Trozzi A 2011. Composition of the volatile fraction of Citrus peel oils. In: Citrus oils: Composition advanced analytical techniques, contaminants and biological activity. Dugo G, Mondello L (eds.). Taylor and Francis group. Boca Raton, FL, USA. p. 1-162.

Dugo G, Sciarrone D, Costa R, Bonaccorsi I, Dugo P, Mondello L, Santi L Fakhry HA 2011a. Characterization of oils from the fruits, leaves and flowers of the bitter orange tree. J Essent Oil Res 23: 1-15.

Dugo G, Bonaccorsi I, Ragonese C, Russo M, Donato P, Santi L, Mondello L 2011b. Analytical characterization of mandarin (Citrus deliciosa Ten) essential oil. Flavour Fragr J 26: 34-46.

Dugo P, Ragonese C, Russo M, Sciarrone D, Santi L, Cotroneo A, Mondello L 2010. Sicilian lemon oil: composition of volatile and oxygen heterocyclic fractions and enantiomeric distribution of volatile components. J



Sep Sci 33: 3374-3385 Dugo P, Giuffrida D 2011. Carotenoids of Citrus oils.

In: Citrus oils: Composition advanced analytical techniques, contaminants and biological activity. Dugo G, Mondello L (eds.). Taylor and Francis group. Boca Raton, FL, USA. p. 445-462.

Dugo P & Russo M 2011. The oxygen heterocyclic components of citrus essential oils. In: Citrus oils: Composition advanced analytical techniques, contaminants and biological activity. Dugo G, Mondello L (eds.). Taylor and Francis group. Boca Raton, FL, USA. p. 405-444.

Dugo G, Bartle KD, Bonaccorsi I, Catalfamo M, Cotroneo A, Dugo P, Lamonica G, McNair H, Mondello L, Previti P, Stagno d’Alcontres I, Trozzi A, Verzera A 1999. Advanced analytical techniques for the analysis of Citrus essential oils. Part. 2. Volatile fraction: LC-HRGC and MDGC. Essenze Derivati Agrumari 69: 159-217.

Frum Y, Viljoen AM 2006. In vitro 5-lipoxygenase activity of three indigenous South African aromatic plants used in traditional healing and the stereospecific activity of limonene in the 5-lipoxygenase assay. J Essent Oil Res 18: 85-88.

Heuberger E, Hongratanaworakit T, Bohm C, Weber R, Buchbauer G 2001. Effects of chiral fragrances on human autonomic nervous system parameters and self-evaluation. Chem Senses 26: 281-292.

Kamatou GPP, Viljoen AM 2008. Linalool- A review of a biologically active compound of commercial importance. Nat Prod Commun 3: 1183-1192.

Mondello L, Costa R, Sciarrone D, Dugo G 2011. The chiral compounds of citrus oils. In: Citrus oils: Composition advanced analytical techniques, contaminants and biological activity. Dugo G, Mondello L (eds.). Taylor and Francis group. Boca Raton, FL, USA. p. 349-404.

Mondello L, Verzera A, Previti P, Crispo F, Dugo G 1998. Multidimensional capillary GC-GC for the analysis of complex samples. 5. Enantiomeric distribution of monoterpene hydrocarbons, monoterpene alcohols and

linalyl acetate of bergamot (Citrus bergamia Risso et Poiteau) oils. J Agr Food Chem 46: 4275-4282.

Mondello L, Dugo G, Dugo P, Bartle KD 1996. On-line HPLC-HRGC in the analytical chemistry of Citrus essential oils. Perfumer & Flavorist 21: 25-49.

Mosandl A, Juchelka D 1997. The bitter orange tree-a source of different essential oils. In: Flavour Perception. Kruse H-P, Rothe M (eds.). Eigenverlag Deutsch Inst. F. Ernahrungsforsch. p. 321-331.

Ozek T, Tabanca N, Demrci F, Wedge DE, Husnu Can Baser K 2010. Enantiomeric distribution of some linalool containing essential oils and their biological activities. Records Nat Prod 4: 180-192.

Peana AT, D’Aquila PS, Panin F, Serra G, Pippia P, Moretti MDL 2002. Anti-inflammatory activity of linalool and linalyl acetate constituents of essential oils. Phytomedicine 9: 721-726.

Peana AT, Rubattu P, Piga GG, Fumagalli S, Boato G, Pipia P, De Montis MG 2006. Involvement of adenosine A1 and A2A receptors in (-)-linalool-induced antinociception. Life Sci 78: 2471-2474.

Ruberto G, Baratta MT 2000. Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem 69: 167-174.

Sciarrone D, Schipilliti L, Ragonese C, Tranchida PQ, Dugo P, Dugo G, Mondello L 2010. Thorough evaluation of the validity of conventional enantio-gas chromatography in the analysis of volatile chiral compounds in mandarin essential oil: A comparative investigation with multidimensional gas chromatography. J Chromatogr A 1217: 1101-1105.

*Correspondence

Ivana BonaccorsiDipartimento Farmaco-chimico, Università di MessinaV.le SS. Annunziata, 98168 Messina, [email protected]. +39 090 6766572Fax: +39 090 358220

850

Article


Received 27 Dec 2010Accepted 25 May 2011Available online 12 Aug 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 850-855, Sep./Oct. 2011 Phytochemical researches and antimicrobial

activity of Clinopodium nubigenum Kunth (Kuntze) raw extracts

Gianluca Gilardoni,1 Omar Malagon,4 Vladimir Morocho,4 Riccardo Negri,1 Solveig Tosi,2,3 Maria Guglielminetti,3 Giovanni Vidari,*,1,2 Paola Vita Finzi1,2

1Università degli Studi di Pavia, Dipartimento di Chimica, Italia,2Università degli Studi di Pavia, Centro Interdipartimentale di Studi e Ricerche sull’Etnobiofarmacia, Italia,3Università degli Studi di Pavia, Laboratorio di Micologia, Dipartimento di Scienze della Terra e dell’Ambiente, Italia,4Universidad Técnica Particular de Loja, Instituto de Química Aplicada, Loja, Ecuador.

Abstract: The essential oil of the species Clinopodium nubigenum (Kunth) Kuntze, Lamiaceae, was analyzed by GC-MS and GC-FID, taking into account the more recent literature. Among the seventy compounds identified, the majority are oxygenated monoterpenoids. The essential oil, tested for antimicrobial activity, resulted effective in vitro against Candida albicans. From the aqueous MeOH extract of the aerial parts of the plant two nonvolatile compounds, named schizonepetoside A and schizonepedoside C, have been isolated. They are rare glycosyl terpenoids, which were previously isolated from only one plant, but never found before in the genus Clinopodium.

Keywords:antimicrobial activityClinopodium nubigenumessential oilGC-MSschizonepetosidetipo de cerro

Introduction

Clinopodium nubigenum (Kunth) Kuntze, an aromatic plant belonging to the family Lamiaceae, is also known with the synonymous Thymus nubigenus Kunth, Micromeria nubigena (Kunth) Benth, and Satureja nubigena (Kunth) Briq (Index Kewensis, 2010). It is widely growing in Latin America between 3000 and 4000 m a.s.l. The plant is popularly known by indigenous people as “tipo de cerro” and, in Ecuador, it is used as a traditional remedy by different communities. The Saraguro people use an aqueous infusion of the plant to treat colds (Andrade et al., 2009); in the region of Azuay, the plant is used as a remedy for flu (Rios et al., 2007); Quechua peoples in the High Sierra apply a decoction of C. nubigenum to cure stomach ache; Cañar communities use a plant infusion to avoid urinary incontinence in children; in Tungurahua, Chimborazo, Cañar, and Azuay provinces of Ecuador the plant finds applications also as a digestive, stomachic, a tonic remedy, and against dysentery and menstrual syndromes (de la Torre et al., 2008). Aromatic properties of C. nubigenum are due to the presence of an essential oil, easily obtainable by steam distillation, which has been analysed in this study. In addition, we examined the non volatile fraction of a H2O-MeOH extract.


Plant material

The aerial parts of the plant were collected in January 2009, during the non-flowering period, at Aguarongo (3242 m a.s.l. coordinates 17700288E-9585973N), in the San Lucas Parish, inside the Saraguro territory, Loja province, Ecuador. It has been identified by one of the authors (V. M.). A voucher sample is conserved at UTPL (Universitad Tecnica Particular de Loja), Loja, Ecuador, with the identification number PPN-la-018.

Instruments and materials

GC-MS analyses were performed on an Agilent Technologies 6890N gas chromatograph, coupled with an Agilent Technologies 5973N (electronic impact source) System ATB-1550; GC-FID analyses were performed on a Perkin Elmer Auto System gas chromatograph; NMR spectra were determined in MeOH-d4 obtained from Sigma-Aldrich; ACS solvents for extraction processes and HPLC grade solvents for chromatographic purifications were purchased from Carlo Erba Reagenti (Milan, Italy); C-18 reversed phase

Phytochemical researches and antimicrobial activity of Clinopodium nubigenum Kunth (Kuntze) raw extracts

Gianluca Gilardoni et al.


LiChroprep RP-18 (25-40 µm) and silica gel Kieselgel 60 (230-400 mesh) were purchased from Merck.

Essential oil distillation and analysis

Two samples (200 g each) of air-dried and fresh aerial parts of the plant were separately hydrodistilled, producing two essential oils, A and B, respectively, spontaneously separating from the collected aqueous layers, with a yield of about 1% w/w. The fresh plant batch was distilled in Loja (Ecuador) immediately after collection; the dry batch was distilled in Pavia (Italy) after four weeks. The collected essential oils, after drying over anhydrous sodium sulfate, were clear, light yellow and mobile liquids, characterized by a pleasant, fresh and minty odor. Two samples (1 µL) of a 5% v/v solution of each oil in dichloromethane were separately injected in GC-MS and analyzed under the same conditions, the second sample after addition to a mixture of n-alkane homologues from heptane to nonadecane. The latter hydrocarbon mixture served to calculate the Linear Retention Index (LRI) value of each oil constituent, according to Van Den Dool & Kratz (1963). The GC-MS instrument was equipped with a HP-5 capillary column (length: 30 m, internal diameter: 0.25 mm, thickness of the stationary phase: 0.25 µm); helium (1 mL/min) was the carrier gas; the injector was operated in the split mode, with a split ratio of 19 and at the temperature of 250 °C; the GC analysis was performed with the following oven temperature program: temperature initially kept at 60 °C for 1 min, then increased to 260 °C with a gradient rate of 5 °C/min, and kept at 260 °C for an additional 10 min. The MS spectra were acquired in the Scan Mode, with a m/z range of 41-350 amu; solvent delay: 2 min. The GC-FID instrument was equipped with a HP-5 capillary column (length: 25 m, internal diameter: 0.25 mm, thickness of the stationary phase: 0.25 µm), using nitrogen as the carrier gas at 1 mL/min; the injector was operated in the split mode, with a split ratio of 25 and at the temperature of 250 °C, detector temperature was set at 280 °C; the GC analysis was performed with the following oven temperature program: temperature initially kept at 60 °C for 1 min, then increased to 200 °C with a gradient rate of 5 °C/min, then to 280 °C with a gradient rate of 15 °C/min. and kept at 280 °C for an additional 5 min.

Solvent extraction and glycosides purification

Dried aerial parts (300 g) were subjected to solvent extraction by maceration for 1 h at room temperature; the operation was repeated with three solvents of increasing polarity: n-hexane, MeOH, 90%

aqueous MeOH. After extraction, each mixture was filtered and the filtrates were separately evaporated under vacuum at 40 °C to afford three residues, weighing 3.6 g (n-hexane), 16.0 g (MeOH), and 1.4 g (90% MeOH), respectively. The last extract was subjected to preparative liquid chromatography on a C-18 column, eluted with an increasing gradient of MeOH in H2O. Two fractions from this separation were further purified by preparative liquid chromatography on silica gel. Elution with an increasing gradient of MeOH in dichloromethane gave two pure glycosylated monoterpenoids. The compounds were identified by 1H-NMR and 13C-NMR spectroscopy, including DEPT and COSY experiments, as schizonepetoside A (1) and schizonepetoside C (2). The data were identical with those reported in the literature (Kubo et al., 1986; Lee et al., 2008).

Biological assays

The antimicrobial activity of essential oils A and B was evaluated against different species of bacteria (Bacillus subtilis ATCC 6633, Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 6538) and fungi, isolated from patients (Candida albicans (C.P. Robin) Berkhout, Trichophyton mentagrophytes (C.P. Robin) Sabour.), and from environment (Aspergillus niger Tiegh. ATCC 16404 recently reclassified as Aspergillus brasiliensis Varga, Frisvad & Samson). Tests were performed in triplicates based mainly on the CLSI procedures (NCCLS, 2006; 2008), with some modifications reported in this paragraph. Along the experiment, Luria Bertani and Sabouraud were used as culture media for bacteria and fungi, respectively; incubating temperatures was 37 °C for C. albicans, E. coli and S. aureus, The other microorganisms were cultivated at 28 °C. This temperature was chosen for T. mentagrophytes accordingly to Fernàndez-Torres et al. (2002). Controls were prepared with culture media or culture media with solvent. Oil activity was at first qualitatively detected in Petri dishes (140 mm) by means of the agar diffusion method The mother suspensions were prepared from cultures growing on solid media 5 cm Petri dishes. Cultures of C. albicans, S. aureus, E. coli, and B. subtilis were 48 h old, while cultures of A. niger and T. mentagrophytes were 7 days old. For filamentous fungi, i.e. T. mentagrophytes and A. niger, pre-suspensions were prepared transferring part of the colony in a tube containing NaCl 0.85% water with broken cover slides and then stirred for 1 min by vortex. Inoculum suspensions were prepared transferring microorganisms in 2 mL sterile water with 0.85% NaCl (api bioMerieux) adjusted at 0.5 McFarland by nephelometric measurement; 1 mL of the suspension

Phytochemical researches and antimicrobial activity of Clinopodium nubigenum Kunth (Kuntze) raw extractsGianluca Gilardoni et al.


for each microorganism was uniformly distributed on the agar surface of 9 cm Petri dishes. Results were recorded after 24 and 48 h for all the microorganisms, with the exception of T. mentagrophytes that was examined after five days. The activity of the oil against fungi and bacteria was evaluated by measuring the inhibition zone diameter using filter paper disks (diameter of 0.5 cm) impregnated with 9 µL of essential oil and 1 µL of DMSO (Sigma-Aldrich). Penicillin G Na salt and amphotericin B (both Sigma-Aldrich) were the reference compounds for bacteria and fungi, respectively. Minimum inhibitory concentration (MIC) was determined only for microorganisms which showed a inhibition halo >1 cm. Essential oil was added to liquid culture medium in 24 microwell plates at final concentration from 1.5 to 20 µL/mL. Tween 80 0.002% (v/v) was included to enhance oil solubility. To determine the lowest concentration required to kill the test organism (minimum fungicidal or bactericidal concentration, MFC or MBC, respectively), the method described by Gadd (1986) was followed. The initial inoculum was subcultured from 24-48 h-old microwell plates contained the oil onto a fresh cultural medium free of the toxicant and examined after 24, 48, and 72 h, respectively.


The essential oil A, obtained from air-dried aerial parts of C. nubigenum, was mainly composed of monoterpenes and sesquiterpenes. Seventy components of this complex mixture were identified by comparison of their EI-MS spectra and calculated linear retention indexes (LRI) with recently reported data (Adams, 2007); the identity of compounds not listed in the reference text was established by means of Wiley and NIST electronic EI-MS libraries. The complete qualitative analysis is reported in Table 1, including compound percentage quantification by GC-FID and GC-MS peak integration. The composition determined for this essential oil corresponds to 97.6% and 88.7% of the entire GC-FID and GC-MS chromatogram, respectively; major compounds are pulegone, menthofuran, isopulegone, α-copaene, zonarene, 1-octen-3-yl acetate, limonene, linalool, p-cymene, piperitenone, β-pinene, and 1,6-octadien-3,7-dimethyl-3-ol. The composition of the essential oil B, obtained by hydrodistillation of fresh aerial parts, resulted to be virtually identical to the other one, except for the relative amounts of menthofuran and isopulegone. In fact, 1.56% of isopulegone and 11.57% of an inseparable mixture of menthofuran and menthone were detected in the GC-FID of the oil A (Figure 1), with menthofuran largely prevailing (GC-MS), whereas menthofuran was

almost absent in the oil B, with isopulegone occurring in a relatively high amount (Figure 2). The GC-MS comparison of the two essential oils A and B is shown in Figure 3. It is known that menthofuran biosynthesis proceeds from isopulegone through cytochrome P-450 dependent oxidation to 9-hydroxypulegone (Dewick, 2009; Mc Clanahan et al., 1988); isolation of the two glycosylated compounds 1 and 2 from C. nubigenum (vide infra) is a further proof of the pathway. Thus, some glucose cleavage from 1 and 2 probably occurred during the plant drying process, storage, and transport from Ecuador to Italy, explaining the differences between the two oils.

Figure 1. GC-FID analysis of the essential oil A from air-dried aerial parts of C. nubigenum.

Figure 2. GC-FID analysis of the essential oil B from fresh aerial parts of C. nubigenum.

A few years ago, El Seedi and collaborators reported the essential oil analysis of Micromeria nubigena H.B.K. (El-Seedi et al., 2008). Interestingly, no plant appears with such authority in the Index Kewensis, contrary to the name Micromeria nubigena (Kunth) Benth, which is considered a synonymous of C. nubigenum Kunth (Kuntze) (Index Kewensis, 2010). The doubt thus exists that the two plants are different species. Indeed, comparison of our findings with the data reported by El Seedi clearly shows dramatic differences in the composition of the essential oils. For example, about 54% thymol and carvacrol were identified in the




Table 1. Chemical analysis of the essential oil of C. nubigenum. LRI: Linear Retention Index (Van den Dool & Kratz, 1963). COMPOUNDS %A %B LRI COMPOUNDS % A % B LRI

1-Butanol-2-methyl acetate* 0.01 <0.01 876 Isobornyl acetate 0.1 <0.01 1288

α-Pinene 0.85 0.45 934 trans-Sabinyl acetate 0.23 0.10 1294

Camphene 0.12 0.03 949 Nonanyl acetate 0.18 0.02 1312

3-Methyl-cyclohexanone 951 Myrtenyl acetate 0.89 0.05 1328

Sabinene 3.26 0.33 974 Piperitenone 1.52 0.34 1345

β-Pinene 978 Citronellyl acetate 1.06 0.46 1355

2-Pentyl furan 0.11 0.06 992 Eugenol 1.11 0.51 1361

3-Octanol 0.11 0.04 996 Piperitenone oxide 0.21 0.11 1370

p-Mentha-1(7),8-diene 0.05 0.03 1005 α-Copaene 7.03 2 .09 1381

Limonene 6.46 2.07 1030 trans-Myrtanol acetate 0.22 0.08 1387

trans-β-Ocimene 0.06 0.01 1048 β-Bourbonene 0.76 0.29 1389

γ-Terpinene 0.01 0.01 1059 β-Cubebene 1393

p-Mentha-3,8-diene 0.02 0.02 1071 Elemene<β-> 0.32 <0.01 1395

Terpinolene 0.09 0.01 1090 (E)-Caryophyllene 0.18 0 .06 1423

p-Cymene 2.08 1.73 1091 β-Copaene 0.50 0 .16 1433

1,6-Octadien-3-ol,3,7-dimethyl-* 7.31 7.00 1101 α-Humulene 1458

Linalool 0.52 0.81 1101 γ-Muurolene 1.06 0 .38 1480

Nonanal 0.06 0.02 1105 Germacrene D 1485

1-Octen-3-yl acetate 0.46 0.02 1113 (E,E)-α-Farnesene 0.39 0 .29 1510

3-Octanol acetate 0.25 0.02 1124 Zonarene 5.76 2 .08 1529

α-Campholenal 1128 α-Calacorene 0.41 0 .05 1547

cis-Limonene oxide 1135 β-Calacorene 0.57 0 .05 1568

cis-p-Mentha-2,8-dien-1-ol 1137 1α,10α-epoxy-Amorph-4-ene 0.25 0.06 1576

p-Menth-3-en-8-ol 0.14 0.01 1150 β-Copaen-4-α-ol<β-> 1592

Menthone 11.57 0.15 1156 α-Corocalene 0.06 0.02 1629

Menthofuran 1167 Muurola-4,10(14)-dien-1-β-ol 0.04 0.01 1635

cis-Isopulegone* 1.56 4.74 1178 α-Muurolol 0.02 <0.01 1649

α-Terpineol 0.05 0.10 1194 cis-Calamenen-10-ol 0.08 0 .06 1668

Decanal 0.06 0.08 1207 Cadalene 1683

Acetic acid, non-3-enyl ester* 0.80 0.13 1209 Mostakone 0.59 0.01 1687

Benzofuran-4,7-dimethyl* 0.33 0.08 1216 Amorpha-4,9-dien-2-ol 0.26 1705

Coahuilensol, methyl ether 0.30 0.01 1226 10-nor-Calamenen-10-one 1711

Pulegone* 37.11 72.79 1249 Pentadecanal* 1718

Linalyl acetate 0.05 0.01 1257 Hexadecanal* 1783

Cinnamaldehyde<(E)-> 0.03 0.02 1274 2-Pentadecanone,6,10,14-trimethyl-* 1849

Tot. % 97.59 98 .09 A: from air-dried aerial parts. B: from fresh aerial parts. *Identified only by Wiley and NIST MS libraries.

hydrodistillate from M. nubigena H.B.K., while these phenols were not detected as significant components of the oils A and B in this study. In addition to species differences or the existence of botanical varieties, other factors, among which important are the climate, the soil, the harvest period and the vegetative cycle, and the method of plant and essential oil preservation, can explain the different chemical contents of the oils. Furthermore, it is worth noting that we collected the

plant in the non-flowering period, while the previous essential oil was obtained by hydrodistillation of leaves and flowers (El-Seedi et al., 2008). In addition to the essential oil, schizonepetoside A (1) and C (2) were isolated from the aqueous MeOH extract of the air-dried aerial parts of C. nubigenum. These compounds are rare glycosylated monoterpenoids, previously isolated, to the best of our knowledge, only from the plant Schizonepeta tenuifolia Briq. (Kubo et

Phytochemical researches and antimicrobial activity of Clinopodium nubigenum Kunth (Kuntze) raw extractsGianluca Gilardoni et al.


The most sensitive microorganism was C. albicans with a inhibition halo >1 cm. The MIC of the essential oils A and B against C. albicans are showed in Table 3. Fungicide activity (MFC) against the yeast was detected after 48 h treatment with a concentration of 8 µL/mL and 5 µL/mL for oil A and B, respectively. Interestingly, only fungistatic activity resulted after 24 h treatment. The activity data reported in this paper are consistent with the antimicrobial activity shown by many genera and species belonging to the family Lamiaceae (De Martino et al., 2009). Antimicrobial properties of the genus Clinopodium were mainly investigated for Old World species. Most of them were demonstrated to be antimicrobial either against different bacteria or fungi (Stojanović et al., 2006; Castilho et al., 2007; Stojanović et al., 2009). Almost sixty species of the genus Clinopodium grow in tropical America (Harley, 2000), but biological activities of only few of them have been tested so far (El-Seedi et al., 2008; Estrada-Reyes et al., 2010). Antimicrobial activity of the

al., 1986; Lee et al., 2008), after which they have been named. They were identified by identical NMR data with those reported in the literature.

O

OO

OH

OH

HOHO

1

O

OO

OH

OH

HOHO

2

Table 2 shows the activity data of the C. nubigenum essential oils A and B against tested microorganisms. No differences between the replicate data were detectable to the naked eye. Difference between A and B was detectable only against A. niger after 24 h of incubation, but both oils showed no activity after 48 h.

Figure 3. GC-MS comparison of the essential oil A, from air-dried aerial parts, with essential oil B, from fresh aerial parts of C. nubigenum.

Table 2. Activity of the essential oils A and B of C. nubigenum against bacterial and fungal microorganisms, as shown by the inhibition zone diameter (cm).

SampleFungi Bacteria

Aspergillus niger Candida albicans

Trichophyton mentagrophytes Bacillus subtilis Escherichia

coliStaphylococcus

aureus

Essential oil A 9 µL 1.5 after 24 h, 0 after 48 h

1.2 0.6 0.7 0 0.8

Essential oil B 9 µL 2 after 24 h, 0 after 48 h

1.2 0.6 0.7 0 0.8

Penicillin G Na salt 9 µg - - - 2.4 2.7 2.7

Amphotericin B 10 µg 1.3 1.4 1.7 - - 0




essential oil of Micromeria nubigena H.B.K. was detected by El-Seedi et al. (2008), and data concerning C. albicans and S. aureus are comparable with those reported in this paper for C. nubigenum. In addition, we report for the first time the anticandidal MIC and MFC values of the essential oil, while demonstrating its fungicide activity. This result is very important for finding new remedy against C. albicans, which is the predominant species causing fungal infections

Acknowledgment

This article is dedicated to the memory of Riccardo Negri. We thank the Italian MIUR for financial support (Funds PRIN).

References

Adams R.P. 1995. Identification of essential oil components by mass chromatography/mass spectroscopy. Carol Stream, IL, USA: Allured Pub. Corp.

Adams RP 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed. Carol Stream, IL, USA: Allured Pub. Corp.

Andrade JM, Armijos C, Malagón O, Lucero HY 2009. Plantas medicinales silvestres empleadas por la etnia Saraguro en la Parroquia San Lucas. Loja, Ecuador.

Castilho P, Liu K, Rodrigues AI, Feio S, Tomi F, Casanova J 2007. Composition and antimicrobial activity of the essential oil of Clinopodium ascendens (Jordan) Sampaio from Madeira. Flavour Fragr J 22: 139-144.

de la Torre L, Navarrete H, Muriel P, Macía M, Balslev H 2008. Enciclopedia de las Plantas Útiles de Ecuador. Quito & Aarhus: Herbario QCA de la Escuela de Ciencias Biológicas de la Pontificia Universidad Católica del Ecuador & Herbario AAU del Departamento de Ciencias Biológicas de la Universidad de Aarhus.

De Martino L, De Feo V, Nazzaro F 2009. Chemical composition and in vitro antimicrobial and mutagenic activities of seven Lamiaceae essential oils. Molecules 14: 4213-4230.

Dewick PM 2009. Medicinal Natural Products, a Biosynthetic Approach. 3rd Ed. Chichester, West Susex, UK: John Wiley & Sons, Ltd.

El-Seedi HR, Khattab A, Gaara AHM, Mohamed TK, Hassan NA, El-Kattan AE 2008. Essential oil analysis of Micromeria

nubigena H.B.K. and its antimicrobial activity. J Essent Oil Res 20: 452-456.

Estrada-Reyes R, Martínez-Vázquez M, Gallegos-Solís A, Heinze G, Moreno J 2010. Depressant effects of Clinopodium mexicanum Benth. Govaerts (Lamiaceae) on the central nervous system. J Ethnopharmacol 130: 1-8.

Fernàndez-Torres B, Cabanes FJ, Carrillo-Munoz AJ, Esteban A, Inza I, Abarca L, Guarro J 2002. Collaborative evaluation of optimal antifungal susceptibility testing condition for dermatophytes. J Clin Microbiol 40: 3999-4003.

Gadd GM 1986. Toxicity screening using fungi and yeast. In “Toxicity testing using microorganisms”, vol. II, Dutka BJ & Bitton G (Eds.), CRC Press, Florida: p. 43-77.

Harley RM, Paucer AG 2000. List of species of tropical American Clinopodium (Labiatae) with new combination. Kew Bulletin 55: 917-927.

Index Kewensis 2010. http://www.ipni.org/, access in autumn 2010.

Kubo M, Sasaki H, Endo T, Taguchi H, Yosioka I 1986. The constituents of Schizonepeta tenuifolia Briquet: Structure of a new monoterpene glucoside, schizonepetoside C. Chem Pharm Bull 34: 3097-3101.

Lee IK, Kim MA, Lee SY, Hong JK, Lee JH, Lee KR 2008. Phytochemical constituents of Schizonepeta tenuifolia Briquet. Nat Prod Sci 14: 100-106.

Mc Clanahan RH, Huitric AC, Pearson PG, Desper JC, Nelson SD 1988. Evidence for a Cytochrome P-450 catalyzed allylic rearrangement with double bond topomerization. J Am Chem Soc 110: 1979-1981.

NCCLS 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard - seventh edition: M7-A7. Wayne, PA, USA: Clinical and Laboratoty Standards Institute.

NCCLS 2008. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standards - third edition: M27-A3. Wayne, PA, USA: Clinical and Laboratory Standards Institute.

Rios M, Koziol J, Brorgtoft Pedersen H & Granda G (Eds) 2007. Plantas útiles del Ecuador: aplicaciones, retos y perspectivas. Quito, Ecuador: Ediciones Abya-Yala.

Stojanović G, Palić I, Ursić-Janković J 2006. Composition and antimicrobial activity of the essential oil of Micromeria cristata and Micromeria juliana. Flavour Fragr J 21: 77-79.

Stojanović G, Golubović T, _Kitić D, Palić R 2009. Acinos species: Chemical composition, antimicrobial and antioxidative activity. J Med Plants Res 3: 1240-1247.

Van Den Dool H, Kratz PD 1963. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J Chromatogr 11: 463-471.

*Correspondence

Giovanni VidariUniversità degli Studi di Pavia, Dipartimento di Chimicavia Taramelli 12, 27100 Pavia, [email protected]. +39 0382987322Fax. +39 0382987323

Table 3. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of the essential oils A and B from C. nubigenum.

SampleCandida albicans

MIC after 24 h µL/mL

MFC after 48 h µL/mL

Essential oil A 8 8

Essential oil B 5 5

Amphotericin B 1 undUnd: undetermined.

856

Article


Received 20 Dec 2010Accepted 15 Mar 2011Available online 12 Aug 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 856-863, Sep./Oct. 2011 Data collection and advanced statistical analysis

in phytotoxic activity of aerial parts exudates of Salvia spp.

M. Giacomini,*,1 A. Bisio,2 E. Giacomelli,2 S. Pivetti,1 S. Bertolini,1

D. Fraternale,3 D. Ricci,3 G. Romussi,2 N. De Tommasi4

1Dipartimento di Informatica, Sistemistica e Telematica, Università di Genova, Italia,2Dipartimento di Chimica e Tecnologie Farmaceutiche e Alimentari, Università di Genova, Italia,3Dipartimento di Scienze dell’Uomo, dell’Ambiente e della Natura, Università di Urbino, Italia,4Dipartimento di Scienze Farmaceutiche,Università di Salerno, Italia.

Abstract: In order to define the phytotoxic potential of Salvia species a database was developed for fast and efficient data collection in screening studies of the inhibitory activity of Salvia exudates on the germination of Papaver rhoeas L. and Avena sativa L.. The structure of the database is associated with the use of algorithms for calculating the usual germination indices reported in the literature, plus the newly defined indices (Weighted Average Damage, Differential Weighted Average Damage, Germination Weighted Average Velocity) and other variables usually recorded in experiments of phytotoxicity (LC50, LC90). Furthermore, other algorithms were designed to calculate the one-way ANOVA followed by Duncan's multiple range test to highlight automatically significant differences between the species. The database model was designed in order to be suitable also for the development of further analysis based on the artificial neural network approach, using Self-Organising Maps (SOM).

Keywords:Data AnalysisPhytotoxicityGermination IndicesWeb Based Database for Long Distance InteractionsNeural NetworksSelf-Organising Maps

Introduction

The use of crop residues, cover crops and plant residues with a high content of allelochemicals in the soil, as well as extracts of allelopathic plants, can provide new tools for the selective weed control through the release of allelochemicals (Bhowmik et al., 2003; Dayan et al., 2000; Xuan et al., 2005) thus contributing to the maintenance and the development of sustainable agro-ecosystems. As Salvia exudates have been shown to possess inhibitory activity on the germination of Papaver rhoeas L. and Avena sativa L. in a screening work (Bisio et al., 2010a), further studies were planned for the isolation and identification of the active metabolites, as well as their mode of action and their interactions with soil components and organisms. Some tools are now available to support web based collaboration and most of them are in the field of common report production or in general multiple author writing (Bramley et al., 2006; Brazma et al., 2003; Camon et al., 2004; Gogoulou et al., 2005; Halsall, 2005; Thorisson et al., 2005). These web-based models can act as a versatile and low-cost way to permit distributed collaborative work (Giacomini et al., 2009;

Giacomini & Cafferata, 2009). Then for the definition of the phytotoxic potential of Salvia species a database for fast and efficient data collection has been developed among the various collaborating groups to be used in the following research steps. In relational database design, the process of organising data to minimise redundancy is called normalisation. The goal of database normalisation is to decompose relations with anomalies in order to produce smaller, well-structured relations. So structured data can be exchanged in a secure and safe way through web-based tools in order to quicken the process. The normalised structure of the data collected into the database simplifies their use for the automatic calculation of several inferential statistical parameters. It has been shown (Bisio et al., 2010b) that these parameters cannot completely describe the differences in the activity of the several Salvia species involved in this experiment. For this reason, a set of more advanced analytical approaches can be applied. Also in this case, the structured features of the collected data will enable a quick execution of the advanced analysis algorithms. Similar cases have been considered in many fields correlated to life sciences and in these

Data collection and advanced statistical analysis in phytotoxic activity of aerial parts exudates of Salvia spp.

M. Giacomini et al.


cases a combination of connectionist and symbolic artificial intelligent approach have been proven to be successful (Basheer & Hajmeer, 2000; Bertone et al., 1996; Giacomini & Ruggiero, 2001; Giacomini et al., 2000; 1997; 1996). The connectionist methods aim to mimic the example based method that is typical of the human brain. There are two main possible connectionist approaches, one based on the supervised and the other based on the unsupervised artificial neural networks. The supervised nets are very efficient, but a complete and fully described set of examples has to be available at the beginning of the analysis (Rumelhart et al., 1986).When this is not possible and when fully unknown situations are feasible, the unsupervised Self Organising Maps (Kohonen, 2001) can be successfully adopted. These maps place the input patterns on a two dimensional finite plane divided into a finite number of areas (shown as hexagonal spaces, as in Figure 4). Similar input examples are placed in the same output element, or in the very near elements. In this way, after the learning process, areas allocated to specific classes can be singled out. A pattern coming from an unclassified example is classified according to the zone in which this pattern is placed by the network (Giacomini et al., 2000). To highlight the importance of neural networks for analysing chemical data, it may suffice to mention that each year more than 1000 publications appear on the use of neural networks in chemistry (Fink & Reymond, 2007; Gasteiger et al., 2003; Krasznai et al., 2010; Selzer & Ertl, 2006). In comparison to other neural network methods the Self-Organising Map (SOM) are transparent and the results are easily interpretable (Vracko, 2005). In fact the SOM can give a transparent design space to engineer and also help designers to analyse and reduce design space. It can save much product design time, and make optimisation easier (Chu et al., 2010). This reduction allows the SOM results to be represented by a two dimension segmented map on which clusters can be easily identified just looking at this map.


Plant material

Fresh aerial parts of Salvia namaensis Schinz (1), S. fallax Fernald (2), S. disermas L. (3), S. chamaedryoides Cav. (4), S. confertiflora Pohl. (5), S. x jamensis J.Compton (6), S. buchananii Hedge (7), S. wagneriana Polak (8), S. scabra Linn.fil. (9), S. miniata Fernald (10), S. cacaliaefolia Benth.(11), S. adenophora Fernald (12), S. rutilans Carrière (13) were obtained from The Regional Center For Agricultural Experimentation and Assistance (Albenga, Italy) and

Research Unit for Floriculture and Ornamental Species (Sanremo, Italy). The species were identified by Dr. Gemma Bramley and voucher specimens are deposited in Kew Herbarium (K) (Kew, Surrey, UK). Commercial seeds, of Papaver rhoeas L.(La Semeria-www.Lasemeria.it-Italy) and Avena sativa L.(Il Monastero-Sementi-Italy) were used for both Petri dish and pot assays.

Assays

Extraction of exudates, germination and growth tests

The exudates of the fresh aerial parts of the Salvia spp. were extracted and used for the germination and growth tests as previously reported (Bisio et al., 2010b) for both the Petri dish and the pot experiments. Four concentrations of testing solutions of Salvia exudates were used. The assay was performed in triplicate. Emergence of the radicle (≥1 mm) was used as an index of germination and it was observed daily for a period of 10 days in each replicate. Seedling growth, i.e. seedling height, cotyledon length and root length, were recorded with callipers. Fresh and dry weights of the seedlings were also measured.

Germination and phytotoxic activity indices

The phytotoxic effects of the tested exudates on germination were described by the use of germination indices (Chiapusio et al., 1997; Ranal & Garcia De Santana, 2006): Total Germination (GT) (Jefferson & Pennacchio, 2003; Ranal & Garcia De Santana, 2006), Speed of Germination (S), Speed of Accumulated Germination (AS) and Coefficient of the Rate of Germination (CRG) (Chiapusio et al., 1997; Ranal & Garcia De Santana, 2006). In addition, new formulas describing the phytotoxic damage were used: Weighted Average Damage (WAD), Differential Weighted Average Damage (DWAD), Germination Weighted Average Velocity (GWAV) (Bisio et al., 2010b). In order to provide a clearer description of these new indices, their mathematical formulations are listed below:

WAD is an index of phytotoxic activity against the target Papaver, Avena and Papaver and Avena together. WADs is the Weighted Average Damage caused by the considered species of Salvia against a

Data collection and advanced statistical analysis in phytotoxic activity of aerial parts exudates of Salvia spp.M. Giacomini et al.


model species taken separately or the model species taken all together.

Where p = Papaver and a = Avena. DWAD is an index of phytotoxic activity against Papaver vs. Avena. This is the Differential Weighted Average Damage (DWAD), which has been used to evaluate which species is most active against Papaver and less active against Avena.

Where GWAV is the Germination Weighted Average Velocity, d = 1, 2, 3 … Nd days of experiment (Nd= 10). The position of d in the formula above indicates that the germination time is a linear penalizing factor, like c. GWAV is an index against the target Papaver, Avena and Papaver and Avena together. The lethal concentrations needed to reduce germination by 50% (LC50) and by 90% (LC90) were also considered (Bisio et al., 2010b).

Database design methodologies

The entity-relation (E-R) approach was adopted (Batini et al., 1992) for the conceptual representation of information, whereas the relational database approach was used to describe the logic scheme of this system. Another possibility to describe the concepts at the basis of these tools is the ontological modelling (Kalinichenko et al., 2003), but in this project the contributions come from a quite broad spectrum of knowledge make the formation of a solid and shared ontology extremely difficult (Webster, 2002). Moreover, the E-R model is closer than other models to an almost automatic translation into logical schema within a real Data Base Management System (DBMS) (Giacomini et al., 2009).


Three replicates were maintained for each treatment in a completely randomised manner. Untransformed data was used to calculate the average and the standard deviation. For the analysis of the

variance (one-way ANOVA), the percentage germination data was transformed into arcsine-square root to meet the requirements of the test (Laterra & Bazzalo, 1999). The statistical significance was set at p<0.05 for all the tests, in order to determine significant differences among the averages.

Information engineering methodologies

Experimental data organisation was designed through the use of the Microsoft® SQL Server 2008, a relational model database server produced by Microsoft®. In addition, the Microsoft® Visual Studio .NET 2010 was used to produce the web application. A program written using MATLAB® environment, in particular the SOM Toolbox, was developed for the artificial neural networks analysis. This function package implements the Kohonen’s Self-Organising Map (SOM) algorithm, which was created by the Laboratory of Computer and Information Science (CIS) of the Department of Computer Science and Engineering at the Helsinki University of Technology. The SOM Toolbox functions were used to initialise and train the network and then to display the SOM results. Another essential part of this package is based on the k-means function:

),( kXkmeansIDX =

The application of k-means clustering provide a partition of the points in the n-by-p data matrix X into k clusters. The rows of X correspond to the points, while the columns correspond to variables. The kmeans returns a n-by-1 vector IDX containing the cluster indices of each point. By default, the kmeans uses squared Euclidean distances. When X is a vector, the kmeans treats it as an n-by-1 data matrix, regardless of its orientation (The MathWorks, 1994-2011).


Figures 1 and 2 show respectively the database Entity-Relation model and the related database diagram. The entity-relation (E-R) model is closer than other models to an almost automatic translation into logical schema within a real Data Base Management System (DBMS). Therefore the E-R approach (Batini et al., 1992) was adopted for the conceptual representation of information, whereas the relational database approach was used to describe its scheme. The GENUS and SPECIES ranks were considered in relation to the taxonomic data. The main entity is the EXPERIMENT, that collects all the experimental parameters, e.g. the pair of species (a Salvia and Papaver rhoeas L. species or a Salvia and


M. Giacomini et al.


Avena sativa L. species) involved in the test and the type of culture used (Petri dish or pot). The VALUE entity was created to collect the germination and growth experiment results. The database diagram (Figure 2) shows the relationships and the transposition of model entities into tables. The redundancy of data was minimised due to this organisation. The results of the experimental germination are automatically placed in the database structure, together with the morphological data collected on the subsequent growth of seedlings (seedling height, cotyledon length, root length, seedling weight). In addition, the database was associated to a specific website through which the user can view the results of the germination indices usually reported in the literature (Total Germination - GT, Speed of Germination - S, Speed of Accumulated Germination - AS, Coefficient of the Rate of Germination - CRG),and the newly defined indices (Weighted Average Damage - WAD, Differential Weighted Average Damage - DWAD, Germination Weighted Average Velocity - GWAV) (Bisio et al., 2010b) and other variables usually recorded in experiments of phytotoxicity (LC50, LC90). This specific web-based tool was designed to highlight automatically significant differences between the species, it implements the algorithms to calculate the one-way ANOVA followed by Duncan's multiple range test (Duncan, 1955). The analysis of the variance (ANOVA) is used to express the observed variance as a sum of several values, each one referable to different causes. Thus providing point estimates of the unknown population variance. The k populations considered were independent, normal and homoscedastic. The results are useful in order to perform a test of statistical validity for the difference of the k averages. The one-way analysis of variance involves a single independent

variable (or treatment) that is presumed to have some effect on the dependent measured variable. In applying the one-way ANOVA k populations (or groups) of equal size n were considered; on each element of a group the same treatment was performed and then the associated dependent variable was measured. A test of statistical validity can be performed on the ANOVA results, in which the null hypothesis is based on the assumption that the means of the k populations are equal. Duncan’s multiple-range test is performed if the ANOVA has produced a rejection of H0. It has the same assumptions as the ANOVA, and requires equal sample sizes. The test compares the range of any subset p of the sample means with the calculated statistic called the least significant range, denoted by Rp:

n

MSWrR pp =

where rp is called the least significant Studentised range, MSW is the mean square within the ANOVA, and n is the sample size. If the range of the subset is less than Rp, then all the sample means in the subset are taken to be equal. On the contrary, if the range of the subset is greater than Rp, then the sample means at the extremes of the range are considered to be significantly different, and thus their associated population means are regarded to be different. The tests should be done in a standard order. First, the range from the largest to smallest mean is compared with the appropriate Rp value. Then the range from the largest to the second smallest; next from largest to the third smallest; and so on, up to the comparison for the range from largest to second largest. The next comparison should be the range from the second largest

Figure 1. Database Entity-Relation model. It shows the main entities and relations of the designed database.



10% (within one element), and of a correlation greater than 90% with another element (only one element over two are maintained in the set). Amongst all possible data mining algorithms, that are present in literature, neural networks have been applied in the form of Self-Organising Maps, also called Kohonen maps. The SOMs are single layer feed-forward neural networks where the output neurons are organised in grids and represent a class of neural-network algorithms in the unsupervised-learning category (Kohonen, 2001). The SOM has spread into numerous fields of science and technology as an analysis method, for example, visualisation of statistical data and document collections, process analysis, diagnostics, monitoring and biomedical applications, including diagnostic methods and data analysis in bioinformatics (Kohonen, 2001). After the application of SOMs, k-means algorithm was performed to define neuron clusters. It is well established, from literature, that the result of applying k-means algorithm depends greatly on the centroid initial choice (Bradley & Fayyad, 1998). For this reason, five neurons were randomly selected as centroids (i.e. maximum five clusters), to avoid any bias. This clustering procedure was repeated 100 times using the skimmed files, setting the epochs number to 200 for the first phase (rough training phase) and to 1000 for the second phase (fine tuning phase). The number of neurons was set at 90 and the maximum

to the smallest and so on. These range comparisons with Rp are continued until all the relationships between the means are determined (Bernstein & Bernstein, 1999). In Figure 3 an example of a test result is shown: a Petri dish germination experiment is reported. As the results of this set of analysis did not show significant differences among the active species, a new approach not based on inferential statistics was taken into consideration. Further analysis was based on the artificial neural network methodology. In addition previous experience of some authors (Bertone et al., 1996; Giacomini et al., 2000; 1997) evidenced that this approach can be very valuable in scenarios where a great amount of quantitative data is used to yield evidence based conclusions. Due to the nature of the relational database it is possible to apply native methods of data mining to identify the hidden relationships and connections within the full set of data; providing that these algorithms are fed by highly accurate data, from the information content point of view. For this reason an algorithm associated with the database has been created to filter the incoming data according to their significance information, both within and among the attributes. The filtering criteria are based on the awareness that the first days after sowing germinal values are considered null and that the variables fresh weight and dry weight are highly correlated. As a result, some rows and columns were excluded because of a variation coefficient smaller than

Figure 2. Database design diagram.


M. Giacomini et al.


Figure 3. The growth rate ( ) ANOVA analysis of each Salvia species in Petri dish experiments. The evaluation of

germination speed of Papaver and Avena is considered in response to the various Salvia exudates (Salvia namaensis, 1; S. fallax, 2; S. disermas, 3; S. chamaedryoides, 4; S. confertiflora, 5; S. x jamensis, 6; S. buchananii, 7; S. wagneriana, 8; S. scabra, 9; S. miniata, 10; S. cacaliaefolia, 11; S. adenophora, 12; S. rutilans, 13). The mean GWAV plus standard deviation is reported. The letters A, B, C, D, F and M indicates the groups of species defined by the ANOVA application followed by the Duncan multiple range test.

Figure 4. A Kohonen map: an example of a clustering result. This map shows a range of grey-scale colours, but normally each cluster has a random colour. The SOM algorithm randomly assigns the cluster, so the specific colour is not relevant, but the boundaries of each area are important because they represent the clustering.

Table 1. Percentage of cluster distribution among the 100 performed tests.

No. of clusters

Experiment type

Avena (Petri) Avena (pot) Papaver (Petri) Papaver (pot)

2 - 96% 80% -

3 26% - 14% -

4 17% - 1% 17%

5 57% 4% 5% 83%

number of clusters to search at five. An example of the clustering test results is shown in Figure 4. Since five was the maximum number of clusters that was searched for, the algorithm in some cases found three or four clusters, when convergence was reached in advance. Table 1 provides a summary of clustering tests results for the types of culture and species used. The clustering was found to be fairly homogeneous although the tests are independent of each other. As referred to previously, the order of neurons is randomised for each test to ensure that the tests have the same input data but in random order, so that the operation of clustering does not depend on the order of the data input. As shown in Table 1 most of the tests related to Avena pot and Papaver Petri dish experiments gave only two clusters. This limited statistical evidence of the real presence of different clusters, has led to the development of an algorithm based on the Kohonen map topology

and inspired by the Indicator Value Analysis method of Dufrène and Legendre (Dufrène & Legendre, 1997) in order to obtain a ranking of the activity of the various Salvia species. The Indicator Value Analysis is a statistical analysis widely applied in biogeography, that looks for the species typical of areas of endemism. The Indicator Value Analysis introduces an index that combines the specificity and fidelity method to quantify the Indicator Species significance: indicator species are the ones mainly found in a single area of endemism and present in the major part of the subzones constituting the area (Dufrène & Legendre, 1997).

where IndVali is the Indicator Value for the species i; j is the Operational Geographic Unit (OGU); Nsites is the number of sites. Previous experience of some authors, shows how Indicator Species Analysis can be applied to obtain a quantitative estimate for the endemic relevance of a species in a certain area of endemism. Due to the analogy between the two fields, this methodology has been applied also to

mL day . μg



the phytotoxicity study explained in this work (Pivetti et al., 2009). The obtained results are reported in Chart 1. Also this new set of calculation rely on the relational structure of the DB. The present tool can be considered as a prototype for a fast and efficient management of such type of data. This further application of the tool will be able to also prove its versatility.

Conclusions

The system presented is based on a database structure that has been applied to this particular study. Therefore it represents the basis for a valid interaction between the users from different backgrounds. A clear structure and organisation of the information are highly important features in order to avoid redundancy, assure data consistency and encourage data communication. The authors sustain that this system is suitable also for other similar cases in the context of botany. In fact, for several years, these tools have been used in this field, for example in synthesis of nanostructures (Giacomini et al., 2009). The first approaches on this study were published in the Crop Protection journal (Bisio et al., 2010b). Consequently the system proposed allows the user to appreciate directly obtained results, through the use of a very complicated statistical tool, as well as the ANOVA or the SOM, without the need for a thorough knowledge of these tools. In fact this tool provides an equally high level of security as MATLAB®, for example, without interacting with highly sophisticated functions. This interface is used in order to explain the results obtained by these tools.

Acknowledgment

The work was developed within the EU

INTERREG ALCOTRA project “AROMA” n.68. The authors want to thanks Dr. Moyra Watson for her revision of English expressions and Dr. Mariangela Mota for her translation in Portuguese of the title, abstract and keywords.

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Chart 1. Ranking of Salvia species according to the method of Dufrène & Legendre (1997).Avena Papaver

Petri dish Pot Petri dish Pot

S. jamensis S. namaensis S. chamaedryoides S. confertiflora

S. chamaedryoides S. jamensis S. cacaliaefolia S. buchananii

S. confertiflora S. fallax S. namaensis S. rutilans

S. namaensis S. chamaedryoides S. disermas S. jamensis

S. cacaliaefolia S. confertiflora S. wagneriana S. cacaliaefolia

S. miniata S. scabra S. miniata S. wagneriana

S. rutilans S. miniata S. adenophora S. adenophora

S. buchananii S. rutilans S. fallax S. disermas

S. scabra S. adenophora S. jamensis S. scabra

S. adenophora S. wagneriana S. scabra S. namaensis

S. disermas S. disermas S. confertiflora S. miniata

S. wagneriana S. buchananii S. buchananii S. chamaedryoides

S. fallax S. cacaliaefolia S. rutilans S. fallax

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*Correspondence

M. GiacominiDipartimento di Informatica, Sistemistica e Telematica, Università di GenovaVia all’Opera Pia 16145 Genova, [email protected]. +39 010 353 6546Fax: +39 010 353 2154

864

Article


Received 29 Nov 2010Accepted 8 Mar 2011Available online 6 May 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 864-868, Sep./Oct. 2011 Chemical composition and cytotoxic activity

of the essential oil from the leaves of Casearia lasiophylla

Marcos José Salvador,*,1 João Ernesto de Carvalho,2 Alberto Wisniewski-Jr,3 Candida A. L. Kassuya,4 Élide P. Santos,5 Dilamara Riva,6 Maria Élida Alves Stefanello6

1Curso de Farmácia, Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Brazil,2Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Universidade Estadual de Campinas, Brazil,3Departamento de Química,Universidade Federal de Sergipe, Brazil,4Faculdade de Ciências da Saúde, Universidade Federal da Grande Dourados, Brazil,5Departamento de Botânica, Universidade Federal do Paraná, Brazil,6Departamento de Química, Universidade Federal do Paraná, Brazil.

Abstract: The essential oil obtained by hydrodistillation from fresh leaves of Casearia lasiophylla Eichler, Salicaceae, was analyzed by gas capillary (GC/FID and GC/MS). The cytotoxicity of the leaves essential oil was tested in vitro against U251 (glioma), UACC-62 (melanoma), MCF-7 (breast), NC1-ADR/RES (ovarian-resistant), NCI-H460 (lung), PC03 (prostate), OVCAR-3 (ovarian), HT-29 (colon) and K562 (leukemia) human cancer cells and against VERO (no cancer cell). The yield of oil was 0.02%. Fifty two compounds were identified, representing 87.1% of the total of the oil. The main components were identified as germacrene D (18.6%), β-caryophyllene (14.7%), δ-cadinene (6.2%), and α-cadinol (5.4%). The oil exhibited antiproliferative activity against all cell lines (TGI<100 μg/mL), with exception of NCI-H460 cell line (TGI 191.31μg/mL). The highest activity was observed against UACC-62 (TGI 7.30 μg/mL), and K562 (TGI 7.56 μg/mL) cell lines. The observed activity could be related to high content of germacrene D and β-caryophyllene, compounds known as cytotoxic.

Keywords:Casearia lasiophyllaSalicaceaeessential oilgermacrene Dβ-caryophylleneantitumor

Introduction

Casearia species (Salicaceae) have been used in folk medicine in Brazil against snakebite and for the treatment of skin diseases and ulcers. Several pharmacological activities have been demonstrated to species of this genus, including anti-inflammatory, anti-ulcer and antitumor (Santos et al., 2010; Vieira Jr et al., 2009; Flausino Jr et al., 2009). Previous chemical analyses of Casearia have demonstrated the occurrence of clerodane diterpenoids with cytotoxicity against several human cancer cell lines (Santos et al., 2010; Vieira Jr et al., 2009; Silva et al., 2009; Chen et al., 2008; Sertiè et al., 2000). The essential oils of Casearia spp analyzed to date are rich in sesquiterpenes (De Morais et al., 1997; Esteves et al., 2005; Schneider et al., 2006; Sousa et al., 2007; Silva et al., 2008; Stefanello et al., 2010) and one sample showed cytotoxic activity (Silva et al., 2008). Casearia lasiophylla Eichler, known as “guaçatunga-

graúda” is a tree native from Brazil for which no chemical or pharmacological evaluation has previously been reported. It has been used in folk medicine to treat wounds (Pedroso et al., 2007; Keller & Tressens, 2007). This prompted us to investigate the chemical and biological properties of this plant. As part of a research project focusing on phytochemical investigation of Brazilian medicinal plants searching for new bioactivity of natural products, in this study, we report the chemical analysis of the essential oil of C. lasiophylla by capillary GC/FID and GC/MS, with the identification of fifty-two compounds. Moreover, the cytotoxic activity of this essential oil against human cancer cell lines is also reported.


Plant material

The leaves of Casearia lasiophylla Eichler,

Chemical composition and cytotoxic activity of the essential oil from the leaves of Casearia lasiophylla

Marcos José Salvador et al.


Salicaceae, were collected in Colombo, Paraná State, Brazil. A voucher specimen (Santos 1250 UPCB) is deposited in the herbarium of Universidade Federal do Paraná.

Isolation and chemical analysis of the essential oil

Fresh leaves were subjected to hydrodistillation for 2 h. The oil was recovered with diethyl ether and dried over anhydrous sodium sulfate. The solvent was removed under vacuum, providing colorless oil with yield (w/w) of 0.02%. The oil was maintained under refrigeration before analysis. The GC analysis was performed on a Shimadzu GC-17A gas chromatograph with FID detector, in a split injector mode. A Durabond-DB5 capillary column (30 m x 0.25 mm, 0.25 μm film thickness) was operated at 60 ºC for 3 min, and then programmed from 60-220 ºC at 5 ºC/min, after which it was kept isothermal at 220 ºC for 5 min. The carrier gas was helium (1 mL/min) and the injector temperature was 250 ºC. The relative percentage of individual components is based on the peak areas obtained by electronic integration without FID response factor correction. The GC/EIMS (70 eV) analysis was performed on a Varian Saturn 2000 GC/ MS spectrometer equipped with a CP-Sil-8CB capillary column (30 m x 0.25 mm, 0.25 μm film thickness) under the same conditions described above. The essential oil components were identified by comparison of their retention indices relative to n-alkanes (Kovat’s index) and mass spectra with those found in the literature (Adams, 2007) and stored on the spectrometer database (NIST 1998). In addition, the compounds limonene, eugenol, methyl salicilate, β-caryophyllene, α-humulene and spathulenol were confirmed by comparison with standards.

Antiproliferative assay

Cell lines: U251 (glioma, CNS), UACC-62 (melanoma), MCF-7 (mama), NCI-ADR/RES (ovarian-resistent), NCI-H460 (lung, no small cells), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon), K562 (leukemia) and VERO cell lines, from American Type Culture Collection (ATCC), were used. The assay was done as described previously (Skehan et al., 1990). Briefly, the cells were distributed in 96-well plates (100 μL cells/well) and exposed to various concentrations of essential oil (0.25, 2.5, 25.0 and 250.0 μg/mL) in DMSO (0.1%) at 37 oC, with 5% of CO2, for 48 h. The final concentration of DMSO did not affect the cell viability. A 50% trichloroacetic acid solution was added and after incubation for 30 min at 4 oC, the cells were washed and dried. Cell proliferation was determined by spectrophotometric quantification (at 540 nm) of the cellular protein content using sulforhodamine B. The

experiments were carried, at least, in triplicate and the concentration necessary to total growth inhibition (TGI) was calculated in μg/mL. Doxorubicin was used as positive control.


Triplicates from at least three separate experiments were employed in each of the antiproliferative assays. An exploratory data analysis was performed initially to determine the most appropriate statistical test; the assumptions of equality of variances and normal distribution of errors were also checked. The relative potency was based on the drug concentration that inhibited cell growth by 50% (IC50) [calculated from [(T-T0)/(C-T0)]×100=50, which is the drug concentration resulting in a 50% reduction in the net cell number of drug-treated cultures relative to the increase in control cultures during the drug incubation period]. The data were analyzed using ANOVA and the F-test used to determine any difference among the groups. When significant differences were detected, pair wise comparisons were made between all the groups using Tukey’s method to adjust for multiple comparisons. Statistical software GraphPad prism 2.01 (GraphPad Software, San Diego, CA) was used to perform the analyses. The level of significance was set at 5%.


The oil obtained by hydrodistillation from fresh leaves of C. lasiophylla was characterized by high content of cyclic sesquiterpenes (86.4%), mainly hydrocarbons (59.9%). Monoterpenes (limonene), aromatic compounds (mesitylene, methyl salicilate, eugenol), and aliphatic compounds (isopentyl acetate) were found as minor constituents. Altogether sixty-four components were observed, of which fifty two were identified, representing 87.1% of the total of the oil (Table 1). The main constituents were germacrene D (18.6%), E-caryophyllene (14.7%), δ-cadinene (6.2%), and α-cadinol (5.4%). The predominance of sesquiterpene hydrocarbons in the leaves essential oil of Casearia species has been previously observed (Stefanello et al., 2010; Silva et al., 2008; Sousa et al., 2007; Schneider et al. 2006; Esteves et al., 2005; De Morais et al., 1997). In particular, E-caryophyllene was found in all samples as a main component (more than 10%). The oil exhibited antiproliferative activity for almost all cell lines evaluated, with TGI varying from 7.30 to 55.26 μg/mL, with exception of NCI-H460 cell line, for which the TGI was 191.31 μg/mL. The most significant activity was observed against UACC-62

Chemical composition and cytotoxic activity of the essential oil from the leaves of Casearia lasiophyllaMarcos José Salvador et al.


trans-Cadina-1,4-diene 1530 0.3α-Cadinene 1534 0.2Germacrene B 1556 2.4Maaliol 1567 0.6Spathulenol c 1575 1.6Caryophyllene oxide 1578 1.7Globulol 1585 TrNI (m/z 135) 1587 TrSalvial-4(14)-en-1-one 1590 0.1Carotol 1592 2.5Cubeban-11-ol 1594 0.8Guaiol 1600 0.35-epi-7-epi-α-Eudesmol 1605 0.6Humulene epoxide II 1607 0.1NI (m/z 69, 161, 179) 1613 TrNI (m/z 79, 91, 105, 123) 1616 TrJunenol 1619 1.610-epi-γ-Eudesmol 1622 0.81-epi-Cubenol 1626 2.8NI (m/z 91, 105, 119) 1628 Trcis-Cadin-4-en-7-ol 1633 0.6epi-α-Cadinol 1641 1.8epi-α-Muurolol 1644 4.8α-Muurolol 1647 0.1α-Cadinol 1655 5.4Selin-11-en-4α-ol 1659 TrNI (m/z 123) 1672 TrKhusilol 1680 Tr2,3-Dihydrofarnesol 1689 TrEudesm-7(11)-en-4-ol 1696 0.3NI (m/z 71, 81, 95, 123) 2109 1.0NI (m/z 73, 221, 355, 429) 2262 7.6NI (m/z 73, 221, 355, 429) 2576 4.3

Total identified (%) 87.1NI: not identified; tr: trace (<0.05%); aTentative identification based on computer matching of the mass spectra of peaks with NIST 98 library and published data (Adams, 2007), besides comparison of retention indices on published data (Adams, 2007). bRI Retention index relative to n-alkanes on a DB-5 column. cIdentification based on retention time of authentic compounds on the DB-5 column.

Despite the cytotoxic activity of the essential oil of C. lasyophylla is lower than that of the positive control (doxorubicin), the present results reveal the antitumor potential of this species. The presence of significant amounts of unidentified compounds in the oil that could be contributing to bioactivity points

(TGI 7.30 μg/mL, melanoma) and K562 (TGI 7.56 μg/mL, leukemia). For VERO cell (non-cancer cell-line) the TGI estimated was 65.80 mg/mL, about 8.5 times larger than the TGI of the melanoma and leukemia cell lines, for which the essential oil showed the major bioactivity (Table 2). The coefficients of variation obtained in these analyses were below to 5%. Among the identified sesquiterpenes, germacrene D, E-caryophyllene and α-cadinol are recognized as cytotoxic and, could explain the observed activity (He et al., 1997; Silva et al., 2008; Loizzo et al., 2008; Palazzo et al., 2009). The oil of Casearia sylvestris also has high content of E-caryophyllene (18.1%) and showed similar cytotoxic activity agains HT-29 cell line (Silva et al., 2008).

Table 1. Chemical composition of Casearia lasiophylla leaf essential oil.Component a RI b Percentage (%)Isopentyl acetate 879 0.3NI (m/z 91, 105, 120) 892 TrNI (m/z 91, 105, 120) 957 TrMesitylene 994 0.1Limonene c 1027 0.1Methyl salicilate c 1192 0.1NI (m/z 73, 149) 1299 TrNI (m/z 77, 93, 105, 121) 1329 Trδ-Elemene 1332 0.9α-Cubebene 1344 0.1Eugenol c 1353 0.1α-Copaene 1373 0.4β-Bourbonene 1380 0.1β-Cubebene 1385 0.2β-Elemene 1387 1.5E-Caryophyllene c 1417 14.7β-Copaene 1427 2.2α-Guaiene 1432 2.6α-Humulene c 1452 2.8allo-Aromadendrene 1457 0.4cis-Muurola-4(14), 5-diene 1470 Trγ-Gurjunene 1473 1.1Germacrene D 1479 18.6β-Selinene 1484 0.7cis-β-Guaiene 1488 0.8Bicyclogermacrene 1493 1.8α-Muurolene 1496 1.0trans-β-Guaiene 1499 1.0γ-Cadinene 1511 0.5δ-Cadinene 1516 6.2Zonarene 1521 0.4

Chemical composition and cytotoxic activity of the essential oil from the leaves of Casearia lasiophylla

Marcos José Salvador et al.


out the need of phytochemical studies in this species, aiming at the isolation and identification of its secondary metabolites.

Table 2. Antiproliferative activity of Casearia lasiophylla leaves essential oil.

Cell linesTGI (μg/mL)*

Essential oil DoxorubicinU251 27.39 0.12UACC-62 7.30 <0.025MCF-7 40.72 0.25 NCI-ADR/RES 43.32 2.26 NCI-H460 191.31 1.17 PC-3 36.97 0.40 OVCAR-3 29.49 0.28 HT-29 55.26 3.04 K562 7.56 0.15 VERO 65.80 6.12

*TGI: Total growth inhibition - concentration that inhibited cell growth by 100%. The coefficients of variation obtained in these analyses were below to 5%. Cell lines: U251 (glioma); UACC-62 (melanoma); MCF7 (breast); NCI-ADR/RES (ovarian-resistent); NCI-H460 (lung); PC03 (prostate); OVCAR-3 (ovarian); HT-29 (colon); K562 (leukemia); VERO (no cancer cell).

Acknowledgements

The authors are grateful to CNPq, FAPESP and FAEPEX-UNICAMP for financial support.

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Sousa FG, Schneider NFZ, Mendes CE, Moura NF, Denardin RBN, Matuo R, Mantovani MS 2007. Clastogenic and anticlastogenic effect of the essential oil from Casearia sylvestris Swart. J Essent Oil Res 19: 376-378.

Chemical composition and cytotoxic activity of the essential oil from the leaves of Casearia lasiophyllaMarcos José Salvador et al.


Stefanello MEA, Wisniewski-Jr A, Simionatto EL, Cervi AC 2010. Essential oil composition of Casearia decandra Jacq. J Essent Oil Res 22: 157-158.

Vieira Jr GM, Gonçalves TO, Regasini LO, Ferreira PMP, Pessoa CO, Lotufo LVC, Torres RB, Boralle N, Bolzani VS, Cavalheiro AJ 2009. Cytotoxic clerodane diterpenoids from Casearia obliqua. J Nat Prod 72: 1847-1850.

*Correspondence

Marcos José SalvadorCurso de Farmácia, Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de CampinasCaixa Postal 6109, 13083-970 Campinas-SP, [email protected].: +55 19 3521 6167Fax: +55 19 3521 6184

869

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695X2011005000082

Received 6 Jan 2011Accepted 20 Mar 2011



21(5): 869-873, Sep./Oct. 2011Active caspase-3 detection to evaluate apoptosis induced by Verbena officinalis essential oil and citral in chronic lymphocytic leukaemia cells

Laura De Martino,1 Giovanni D’Arena,2 Maria Marta Minervini,2 Silvia Deaglio,3 Nicola Pio Sinisi,2 Nicola Cascavilla,2 Vincenzo De Feo*,1

1Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Salerno, Italy,2Unità di Ematologia e Trapianto di Cellule Staminali, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (Foggia), Italy,3Laboratorio di Immunogenetica, Università di Torino, Torino, Italy.

Abstract: Verbena officinalis L., Verbenaceae, commonly known as vervain, is a plant widely used in medicine. Despite of its widespread use in different traditional practices, the mechanisms of pharmacological actions of the plant and its volatile oil are still unclear. We evaluated the pro-apoptotic activity of V. officinalis essential oil and of its main component, citral, on lymphocytes collected from ten patients with chronic lymphocytic leukaemia (CLL), a disease in which a faulty apoptotic mechanism is still retained one of the primary pathogenic events, by adding to treated mononuclear cells, annexin-V, propidium iodide, and CD19. Apoptosis was also evaluated using anti-active-caspase-3 monoclonal antibody after permeabilization of the cells. Both V. officinalis essential oil and citral were found able to induce apoptosis in CLL cells and to activate caspase-3, which is considered the way by means they active apoptosis in B neoplastic cells. This data further support evidences that indicate natural compounds as possible lead structure to develop new therapeutic agents for CLL.

Keywords:apoptosis

citralcaspase-3

chronic lymphocytic leukaemia flow cytometry

Verbena officinalis

Introduction

Numerous bioactive substances seem to act as cancer-preventing agents by inhibiting the activation of pro-carcinogens, enhancing the detoxification of carcinogens or impeding the progression of carcinogenesis (Hursting et al., 1999; Wattenberg, 1992). Chronic lymphocytic leukaemia (CLL) is the commonest form of leukaemia in Western World and is considered a disease of B-cell in which a faulty apoptotic mechanism is still retained one of the primary pathogenic events (Chiorazzi et al., 2005; Reed & Kitada, 2001). We have recently showed that Verbena officinalis L., Verbenaceae, essential oil and its component citral are able to induce in vitro apoptosis of CLL cells thus suggesting its hypothetical therapeutic role in this disease (De Martino et al., 2009). However, the molecular mechanism, which underlies this process, is still unclear. Results of some studies are consistent with the hypothesis that some essential oil components in cancer cellular lines act on cell cycle and apoptosis (Carnesecchi et al., 2001; Gu et al., 2010). Dudai and

coworkers (2005) suggested that citral displays its proapoptotic activity through a direct procaspase 3 activation in human and mouse leukaemic cell lines. In this study, we report our data of a flow cytometric study aiming to evaluate the ability of V. officinalis essential oil and its component citral to activate caspase-3 of CLL cells in vitro.


Plant material

Verbena offi cinalis L., Verbenaceae, aerial parts were collected in July 2008 from plants growing at the Garden of Medicinal and Aromatic Plants on the Campus of Salerno University. The plant was identifi ed by Prof. Vincenzo De Feo. A voucher specimen of the plant is deposited in Herbarium of the Medical Botany Chair, at the University of Salerno, labeled as DF/154/2008.

Oil isolation and analysis

Active caspase-3 detection to evaluate apoptosis induced by Verbena officinalis essential oil and citral in chronic lymphocytic leukaemia cellsLaura De Martino et al.


One hundred grams of fresh aerial parts were submitted to hydrodistillation, in agreement with procedures of the European Pharmacopoeia (2004). A pale yellow essential oil was recovered in a 0.39% yield. The chemical composition of the oil was obtained by GC and GC-MS. Analytical gas chromatography was carried out on a Perkin-Elmer Sigma 115 gas chromatograph fitted with an HP-5 MS capillary column (30 m x 0.25 mm i.d.; 0.25 μm film thickness). Column temperature was initially kept at 40 °C for 5 min, then gradually increased to 250 °C at 2 °C/min, held for 15 min and finally raised to 270 °C at 10 °C/min. Diluted samples (1/100 v/v, in n-hexane) of 1 μL were injected manually at 250 °C, and in splitless mode. Flame ionization detector (FID) was kept at 280 °C. Analysis was also run by using a fused silica HP Innowax polyethylenglycol capillary column (50 m x 0.20 mm, i.d.; 0.25 μm film thickness). In both cases, carrier gas was He, with flow rate of 1 mL/min. GC-MS analyses were performed on an Agilent 6850 Ser. II apparatus, fitted with a fused silica HP-1 capillary column (30 m x 0.25 mm; 0.33 μm film thickness), coupled to an Agilent Mass Selective Detector MSD 5973; ionization voltage 70 eV; electron multiplier energy 2000 V. Gas chromatographic conditions were as reported above; transfer line temperature, 295 °C. Most constituents were identified through gas chromatography by comparing their retention indices to either those from the literature (Davies, 1990) or with those of authentic compounds available in our laboratories. The retention indices were determined in relation to a homologous series of n-alkanes (C8-C24) under the same operating conditions. Further identification was made by comparing the mass spectra either to those stored in NIST 02 and Wiley 275 libraries or to the mass spectra from literature (Adams, 2007) and our home-made library. Component relative concentrations were calculated based on GC peak areas without using correction factors. Citral was purchased by Sigma-Aldrich Co, Milan, Italy.

Biological assays

Ten patients with untreated CLL were included in this study. Clinical and biological features of these patients at study entry are given in Table 1. A written informed consent was obtained before sample collection from all subjects. Mononuclear cells were isolated from heparinized peripheral blood by density gradient centrifugation and washed twice with phosphate buffered saline (PBS). Briefly, cells were incubated for up to 24 h at a density of 2.5 x 106/µL in RPMI 1640 at 37 °C CO2, with a mixture of 90 µL of PBS and 0.1 µL of vervain essential oil (A) and 9.9 µL of distilled water (to obtain vervain essential oil diluted 1:100) or 10 µL

of pure citral (B) at the concentration of 1.9 mM and with only 100 µL of PBS to also assess spontaneous apoptosis and to use as internal control. The proapoptotic effect of the compound was evaluated after three different times of incubation (4, 8, 24 h) at room temperature in dark conditions, by adding to treated mononuclear cells, after having been washed twice in PBS, annexin V 5 µL and propidium iodide 5 µL [Annexin V-FITC Apoptosis Detection Kit I (Becton Dickinson Biosciences [BDB] Pharmingen) and CD19-APC-Cy7 (BDB). In addition, anti-active caspase-3-FITC (BDB Pharmingen) and CD19-APC-Cy7 (BDB) were added to 1 x 106 treated mononuclear cells, after fixation with 100 µL of Reagent A (Permeabilization Medium, Fix & Perm, Caltag, USA) for 15 min at room temperature in the dark, washing in PBS, permeabilization with 100 µL of Reagent A (Permeabilization Medium), and washing in PBS. Cells were then incubated in the dark for 15 min, finally resuspended in PBS and analyzed by flow cytometry, by acquiring a minimum of 20,000 events for each sample and using an analysis gate on CD19-positive cells to avoid non neoplastic T-cell contamination. As internal control, untreated mononuclear cells were also stained in the similar fashion. A FACSCanto II cytometer equipment (Becton Dickinson) was used. All subsequent analyses were performed using FACS-Diva Software (BDB).

Table 1. Clinical-biological characteristics of CLL patients at study entry.Characteristics Patients (no.)

Sex Male Female

6 4

Age (years) Mean Range

64.5 years

46 - 87 years

Rai clinical stage 0-II III-IV

6 4

B-cell lymphocytosis (/µL) Mean Range

32,000

9,400-65,000

CD38 expression (cut-off 20%) Positive Negative

2 8

ZAP-70 expression (cut-off 20%) Positive Negative

3 7

IgVH mutational status Mutated Unmutated

7 3

Cytogenetic abnormalities (by FISH) Normal del13q14 +12

4 3 3

CD38: Cluster Differentiation number 38; ZAP-70: Zeta chain associated protein kinase; IgVH: Immunoglobulin heavy chain variable region mutational status; FISH: Fluorescence in situ hybridization.

Active caspase-3 detection to evaluate apoptosis induced by Verbena officinalis essential oil and citral in chronic lymphocytic leukaemia cells

Laura De Martino et al.



Table 2 reports the percentage composition of the essential oil of Verbena officinalis L, Verbenaceae. Forty components were identified, accounting for


Results were expressed as the mean of three replicates±SD. The values of apoptotic cells were compared by using Student t test as appropriate.

Table 2. Percentage composition of Verbena officinalis essential oil.Compound Kia Kib %c Identificationd

α-Pinene 938 1032 0.2±0.0 LRI, MS, Co-GC

Sabinene 973 1132 0.5±0.0 LRI, MS, Co-GC

Hepten-3-one 975 0.2±0.1 LRI, MS

β-Pinene 978 1118 T LRI, MS, Co-GC

α-Terpinene 1012 1188 T LRI, MS, Co-GC

o-Cymene 1020 1187 0.1±0.0 LRI, MS, Co-GC

β-Phellandrene 1029 1218 0.7±0.2 LRI, MS, Co-GC

Limonene 1030 1203 2.3±0.9 LRI, MS, Co-GC

1,8-Cineole 1034 1213 0.4±0.1 LRI, MS

(Z)-β-Ocimene 1038 1246 T LRI, MS, Co-GC

(E)-β-Ocimene 1049 1280 0.3±0.1 LRI, MS, Co-GC

γ-Terpinene 1057 1255 0.1±0.0 LRI, MS, Co-GC

Terpinolene 1086 1265 T LRI, MS

Linalool 1097 1553 0.1±0.0 LRI, MS, Co-GC

trans-Pinocarveol 1138 1654 T LRI, MS

iso-Pinocamphone 1153 1566 0.2±0.0 LRI, MS

trans-Pinocamphone 1159 T LRI, MS

Pinocarvone 1165 1587 T LRI, MS

Borneol 1167 1719 0.1±0.0 LRI, MS, Co-GC

Terpinen-4-ol 1176 1611 0.2±0.0 LRI, MS, Co-GC

p-Cymen-8-ol 1185 1864 T LRI, MS

α-Terpineol 1189 1706 0.3±0.1 LRI, MS, Co-GC

Isobornyl formate 1228 44.4±0.9 LRI, MS

cis-Anethole 1262 0.2±0.0 LRI, MS

(E)-Citral 1270 45.5±0.9 LRI, MS, Co-GC

Isobornyl acetate 1277 T LRI, MS

Bornyl acetate 1284 1591 T LRI, MS

α-Copaene 1377 1497 0.2±0.1 LRI, MS

Isoledene 1382 0.1±0.0 LRI, MS

β-Elemene 1387 1600 0.2±0.1 LRI, MS

Longifolene 1411 1576 T LRI, MS

β-Caryophyllene 1418 1612 0.1±0.1 LRI, MS

β-Cedrene 1424 1638 0.4±0.1 LRI, MS

α-Humulene 1455 1689 0.2±0.0 LRI, MS

allo-Aromadendrene 1463 1661 0.1±0.0 LRI, MS

γ-Gurjunene 1473 1687 T LRI, MS

Bicyclogermacrene 1491 1756 0.1±0.0 LRI, MS

cis-Muurola-4(14),5-diene 1510 1675 0.2±0.1 LRI, MS

α-7-epi-Selinene 1518 1740 0.2±0.1 LRI, MS

Total 97.6Kia: Kovats retention index on HP-5 MS column; Kib: Kovats retention index on HP Innowax; c: T trace <0.05; d: LRI linear retention index; MS: identification based on comparison of mass spectra; Co-GC: retention time identical to authentic compounds.

Active caspase-3 detection to evaluate apoptosis induced by Verbena officinalis essential oil and citral in chronic lymphocytic leukaemia cellsLaura De Martino et al.


97.6% of the total oil. The oil is mainly constituted by monoterpenes (95.4%), of which oxygenated compounds constitute 91.2%; citral is the main constituent (45.5%). The composition of the essential oil differs from others reported in literature. In fact, Ardakani and co-workers (2003) reported 1-octen-3-ol and verbenone as the main constituents of the essential oil from V. officinalis collected in Iran. On the other hand, spathulenol, limonene and 1,8-cineole have been reported as principal constituents of an essential oil from Morocco (Chalchat & Garry, 1996). In all patients with CLL, the number of B-cells was found >85% of all lymphocytes. Table 3 shows the pro-apoptotic activities of V. officinalis essential oil and citral, compared to untreated cells, used as internal control to evaluate spontaneous apoptosis, on B-cells collected from CLL patients. The analysis of these ten patients again confirmed our previous observation that V. officinalis essential oil and citral are able to induce apoptosis of neoplastic B-cells in CLL at all different times of incubation. As showed, the percentage values of apoptosis increase in 8 h and reduce after 24 h. This is probably due to the fact that the percentage of necroctic cells steadily increased with time peaking at 24 h in all samples. In addition, no difference was found between the activities of both compounds (p ns). Preliminary literature evidence indicates that isoprenoids, a broad class of mevalonate-derived phytochemicals which are ubiquitous in the plant kingdom, may suppress, with great potency, the proliferation of tumor cells: also in a previous study De Martino and co-workers (2009) demonstrated that both vervain essential oil and citral induced a significant apoptosis in CLL samples compared to controls; Gu and co-workers (2010) demonstrated that linalool, a natural small molecule monoterpene, inhibits growth of leukaemia cells with wt p53 while sparing normal hematopoietic cells. Apoptosis, or programmed cell death, is a highly ordered, genetically controlled process that plays a fundamental role in both normal biological processes and disease status (Kerr et al., 1972). Apoptosis generally occurs as a result of cell insult or activation of death receptors, both of which lead to a cascade of cell signalling and caspase-mediated events culminating in cell death (Cohen, 1997; Danial & Korsemeyer, 2004; Riedl & Shi, 2004). The death receptors include tumor necrosis factor receptor (TNFR), Fas, decoy receptors and death receptors. After ligand binding, these death receptors interact with a variety of death domain adaptor proteins which subsequently activate the caspases and various signalling pathways. The caspase family is involved in a series of cleavage events that results in the initiation and execution of apoptosis. In addition to the death receptors and caspases, members of the

Bcl-2 protein family are also critical for the regulation of apoptosis, largely by controlling the permeability of the outer mitochondrial membrane to proteins such as cytochrome c. Bcl-2 and Cbl-xL are the most prominent anti-apoptotic members of this family, and their functions can be regulated through interactions with pro-apoptotic Bcl-2 family members, such as Bad and Bax.

Table 3. Verbena officinalis essential oil (A) and citral (B) induced-apoptosis in lymphocytes collected from CLL patients and comparison with spontaneous apoptosis. Data are expressed as mean percentage number of CD19-positive cells (± standard deviation) and range (in brackets).

Hours A Spontaneous apoptosis p

4 56.2±10.7 (46-80) 5.4±1.6 (0.9-7.0) <0.0001

8 68.2±8.9 (58-83) 6.7±2.3 (1.3-10) <0.0001

24 32.1±6.5 (24-52) 6.9±1.3 (2.6-10) <0.0001

B

4 57.3±9.2 (48-72) 4.3±1.1 (1.0-5.9) <0.0001

8 65.9±9.6 (50-80) 7.0±1.3 (1.5-9.9) <0.0001

24 32.2±7.9 (23-47) 7.2±2.0 (2.3-9.8) <0.0001

Table 4. Verbena officinalis essential oil (A) and citral (B) induced-caspase-3 activation in lymphocytes collected from CLL patients and comparison with spontaneous apoptosis. Data are expressed as mean percentage number of CD19-positive cells (± standard deviation) and range (in brackets).

Hours A Spontaneous apoptosis p

4 54.3±11.1 (43-78) 2.3±1.1 (1.2-4.5) <0.0001

8 63.7±8.6 (53-81) 3.9±1.5 (1.9-5.9) <0.0001

24 32.1±6.5 (22-42) 6.9±1.9 (2.4-8.9) <0.0001

B

4 61±9.8 (47-79) 2.6±1.2 (1.3-5.1) <0.0001

8 64.5±7.9 (54-79) 4.1±1.6 (2.0-5.2) <0.0001

24 33.3±9.2 (22-49) 7.8±3.2 (2.0-6.9) <0.0001

Caspase-3 is an active cell-death protease involved in the execution phase of apoptosis, where cells undergo morphological changes such as DNA fragmentation, chromatin condensation and apoptotic body formation. Caspase-3 is activated in response to serum withdrawal, activation of Fas, treatment with radiation, pharmacological agents, as well as other upstream caspases like caspase-8 and caspase-9. In this study we showed that active caspase-3 was detected in all samples in vitro treated with both V. officinalis essential oil and citral, without statistical differences between the two compounds (Table 4). Also in this case, it is probable that the percentage of necrotic cells increased with time picking at 24 h. In Figure 1 a representative experiment is showed. So, this work is a confirmation and a sequel of the previous study (De

Active caspase-3 detection to evaluate apoptosis induced by Verbena officinalis essential oil and citral in chronic lymphocytic leukaemia cells

Laura De Martino et al.


Martino et al. 2009): here, we demonstrated that both vervain essential oil and citral induced a significant apoptosis in CLL samples compared to controls and these substances induced a death pathway activating caspase 3, which is considered the way by means they active apoptosis in B neoplastic cells. This data further support evidences that indicate natural compounds as possible lead structure to develop new therapeutic agents for CLL.

Figure 1. Flow cytometric analysis of activated caspase-3 by Verbena officinalis essential oil in a CLL patient sample at 8 h. Untreated cells (grey histogram) and treated cells (red histogram) are shown.

References

Adams RP 2007. Identification of essential oil components by gas chromatography/mass spectroscopy, 4th ed. Allured Publishing Corporation: Carol Stream, IL, USA.

Ardakani MS, Mosadeggh M, Shafaati A 2003. Volatile constituents from the aerial parts of Verbena officinalis L. (Vervain). Iran J Pharm Res 2: 39-42.

Carnesecchi S, Schneider Y, Ceraline J, Duranton J, Duranton B, Gosse F, Seiler N, Raul F 2001. Geraniol, a component of plant essential oils inhibits growth and polyamine biosynthesis in human colon cancer cells. J Pharmacol Exp Ther 298: 197-200.

Chalchat JC, Garry RP 1996. Chemical composition of the leaf oil of Verbena officinalis L. J Essent Oil Res 8: 419-420.

Chiorazzi N, Rai KR, Ferrarini M 2005. Chronic lymphocytic leukemia. N Engl J Med 352: 804-815.

Cohen GM 1997. Caspases: the executioners of apoptosis. Biochem J 326: 1-16.

Danial NN, Korsemeyer SJ 2004. Cell death: critical control points. Cell 116: 205-219.

Davies NW 1990. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. J Chromatogr 503: 1-24.

De Martino L, D’Arena G, Minervini MM, Deaglio S, Fusco BM, Cascavilla N, De Feo V 2009. Verbena officinalis essential oil and its component citral as apoptotic-inducing agent in chronic lymphocytic leukemia. Int J Immunopathol Pharmacol 22: 1097-1104.

Dudai N, Weinstein Y, Krup M, Rabinski T, Ofir R 2005. Citral is a new inducer of caspase-3 in tumor cell lines. Planta Med 71: 484-488.

European Pharmacopoeia 2004. 5th ed., Council of Europe, Strasbourg Cedex, France 2.8.12, p. 217-218.

Gu Y, Zhang T, Qiu X, Zhang X, Gan X, Fang Y, Xu X, Xu R 2010. Linalool preferentially induces robust apoptosis of a variety of leukemia cells via upregulating p53 and cyclin-dependent kinase inhibitors. Toxicology 268: 19-24.

Hursting SD, Fisher SM, Wargovich MJ, Digiovanni J 1999. Nutritional modulation of the carcinogenesis process. In: Heber D, Blackburn G (eds.). Nutritional Oncology, Academic Press: San Diego, CA, p. 91-104.

Kerr JFF, Wylie A, Currie AR 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257.

Reed JC, Kitada S 2001. Apoptosis deregulation in chronic lymphocytic leukemia. In:. Cheson B (Ed), Chronic lymphoid leukemias. Marcel Dekker, Inc., NY, USA, p. 111-126.

Riedl SJ, Shi Y 2004. Molecular mechanisms of caspase regulation during apoptosis. Nature Rev Mol Cell Biol 5: 897-907.

Wattenberg LW 1992. Inhibition of carcinogenesis by minor dietary constituents. Cancer Res 52: 2085-2091.

*Correspondence

Prof. Vincenzo De FeoDipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte don Melillo, 84084 Fisciano (Sa), [email protected]. +39 089 969751Fax: +39 089 969602

874

Article


Received 8 Mar 2011Accepted 17 Jul 2011Available online 2 Sep 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 874-883, Sep./Oct. 2011 Phytochemical profile and analgesic

evaluation of Vitex cymosa leaf extracts

Suzana Guimarães Leitão,*,1 Tereza Cristina dos Santos,#,2

Franco Delle Monache,4 Maria Eline Matheus,3 Patrícia Dias Fernandes,3 Bruno Guimarães Marinho3

1Faculdade de Farmácia, Departamento de Produtos Naturais e Alimentos, Universidade Federal do Rio de Janeiro, Brazil,2Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 3Departamento de Farmacologia, ICB, Universidade Federal do Rio de Janeiro, Brazil,4Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Università La Sapienza. Italy.

Abstract: Vitex cymosa Bertero ex Spreng., Lamiaceae, is found in Central and Amazon regions of Brazil, where it is popularly used as antirheumatic. Extracts from the leaves of V. cymosa were tested in analgesia models such as abdominal contortions induced by acetic acid and formalin to test peripheral analgesia; as well as the tail flick and hot plate models, to test spinal and supraspinal analgesia. A significant reduction was observed in the number of contortions with all extracts and in all doses. In the formalin model, a reduction in the second phase (inflammatory) was observed with all extracts, whereas only the n-butanol extract was able to act in the first, neurogenic, phase. In the tail flick model, all extracts increased latency time. Naloxone treatment reverted analgesic effect of all extracts with the exception of the dichloromethane one. All extracts developed peripheral and central analgesic activity. In the hot plate model no antinociceptive effect was observed for all tested extracts. All these results taken together suggest that V. cymosa leaf extracts were able to promote peripheral and central antinociceptive activity mediated by the opioid system.Twenty three substances were isolated and identified in the extracts and include flavonoids (C-glucosyl flavones, flavones and flavonols), triterpene acids from ursane and oleanane types, iridoids (free and glucosides), as well as simple phenols.

Keywords:Vitex cymosaVerbenaceae sensu lato Lamiaceae sensu lato antinociceptive activitymedicinal plantsflavonoidstriterpene acidsiridoids

Introduction

Vitex genus, Lamiaceae, (the genus has been formerly included in the Verbenaceae s.l. and recently transferred to the Lamiaceae s.l.) is constituted by small trees or shrubs occurring in tropical and subtropical regions. Many species have been studied in relation their chemistry, which is characterized by the presence of C-glucosyl fl avones, ecdysteroids, diterpenes and iridoids (Brito, 1992), as well as in relation to their biological activities. Antiinfl ammatory (Sidhartha et al., 1990; Chawla, et al., 1991), wound healing (Sidhartha et al., 1990) and antiandrogenic (Bhargava, 1989) activities, as well as inhibition of lymphocyte proliferation (You et al., 1998) and smooth muscle relaxant effect (Dayrit et al., 1987; Dayrit & Lagurin, 1994) are among those described in literature. V. agnus-castus is actually used in the treatment of pre-menstrual syndrome and menopausal

complaints (Mills & Bone, 2000). Despite the fact that the genus is well represented in Brazil with c.a. 35 species (Leitão et al., 2008), as far as we know, only three species have been chemically or pharmacologically investigated so far (Leitão et al., 2008, Gallo et al., 2008; 2006, Sá Barreto et al., 2005, Gonçalves et al., 2001). Vitex cymosa Bertero is a small tree widely distributed in the Central and Amazon regions of Brazil, where it is popularly known as “tarumã-do-igapó” and "tarumã-do-alagado” (Correa, 1926). The species are used by the native populations of the State of Maranhão to treat rheumatic pains. In a previous work done by our group (Miranda et al., 2010) extracts from this plant’s barks, collected at Maranhão State, Brazil, were tested in antinociceptive models, such as the tail fl ick test, to evaluate the central analgesic effect. In this work a signifi cant antinociceptive activity for the dichloromethane extract was observed. The (+)-trans-4-hydroxy-6-propyl-1-oxocyclohexan-2-one

Phytochemical profile and analgesic evaluation of Vitex cymosa leaf extractsSuzana Guimarães Leitão et al.


was isolated and determined to be the active principle. In this work we investigated the analgesic properties of the dichloromethane leaf extracts of this plant.


Plant material

Leaves of Vitex cymosa Bertero ex Spreng., Lamiaceae, were collected in Corumbá, Mato Grosso do Sul State, Brazil in December (in fruit). The plant was identified by Dr. Vali Pott, from Embrapa, Corumbá. A voucher specimen was deposited at Universidade Federal de Juiz de Fora herbarium under number CESJ 11,711.

Plant extracts

Dried and pulverized leaves (2.2 kg) were exhaustively extracted with ethanol. After filtration and concentration under reduced pressure the aqueous residue was sequentially extracted with organic solvents of increasing polarities, to afford the new extracts: dichloromethane (VD), ethyl acetate (VA) and n-butanol (VB) extracts, in this order, as well as (LW), which is the water-soluble remaining extract.

Isolation and identification of substances from the extracts

Dichloromethane extract (VD)

Part of VD was first chromatographed on silica gel eluted with CH2Cl2 with increasing amounts of EtOAc. Fractions eluted with CH2Cl2-EtOAc (9:1) were re-chromatographed on Sephadex LH-20 (MeOH) yielding 21. Fractions eluted with CH2Cl2-EtOAc (8:2) were purified by prep. TLC (CH2Cl2-EtOAc, 3:1) to afford 1, and 2. Fractions eluted with CH2Cl2-EtOAc (7:3) were re-chromatographed on Sephadex LH-20 (MeOH), yielding 3. A second fractionation of VD was performed by CC on silica gel eluted with CHCl3 with increasing amounts of EtOAc, and with EtOAc with increasing amounts of MeOH. Fractions eluted with CHCl3 were re-chromatographed on silica gel to afford 4, 13, and a mixture of 15 and 17 (CHCl3-EtOAc 1:1). Fractions eluted with CHCl3-EtOAc 9:1 were re-chromatographed on Sephadex LH-20 (MeOH) affording a mixture of 16 and 18, as well as 5. Fractions eluted with EtOAc-MeOH 8:2 were re-chromatographed in silica gel with the organic phase of a mixture of EtOAc-acetone –H2O (25:8:2), further purified on Sephadex LH-20 and, finally, by prep. TLC to afford 23 and 20. A third fractionation of VD was directly subjected to Sephadex LH-20 (MeOH) and led to the isolation of 19.

Ethyl acetate extract (VA)

VA was chromatographed on silica gel eluted with the organic phase of a mixture of EtOAc-acetone-H2O (25:8:2 to 25:10:5) affording sisteen fractions. From the first two fractions, 12 and 14, were obtained by crystallization, respectively. From fraction 6, 10 and 11 were obtained by prep. HPLC (see conditions below). From fractions 12-13, a mixture of the flavonoids 6, and 7, was obtained by crystallization and, from fraction 15, the flavonoids 8, and 9, were further purified by prep. TLC.

Butanol extract (VB)

VB was fractionated by silica gel CC eluted with CHCl3 with increasing amounts of MeOH affording 21, 22 and 23, as described previously (Santos et al., 2001). From fractions 10-16 (CHCl3-MeOH 9:1) 6, was obtained, and fractions 17-18 afforded 7.

Animals

All experiments were performed with male Swiss mice (20-25 g) obtained from our own animal facility. Animals were maintained in a room with controlled temperature 22±2 °C for 12 h light/dark cycle with free access to food and water. Twelve hours before each experiment animals received only water, in order to avoid food interference with substances absorption. Animal care and research protocols were in accordance with the principles and guidelines adopted by the Brazilian College of Animal Experimentation (COBEA) and approved by the Biomedical Science Institute/UFRJ - Ethical Committee for Animal Research.

Drugs and extracts administration

All extracts used were administered by oral gavage at concentrations of 10, 30 and 100 mg/kg in a final volume of 0.1 mL. Morphine and dipyrone were used as reference drugs and were administered by oral gavage. The control group was composed by PBS with the same amount of DMSO used in the dose of 100 mg/kg of extracts (0.005%) and had no effect per se. Naloxone was administered i.p. 15 min before oral administration of morphine or extracts.

Acute toxicity

Acute toxicity was determined following the experimental model described by Lorke (1983). A single oral dose of extracts (500 mg/kg) was administered to a group of ten mice (five males and five females). Behavior parameters including convulsion, hyperactivity, sedation,



grooming, loss of righting reflex, increased or decreased respiration, and food and water intake were observed over a period of five days. After this period animals were killed by cervical dislocation, stomachs were removed, an incision along the greater curvature was made, and the number of ulcers (single or multiple erosion, ulcer or perforation) and hyperemia was counted.

Acetic acid-induced abdominal writhing

Mice were used according to Matheus et al. (2006). Briefly, the total number of writhings following intraperitoneal (i.p.) administration of 2% (v/v) acetic acid (AA) was recorded over a period of 20 min, starting 5 min after AA injection. The animals were pretreated orally with extracts or vehicle, 60 min before administration of AA.

Formalin test

The procedure was similar to the method described by Gomes et al. (2007). Animals received the injection of 20 µL of formalin (2.5% v/v) into dorsal surface of the left hind paw. Immediately, the time that the animal spent licking the injected paw was recorded. The nociceptive response develops two phases: 0 to 5 min after formalin injection (first phase, response to neurogenic pain), and 15 to 30 min after formalin injection (second phase, response to inflammatory pain). For comparison purposes, the IC50 of morphine was calculated as 1.7 mg/kg. The animals were pre-treated with oral dose of extracts (10 to 100 mg/kg), vehicle or morphine (1.7 mg/kg), 60 min before administration of formalin.

Tail-flick test

The procedure used was similar to Matheus et al. (2006). Briefly, mice tails were immersed on a water bath set at temperature of 50±1 oC. The time necessary for the mice to withdrawal the tail, in seconds (named reaction time) was registered at 20, 40, 60, 80, 100 and 120 min after administration of V. cymosa extracts (10, 30 and 100 mg/kg) or morphine (2.3 mg/kg). For comparison purposes, the IC50 of morphine was calculated as 2.3 mg/kg. Baseline was considered as the mean of reaction time obtained at 40 and 20 min before administration of V. cymosa extracts or morphine, and was defined as normal reaction of animal to the temperature. Increase in baseline (in %) was calculated by the formula: (reaction time x 100)/baseline) - 100.

Hot plate test

Mice were tested according to the method described by Matheus et al. (2006). The animals were

placed on a hot plate set at 55±1 oC. Reaction time was recorded when the animals licked their fore- and hind-paws and jumped, at 30, 60, 90, 120, 150 and 180 min after oral administration of V. cymosa extracts (100 mg/kg) or morphine (1 mg/kg). Baseline was considered as the mean of reaction time obtained at 60 and 30 min before administration of extracts or morphine and was defined as normal reaction of animal to the temperature. Increase in baseline (in %) was calculated by the formula: (reaction time x 100)/baseline) - 100.

Evaluation of the mechanism of action of Vitex cymosa extracts

To evaluate the mechanism of action of V. cymosa extracts, animals were pre-treated with naloxone, a non-selective opioid antagonist. Naloxone (5 mg/kg) was administered i.p. 15 min before oral administration of extracts (30 mg/kg) or morphine (2.3 mg/kg).


All experimental groups were composed by ten animals. The results are presented either as the mean±SD in acetic acid-induced writhing and formalin or as the increase in baseline (%) in tail flick test. Statistical significance between groups was performed by the application of analysis of variance ANOVA followed by Bonferroni’s test. p values less than 0.05 (p<0.05) were used as the significant level.

Results

Phytochemical identification of known compounds

Phytochemical investigation of Vitex cymosa leaf extracts led to the isolation and identification of a number of known iridoids, triterpenes and flavonoids, which are secondary metabolites commonly found in the genus. Their chemical structures were identified by means of one and two dimensional 1H and 13C NMR techniques, as well as by mass spectral data, which were in accordance with previously reported data. From the dichloromethane extract, VD, the iridoids tarumal, 21 (Santos et al., 2001) and agnuside, 23 (Boros & Stermitz, 1990); the flavonoids kampferol, 1, 3’-O-methyl-luteolin, 2, luteolin, 3, pachypodol, 4, and apigenin, 5 (Agrawal, 1989); and the triterpenes 2α,3α-hydroxy-oleanolic acid, 15, 2α,3β-hydroxy-oleanolic acid, 16, 2α,3α-hydroxy-ursolic acid, 17, 2α,3β-hydroxy-ursolic acid, 18, 2β,3β,19α-hydroxy-ursolic acid, 19, (Mahato & Kundu, 1994), and 28-O-glucosyl-2α,3α,19α-hydroxy-ursolic acid ester, 20 (Seto et al, 1984), were isolated. From the ethyl acetate extract, VA, orientin, 6, iso-orientin, 7, vitexin, 8, iso-vitexin,



9, 2”-O-caffeoyl-orientin (Leitão & Delle Monache, 1998), 11, 2”-O-p-hydroxybenzoyl-orientin, 10, (Leitão & Delle Monache, 1998) protocatechuic acid, 12, vanilic acid, 13, and p-hydroxybenzoic acid, 14, were isolated. From the n-butanol extract, VB, orientin, 6, iso-orientin, 7, tarumal, 21, viteoid II, 22 and agnuside, 23, (Santos et al., 2001) were isolated.

Acute toxicity

In order to evaluate the possible toxic effects of V. cymosa extracts, mice were treated orally with 500 mg/kg of each of them. Treated mice did not present any behavioral alterations. Also, stomachs were removed from the animals and examined under dissecting microscope. No lesions or bleedings were observed (data not shown).

Analgesic activity

To test the antinociceptive effects of Vitex cymosa extracts were used models of peripheral, spinal and supra-spinal analgesia.

Acetic acid-induced abdominal writhing

Intraperitoneal injection of acetic acid induced 100.1±3.9 writhings in a period of 20 min. Pre-treatment of mice with V. cymosa extracts in doses of 10, 30 and 100 mg/kg induced a dose-response reduction on total number of contortions. The most potent extract was VB (Figure 1). Even at 10 mg/kg, VB significantly reduced in 25% (100.1±3.9 writhings in control group versus 73.4±4.0 writhings in extract-treated group) the number

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antinociceptive action of morphine lasted about 120 min. Dichloromethane extract (VD) had similar curves to all doses tested, and reached maximal response 80 minutes after administration. Although VD induced an increase in the baseline, this was not a significant effect. VB extract developed a dose-response curve, with very little differences between each tested dose (10, 30, and 100 mg/kg). Twenty minutes after VB administration a significant increase in the baseline could already be observed with all doses. At 120 min, the antinociceptive effect of morphine began to decline, whereas the effect of VB continued at high levels within 120 min, and lasted 2 h after a single oral administration (data not shown). The response to VA was quite different. Significant antinociceptive activity was observed at the dose of 100 mg/kg. This effect appeared after 60 minutes of administration and was maintained until the end of the experiment.

Figure 2. Effects of Vitex cymosa leaf extracts in the formalin test. Animals were pre-treated with different doses of extracts or vehicle by oral administration. Groups are: vehicle ( ▓ ), morphine – 1.7mg/kg (■) or extracts at different doses (□). The results are presented as mean ± S.D. (n=10) of time, in seconds, that the animal spent licking the injected paw calculated as described on methods section. Statistical significance was calculated by ANOVA followed by Bonferroni’s test.*p<0.05 when compared with vehicle-treated mice.

of writhings. At the doses of 30 and 100 mg/kg there was significant effect to all three fractions, VD, VA and VB (Figure 1).

Figure 1. Effects of Vitex cymosa leaf extracts on acetic acid-induced writhings. Animals were pre-treated with extracts or vehicle by oral administration. Groups are: vehicle (■), VD (Δ), VB (□), or VA (○). The results are presented as mean±SD; (n=10) of total writhings calculated as described on methods section. *p<0.05 ANOVA followed by Bonferroni’s test

Formalin test

Intraplantar injection of formalin evoked a characteristic biphasic licking response. For the control group, the duration of licking in the 1st phase was 48.8±11.7 s, and for the 2nd phase was 141.6±33.0 s. As shown in Figure 2, pre-treatment with different doses of VD, VA or VB had no significant effect against the 1st phase. However, 2nd phase was markedly reduced by VD, with maximal effect observed with 100 mg/kg. VA and VB developed significant effect on the 2nd phase only at doses of 30 and 100 mg/ kg. However, VA effects were lesser pronounced than those of VB or VD. Results obtained demonstrated that VD was the most efficient in reducing the time that the animal spent licking the formalin-injected paw.

Tail-flick and hot plate tests

In view of the fact that V. cymosa extracts demonstrated significant antinociceptive activity on acetic acid-induced writhing and formalin tests, we decided to test its action on spinal and supraspinal models of pain - tail flick and hot plate tests. As shown in the Figure 3, all three extracts induced an increase in the time to withdrawal the tail from the bath (demonstrated as increase in baseline). Morphine (2.3 mg/kg) produced its maximum antinociceptive effect between 60 and 100 min after oral administration, with maximal values reaching 50% increase in baseline at 100 min. The

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Figure 3. Effects of Vitex cymosa leaf extracts in the tail flick response. Animals were pre-treated with different doses of extracts or vehicle by oral administration. Groups are: vehicle (▲), morphine (2.3 mg/kg, ■), extracts at 10 (□), 30 (○), or 100 mg/kg (Δ). The results are presented as mean±SD. (n=10) of increase in baseline (%) calculated as described on methods section. Statistical significance was calculated by ANOVA followed by Bonferroni’s test.*p<0.05 when compared with vehicle-treated mice. Where no error bars are shown is because they are smaller than the symbol.

To test the hypothesis that V. cymosa could have supraspinal analgesic effect, the hot plate model was used. Upon oral administration of V. cymosa extracts (at 100 mg/kg), no antinociceptive effect was observed (data not shown).

Mechanism of action

In order to elucidate the mechanism by which V. cymosa extracts induce antinociceptive effect, animals were pre-treated with the non-selective opioid antagonist, naloxone. Figure 4 shows that naloxone (5

mg/kg) completely reverted the morphine (2.3 mg/kg) antinociceptive effect in tail flick model. Naloxone also reverted the antinociceptive activity of all V. cymosa extracts tested (at 30 mg/kg). Results of naloxone against V. cymosa extracts were comparable to those obtained with naloxone against morphine on the spinal model of analgesia.

Figure 4. Effect of naloxone on the antinociceptive activity of Vitex cymosa leaf extracts. Animals were pre-treated with naloxone (5 mg/kg, i.p.) 15 min before oral administration of extracts (30 mg/kg). Groups are: vehicle (◊), morphine (2.3 mg/kg, ▼), morphine + naloxone ( ), VD (▲), VD + naloxone (Δ), VA (●), VA + naloxone (○), VB (■), VB + naloxone (□). The results are presented as mean±S.D. (n=10) of increase in baseline (%) calculated as described on methods section. Statistical significance was calculated by ANOVA followed by Bonferroni’s test. *p<0.05 when comparing extracts+naloxone or morphine+naloxone with extracts or morphine-treated mice. Where no error bars are shown is because they are smaller than the symbol.

Discussion

The purpose of this paper was to establish a scientific basis for one of the traditional uses of Vitex cymosa, which is against rheumatic pains. All tested extracts significantly inhibited the abdominal contortions induced by acetic acid in mice. Acetic acid causes an increase in peritoneal fluid levels of prostaglandins, involving in part, peritoneal receptors (Deraedt et al., 1980) and inflammatory pain by inducing capillary permeability (Amico-Roxas et al., 1984). Although the writhing test has poor specificity (Le Bars et al, 2001), it is a very sensitive method of screening antinociceptive effects of compounds. The acetic acid-induced writhing method is able to determine antinociceptive effects of compounds and dose levels that might appear inactive in other methods (Gene et al., 1998) This results indicate that Vitex cymosa presents antinociceptive activity. Despite the assayed extracts possess very different chemical compositions, they all consist of a varied class

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of compounds for which some kind of antinflammatory and/or analgesic activity has been described in literature. Flavonoids, for example, were detected in all the assayed Vitex cymosa extracts (VD, VA and VB). Several studies have reported the antinociceptive effects of flavonoids (Thirugnanasambantham et al., 1990; Pathak et al., 1991; Ramesh et al., 1998; Toker et al., 2004), and, of particular interest is that one reporting the antinociceptive activity of luteolin (Block et al., 1998), which was isolated from VD extract. The antinociceptive activity of this flavonoid has been evaluated by Block et al. (1998) in the acetic acid-induced abdominal constriction model in mice. Pharmacological analysis revealed that luteolin caused gradual and significant inhibition of abdominal writhings. When compared to acetylsalicylic acid, acetaminophen, dipyrone and indomethacin, by using the same pharmacological procedure, luteolin was about 8-16-fold more active than these standard drugs (Block et al., 1998). Luteolin was also detected, among other flavonoids, in extract of Vitex rotundifolia fruits (Emi et al., 1998). Thirugnanasambantham et al. (1990) showed that various synthetic flavone derivatives, structurally related to luteolin, displayed significant analgesic activity and suggested that some of them could be acting through an opiate-like mechanism. However, whether or not the antinociceptive action of luteolin involves such mechanism requires further investigations. Luteolin, together with apigenin, orientin and vitexin - which are present in Vitex cymosa extracts, were also isolated in a bioassay-guided fractionation of an antinociceptive extract from Zea mays stigmas (corn silk), which activity was evaluated by the p-benzoquinone-induced writhings and the formalin-induced edema models (Abdel-Wahab et al., 2002). Concerning the isolated triterpenes, they are all hydroxyl-derivatives of ursolic and oleanolic acids, for which antinociceptive activity has been demonstrated previously by our group (Costa et al., 2003). The presence of iridoids in the active extracts is also noteworthy. Although there are some reports on the anti-inflammatory activity for this class of compounds (Ghisalberti, 1998; Recio et al., 1994; Singh, et al., 1993) including a report from Vitex genus (Suksamrarn et al., 2002) pharmacological data on analgesic activity for this class of compounds is scarce. There is one report on the analgesic properties of agnuside (Okuyama et al., 1998) but no reports for the iridoids tarumal or viteoid II. It is interesting to note that Vitex cymosa is used in the Brazilian traditional medicine to treat rheumatoid arthritis. Also noteworthy, is the fact that one of the most prescribed phytomedicines to treat degenerative diseases of the musculoskeletal system nowadays is the South African plant Harpagophytum procumbens D.C., known as Devil’s Claw, which active principles are claimed to

be iridoid glucosides (Stewart & Cole, 2005; Baghdikian et al., 1997). The neurogenic and inflammatory pain was evaluated using formalin test. Drugs that act primarily as central analgesics inhibit both phases while peripherally acting drugs inhibit only the phase 2 (Rosland et al., 1990). We observed that none of V. cymosa extracts was able to decrease the time that the animal spent licking the injected paw on the first phase, while the second phase was inhibited by all tested extracts, specially VD. The presence of dihydroxy- and trihydroxy derivatives of oleanolic and ursolic acids in this extract is noteworthy, since, in a previous work from our group, we have demonstrated the analgesic potential of a mixture of ursolic, oleanolic and micrometric acids, in the same models reported here (Costa et al., 2003). Inhibition of the second phase alone, (Rosland et al, 1990), suggests a peripheral antinociceptive activity of Vitex cymosa. These results corroborate with the inhibitory effect of V. cymosa extracts on the acetic acid-induced writhing response. Several studies demonstrated that opioids can produce analgesia through peripheral mechanisms after inflammation of tissues (Stein et al., 1989, 1996). V. cymosa extracts produced antinociceptive effect in the formalin test in mice after oral administration. These findings are in agreement with others studies where antinociceptive effects of peripherally acting µ opioid agonist, loperamide, were detected in the formalin test after subcutaneous administration (Shannon & Lutz, 2002). The peripheral antinociceptive activity produced by opioid system is based on the opening of potassium channels, causing hyperpolarization in nociceptive afferents. (Milan, 1999). In order to verify if V. cymosa extracts could develop central antinociceptive effect, the models of tail flick (spinal analgesia) and hot plate (supra-spinal analgesia) were used. Extracts from the leaves of V. cymosa were able to increase latency time in the tail flick. In this model, after the thermal stimulus applied to the tail, superficial tissue layers are broadly stimulated and a tail withdrawal reflex is evoked, which characterizes spinal mechanism and involves sensory and motor components (Green & Young, 1951). The rapid onset with an early maximum effect, which warred very shortly after drug administration, is characteristic of the time course of action of opioid agonists (e.g. morphine, fentanyl and etorphine) that mediate analgesia via central mechanisms and were described under both normal and inflammatory conditions (Aceto et al., 1997; Millan et al., 1987). Administration of VD, VB, and VA produced different time course of action. VD did not evoke a significant effect and, the antinociceptive effect of VA was observed only with a higher dose. VB, however, showed a rapid onset and longer-lasting action than



the centrally acting opioids, with higher effects. In this way, these results presented in the tail flick test show spinal antinociceptive activity by the V. cymosa extracts. The spinal antinociceptive activity is associated with a reduction in the release of excitatory neurotransmitters (glutamate, substance P etc.) at the spinal cord, and hyperpolarization of ascending neurons (Besson & Chaouch, 1987). In the hot plate test, the antinociceptive activity was not observed probably due to the difficulty in crossing the blood-brain barrier by the active molecules of V. cymosa extracts, revealing a probable supraspinal antinociceptive effect if the active molecules were administered by intracerebroventricular route. (Grisel & Mogil, 2000). The supraspinal antinociceptive activity is related to activation of the endogenous inhibitory control of pain. (Saadé & Jabbur; 2008). The reduction of the antinociceptive effect of the extracts produced by the prior administration of naloxone, a non-selective opioid antagonist, shows the participation of the opioid system in the mechanism of action of extracts of Vitex cymosa, proving the involvement of this system in the peripheral, spinal and supraspinal antinociceptive actions. Together, these data are consistent with the interpretation that systemically administered morphine exerts its analgesic effects by interacting with both central and peripheral opioid receptors (Perrot et al., 2001; Shannon & Lutz, 2002).

Conclusions

The lactone (+)-trans-4-hydroxy-6-propyl-1-oxocyclohexan-2-one which was determined to be the active principle of V. cymosa dichloromethane bark extracts could not be isolated from the dichloromethane extracts from the leaves. The exact mechanism of action and the active principles responsible for the analgesic activities described here remain to be confirmed, but taking all these in vivo results together, it can be suggested that Vitex cymosa leaf extracts were able to promote peripheral and central antinociceptive activity mediated by the opioid system.

Acknowledgements

This work was supported by FAPERJ and CNPq. We are indebted to Dr. Vali Pott for plant collection and identification.

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*Correspondence

Suzana Guimarães LeitãoDepartamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, Universidade Federal do Rio de JaneiroCCS, Bloco A, 2o andar salas 4 e 10, Ilha do Fundão, 21941-590, Rio de Janeiro-RJ, [email protected]/Fax: +55 21 2562 6413/6425

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Received 27 Dec 2010Accepted 22 Mar 2011Available online 17 Jun 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 884-888, Sep./Oct. 2011 Inhibition of human platelet aggregation

in vitro by standardized extract of Wendtia calycina

Milagros Garcia Mesa,1 Anna Lisa Piccinelli,2 María Antonia Alfonso Valiente,1 Aldo Pinto,2 Alessia Fazio,3 Luca Rastrelli*,2

1Departamento de Bioquímica, Instituto Nacional de Angiología y Cirugía Vascular, Cuba,2Department of Pharmaceutical Sciences, School of Pharmacy, University of Salerno, Italy,3Facoltà di Farmacia e di Scienze della Nutrizione e della Salute dell’Università degli Studi della Calabria, Italy.

Abstract: Wendtia calycina (Griseb.) Griseb., Vivianiaceae, is a Paraguayan herbaceous plant commonly known as burrito. Our previous study indicated that burrito leaves are a very good source of phenylpropanoid glycosides, principally verbascoside. From W. calycina leaves, a standardized, water-soluble extract rich in phenylpropanoid glycosides (WSE) has been developed on an industrial scale to be used as a food supplement, cosmetic, phytomedicine, and ingredient of different formulations. In this study, we investigated the effect of the WSE on human platelet aggregation in vitro induced by adenosine diphosphate (ADP), epinephrine (EPN), collagen (COL) or arachidonic acid (AA). WSE, concentration-dependently, inhibited ADP and EP-induced human platelet aggregation (IC50 were 0.82±0.15 mg/mL and 0.41±0.02 mg/mL, respectively). It did not inhibit collagen-induced platelet aggregation, thus suggesting a selectivity for the ADP-induced platelet activation pathways.

Keywords:Wendtia calycina leaves Phenylpropanoid glycosides Human platelet aggregation

Introduction

Blood platelets are absolutely required for haemostatic plug formation when normal vessels are injured. However, the interactions between platelets and collagen can also cause circulatory disorders, such as thrombosis, atherosclerosis, and myocardial infarction (Schwartz et al., 1990). Atherothrombosis is one of the pathological events associated to oxidative reactions. Its most threatening consequences, myocardial infarction, stroke and lower-limb arterial thrombosis, are among the fi rst causes of death and disability all over the World. The activation of blood platelets has a central role in atherothrombotic events. This activation is a consequence of platelet adhesion and aggregation on a damaged vascular endothelium giving the basis for the formation of arterial thrombus with the consequent blood fl aw decrease and tissue ischemia. An important role in the mechanism by which collagen induces platelet aggregation is played by thromboxane A2 (TXA2) formation (Cattaneo et al., 1991), which also contributes to an increase in cytosolic free Ca+2 level ([Ca+2]i) in collagen-activated platelets. An increase in [Ca+2]i activates both the Ca+2/calmodulin-

dependent phosphorylation of myosin light chain (20-kDa) and the diacylglycerol-dependent phosphorylation of cytosolic protein (40- or 47-kDa) to induce platelet aggregation (Nishikawa et al., 1980; Kaibuchi et al., 1982) In addition, diacylglycerol also can be hydrolyzed by diacylglycerol lipase to produce arachidonic acid, a precursor of TXA2, which is a potent platelet aggregation agent generated from arachidonic acid liberated when PIP2 is broken down by stimulation with collagen, thrombin and ADP (Menshikov et al., 1993). Inhibition of the platelet-collagen interaction might be a promising approach to the prevention of thrombosis. The mechanism of action of the antiplatelet drugs currently used in the medical practice are unrelated to the antioxidant action. Theoreticaly, an antioxidant agent may inhibit platelet aggregation, since oxidative reactions are involved in platelet activation and aggregation (Maytin et al., 1999). However, recent studies have demonstrated that the antioxidant activity is involved in the mechanism of antiplatelet action of the fl avonoids quercetin and catechin (Pignatelli et al. 2000; Singh et al., 2006). Epidemiological studies pointed out that diets rich in fruits and vegetables (rich in phenolic compounds)

Inhibition of human platelet aggregation in vitro by standardized extract of Wendtia calycina

Milagros Garcia Mesa et al.


could prevent certain diseases in which free radicals are implicated. Since purified phenolic compounds are difficult to obtain and since extracts sometimes have biological activities higher than those of pure molecules, there is a growing interest for the use of plant extracts. Among them, Pycnogenol from French Pinus maritime bark, oligomeric proanthocyanidins from Vitis vinifera and Ginkgo biloba leaf extract are used in pharmacology and cosmetology or as a food supplement (Calliste et al., 2005). Wendtia calycina (Griseb.) Griseb., Vivianiaceae, is a species that occurs mainly in Paraguay where is known as “burrito”. The W. calycina leaves are used traditionally as a tea substitute, as well as a remedy to treat all kind of gastric disorders, inflammation and arthritis. Our previous study indicated that burrito leaves are a very good source of phenylpropanoid glycosides, principally verbascoside and that its aqueous extract possessess in vitro antioxidant activity suggesting that it may be useful for the prevention of those pathologic events associated with oxidative stress (Piccinelli et al., 2004). From W. calycina leaves, a standardized, water-soluble extract (WSE) rich in phenylpropanoid glycosides has been developed on an industrial scale to be used as a food supplement, cosmetic, phytomedicine, and ingredient of different formulations. The extract is prepared by a standardized procedure that includes ultrasonic extraction with aqueous EtOH (60% v/v), followed by a spray-drying using maltodextrin as excipient. WSE represents a concentrate of phenolic compounds and its main constituents are phenylpropanoid glycosides (PPGs), principally verbascoside (7.5 %) and isoverbascoside (1.1 %). This product also contains various benzoic acid derivatives as minor constituents. A recent study has shown the inhibition of inducible nitric oxide synthase in vitro and in vivo by water-soluble extract of W. calycina (WSE) (Marzocco et al., 2007). This work was aimed to assess the in vitro effect of WSE on human platelet aggregation induced by adenosine diphosphate (ADP), epinephrine (EPN), collagen (COL) or arachidonic acid (AA).


Plant material

The leaves of Wendtia calycina (Griseb.) Griseb., Vivianiaceae, erroneously reported previously as Wendita calysina Martius (Piccinelli et al., 2004; Marzocco et al., 2007) were produced by Coprosa Ltd., Paraguay (Cooperativa multiactiva de Produccion Servicios publicos, Consumo, Ahorro y Credito, San Andres) and furnished by Commercio Altenativo S.r.l. (Ferrara, Italy), in February 2008. A specimen of the plant (No. K00053145) is deposited at the Herbarium Royal Botanic Gardens, Kew (K). The water soluble extract

of W. calycina (WSE) was prepared by a standardized procedure that includes ultrasonic extraction with aqueous EtOH 60%, followed by spray-drying using maltodextrins as excipient. Unless stated otherwise, all reagents and compounds used were obtained from Sigma Chemical Company (Sigma, Milan, Italy).

Blood collection and platelet rich plasma preparation

Blood of patients of both genders, aged from 19 to 55 years, participating in a routine health study, was obtained from Cerro Polyclinic in Havana City, Cuba. All enrolled subjects signed the informed consent and denied to have consumed any drug with known antiplatelet action at least two weeks before the flebotomy. Venous blood was obtained through plastic syringes containing 1/10 (v/v) of 3,8% (w/v) trisodium citrate salt solution to avoid coagulation. Blood was then transferred in plastic tubes and platelet-rich plasma (PRP) was prepared by centrifugation at 150 x g for 10 min at room temperature. After centrifugation the PRP was collected and the remaining blood was respun at 1000 x g for further 20 min at room temperature for preparation of platelet-poor plasma (PPP). A platelet count was performed on the PRP to ensure that the platelet concentration was in the range 220-250 x 103/μL.

Platelet aggregation

W. calycina water-soluble extract (WSE) was diluted with a phosphate-buffered saline (PBS) solution at pH 7.4 to assess its effect on platelet aggregation. A solution of acetylsalycilic acid (ASA) in 1% sodium bicarbonate was used to assess the effect of the drug (5.5 mmol/L in the incubation media) in the same conditions used for the WSE. Adenosine diphosphate (ADP), collagen, epinephrine, and arachidonic acid were obtained from CPM (CPM S.A.S, Rome, Italy) and prepared according to the instructions provided. The turbidimetric method (Born & Cross, 1968) was applied to measure platelet aggregation, using a CLOT 2S Aggregometer (Seac and Radim Group, Rome, Italy). Five microliters of different dilutions of WSE in PBS (0.33, 0.66 and 1.32 mg/mL) or PBS (control) was added to 285 μL aliquots of PRP in aggregometer cuvettes. Successively, 15 μL of ADP, epinephrine, collagen, or arachidonic acid (final concentrations in the incubation media being 5 μmol/L, 5 μmol/L, 3.2 μmol/mL, and 0.6 mmol/L, respectively) were added after 2 min of preincubation at 37 °C. Platelet aggregation was monitored for 5 min. The results are expressed as percentages of aggregation as provided by the instrument. The percentage inhibition of platelet aggregation was calculated as follows: percentage inhibition (%) [1 - (platelet aggregation of sample/platelet aggregation of control)]×100%. Each sample was

Inhibition of human platelet aggregation in vitro by standardized extract of Wendtia calycinaMilagros Garcia Mesa et al.


ADP

C0.3

30.6

61.3

2

0

20

40

60

WSE

*

***

% o

f pla

tele

t agg

rega

tion

Figure 1. Effect of WSE on ADP (5 μmol/L) induced aggregation. For platelet preparation see Material and Methods section. Results are expressed as mean+sem. (n=9), and *p<0.05, ***p<0.001.

EP

C0.3

30.6

61.3

2

0

20

40

60

WSE

*

***

% o

f pla

tele

t agg

rega

tion

Figure 2. Effect of WSE on epinephrine (EP5 μmol/L) induced aggregation. For platelet preparation see Material and Methods section. Results are expressed as mean+sem. (n=9) and *p<0.05, ***p<0.001.

collagen

C0.3

30.6

61.3

2

0

20

40

60

WSE

% o

f pla

tele

t agg

rega

tion

Figure 3. Effect of WSE on collagen (3.2 μmol/mL) induced aggregation. For platelet preparation see Material and Methods section. Results are expressed as mean+sem (n=9).

measured in triplicate. IC50 values (the concentration necessary to reduce the induced platelet aggregation by 50% with respect to control) were obtained from concentration-effect curves.


Data are expressed as percent of platelet aggregation. The value of WSE IC50 (the concentration necessary to reduce the platelet aggregation by 50%) was obtained from concentration-effect curves of WSE. Data are expressed as the mean and the interval of confidence. The statistical analysis was performed by analysis of variance (ANOVA) by Kruskal-Wallis ranks and the Mann-Whitney U test. The maximum antiplatelet effect of WSE and ASA were compared by the Student's t test for independent data.


WSE in a concentration-dependent (0.33, 0.66 or 1.32 mg/mL) manner inhibited ADP-induced (5 µmol/L) human platelet aggregation (Figure 1). A statistically significant reduction, compared to vehicle-treated PRP, was observed to the concentrations of 0.66 and 1.32 mg/mL (p<0.05 and p<0.001 respectively). The calculated IC50 was 0.82±0.15 mg/mL (IC 0.52-1.11) (Table 1). In similar way, WSE treatment of human platelet in vitro produced aggregation reduction induced by EP (5 µmol/L) that was related in a concentration-dependent manner (Figure 2). Even though the WSE concentration of 0.33 mg/mL reduced of about 10% the platelet aggregation it was not statistically (p>0.05) significant from control, whereas the concentration of 0.66 and 1.32 mg/mL significantly inhibited human platelet aggregation (p<0.05 and p<0.001 respectively). The IC50 of W. calycina water-soluble extract in EP-induced aggregation was of 0.41±0.02 mg/mL in a range of IC 0.37-0.45 mg/mL. When the human platelet aggregation was induced by collagen (2 μg/mL) or AA (0.5 mmol/L) was not significantly impaired by the presence of WSE, at any tested concentrations (Figure 3 and 4 respectively).

Table 1. WSE concentration required to produced 50% inhibition (IC50) of human platelet aggregation induced by various aggregating agents.

Aggregating agents (concentration)IC50 (mg/mL)

mean sem cl

ADP (5 µmol/L) 0.82 0.15 0.52-1.11

-Epinephrine (5 µmol/L) 0.41 0.02 0.37-0.45

Collagen (2 µg/mL) >1.32a - -

Arachidonic acid (0,5 mmol/L) >1.32a - -IC50 values were determined from each experiment (n=9), through extrapolation from the curve percentage of inhibition versus log WSE concentration and expressed as mean+sem and confidence limit (cl) at 95%.

Inhibition of human platelet aggregation in vitro by standardized extract of Wendtia calycina

Milagros Garcia Mesa et al.


AA

C0.3

30.6

61.3

2

0

20

40

60

WSE

% o

f pla

tele

t agg

rega

tion

Figure 4. Effect of WSE on arachidonic acid (AA; 0.63 μmol/mL) induced aggregation. For platelet preparation see Material and Methods section. Results are expressed as mean+sem (n=9).

The effect of ASA on human platelet aggregation induced by ADP, EP, collagen or AA was performed as reference drug. In our experimental model of human platelet aggregation, ASA (5.5 mmol/L) significantly (p<0.001) inhibited the human platelet aggregation induced by collagen (2 µg/mL) and AA (0.5 mmol/L), but it was ineffective on the aggregation induced by ADP (5 µmol/L) or EP (5 µmol/L) (Figure 5).

ASA

CADP EP

Collagen AA

010203040506070

% o

f pla

tele

t agg

rega

tion

*** ***

Figure 5. Effect of acetylsalycilic acid (5.5 mmol/L) on ADP (5 μmol/L), epinephrine (EP; 5 μmol/L), collagen (3.2 μmol/mL), or arachidonic acid (AA; 0.6 mmol/L). For platelet preparation see Material and Methods section. Results are expressed as mean+sem (n=4) and ***p<0.001.

Different plant extracts have been used in the traditional medicines as remedies for various diseases. Generally, polyphenol-rich extracts have strong antioxidant activity, which can translate into several different beneficial human health effects. W. calycina leaves contain various phenolic compounds, including

dihydroquercetin, methoxyflavones, methoxyflavonols, phenylpropanoid glycosides, and benzoic acid derivatives (Piccinelli et al., 2004). These last two classes of compounds are water-soluble natural products and were found to be the main components of W. calycina water-soluble extract (WSE) particularly phenylpropanoid glycosides content (15.1 mg/l00 mL of aqueous infusion) appears to be very remarkable (Piccinelli et al., 2004). In this study, we have used ADP and collagen as aggregating agonists to assess the effect of WSE on human platelet aggregation in vitro. ADP and collagen have some differences as platelet aggregation stimuli. ADP stimulates blood platelets through the activation of specific purinergic receptors. GPVI is considered one of the glycoprotein which function as collagen receptors on platelets.The activation of these receptors is a signal for the increase of the cytoplasmic calcium, leading to platelet aggregation, thromboxane A2 formation and granules mediators liberation (known as the release reaction) such as ADP and serotonine, which amplify the response to the initial stimuli. Two aggregation waves may be clearly identified on ADP-aggregation curves: the first one (reversible) corresponds to the receptor activation signal and the second one (irreversible) corresponds to the effect of TXA2. On the other hand, collagen aggregation curves have a latency period, followed by a slow aggregate formation which depends on TXA2 formation. Therefore, agents which specifically block ADP receptors are good inhibitors of ADP- but weak inhibitors of collagen- induced platelet aggregation, this the case of the thienopyridin drugs, ticlopidine and c1opidogrel. Aspirin-like drugs, which inhibit platelet cyclooxygenase and TXA2 formation, are good inhibitors of collagen-induced aggregation and inhibit the ADP second WSE, concentration-dependently, inhibited ADP-induced platelet aggregation human PRP. It was an inhibitor as potent as ASA against ADP, however, It did not inhibit collagen-induced platelet aggregation, thus suggesting a selectivity for the ADP-induced platelet activation pathways. The assayed WSE concentrations may be kind of high, however this not deny the importance of the pharmacological finding, since it is known that the degree of inhibition of platelet aggregation depends not only of the concentration of the antiplatelet agent, but also on the concentration of the aggregating agonist. We used the concentrations of ADP and collagen that produced submaximum aggregation in each plasma sample, consequently, platelet aggregation rates were always higher than 50 % for both stimuli in control conditions. Besides, in our experimental conditions the ASA effective concentration against collagen was higher than expected according to the literature. Thus,

Inhibition of human platelet aggregation in vitro by standardized extract of Wendtia calycinaMilagros Garcia Mesa et al.


it is possible that a effect of lower WSE concentration may be seen at milder conditions. WSE is a W. calycina extract that have been prepared in similar way to that traditionally used in Paraguay. The extrapolation of in vitro to in vivo situations is quite difficult. However, our data suggest that those people who regularly consume this vegetable remedy may have a decreased platelet reactivity leading to a reduced susceptibility to atherothrombotic events. Future clinical assays will confirm or deny this hypothesis. In conclusion, the results of this study confirm the hypothesis that a water-soluble extract of W. calycina has an antiplatelet action. This is the first experimental evidence of the antiplatelet potential of this plant. The future investigation will be directed to the characterization of this pharmacological action.

References

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Cattaneo M, Tenconi PM, Lecchi A, Mannucci PM 1991. In vitro effects of picotamide on human platelet aggregation, the release reaction and thromboxane B2 production. Thromb Res 62: 717-724.

Kaibuchi K, Sano K, Hoshijima M, Takai Y, Nishizuka Y 1982. Phosphatidylinositol turnover in platelet activation; Calcium mobilization and protein phosphorylation. Cell Calcium 3: 323-335.

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S, Autore G 2007. Inhibition of inducible nitric oxide synthase in vitro and in vivo by a water-soluble extract of Wendtia calycina leave. Naunyn-Schmiedeberg’s Arch Pharmacol 375: 349-358.

Menshikov MYu, Ivanova K, Schaefer M, Drummer C, Gerzer R 1993. Influence of the cGMP analog 8-PCPT-cGMP on agonist-induced increases in cytosolic ionized Ca2+ and on aggregation of human platelets. Eur J Pharmacol 245: 281-284.

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Piccinelli AL, De Simone F, Passi S, Rastrelli L 2004.) Phenolic constituents and antioxidant activity of Wendtia calycina leaves (burrito), a folk paraguayan tea. J Agric Food Chem 52: 5863-5868.

Pignatelli P, Pulcinelli FM, Celestini A, Lenti L, Ghiselli A, Gazzaniga PP, Violi F 2000. The flavonoids quercetin and catechin synergistically inhibit platelet function by antagonizing the intracellular production of hydrogen peroxide. Am J Clin Nutr 72: 1150-1155.

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Singh I, Mok M, Christensen AM, Turner AH, Hawley JA 2008. The effects of polyphenols in olive leaves on platelet function. Nutr Metab Cardiovasc Dis 18: 127-132.

*Correspondence

Luca RastrelliDepartment of Pharmaceutical Sciences, School of Pharmacy, University of Salerno Via Ponte don Melillo, 84084 Fisciano, [email protected]. +39 89 969766Fax: +39 89 969602

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21(5): 889-900, Sep./Oct. 2011Antispasmodic effects of Aloysia polystachya and A. gratissima tinctures and extracts are due to non-competitive inhibition of intestinal contractility induced by acethylcholine and calcium

Alicia E. Consolini,* Andrea Berardi, María A. Rosella, María G. Volonté

Área Ciencias Farmacéuticas, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina.

Abstract: The antispasmodic effects of acqueous extracts (AE) and tinctures (T) of Aloysia polystachya (Griseb.) Moldenke and Aloysia gratissima (Gillies & Hook.) Tronc., Verbenaceae, were studied on rat isolated ileum and duodenum. These plants are used for gastrointestinal disorders and as eupeptic in South America. Both AE non-competitively inhibited the dose-response curves (DRC) of ACh and the DRC of Ca2+ in high-[K+]o, as well as the T. The T of A. polystachya and A. gratissima respectively inhibited the ACh-DRC at the IC50 of 3.15±0.57 and 6.46±2.28 mg leaves/mL. The Ca2+-antagonist activity of both T occurred with IC50 respectively similar to those of the ACh-DRC, and was potentiated by the depolarization produced by 10 mM TEA, a blocker of K+-channels. The spasmolytic effect of T does not involve DA release and binding to D2, since it was not reduced by 10 µM metoclopramide. Also, T induced dose-dependent relaxation on the tonic contracture produced by high-[K+]o and ACh. By TLC there were detected in the leaves the presence of carvone, and flavonoids such as quercetin and hesperidin. By HPLC there were not found vitexin nor isovitexin, identified in A. citriodora. The monoterpene (-)-carvone non-competitively inhibited the ACh-DRC (pD´2 of 4.0±0.1) and the DRC of Ca2+ (pD´2 of 3.86±0.19), suggesting that the Ca2+-influx blockade is the mechanism of its antispasmodic effect. Results suggest that the antispasmodic effect of A. polystachya and A. gratissima are mostly explained by the non-competitive blockade of Ca+2 influx. It could be associated to the presence of flavonoids, and in the tinctures to some spasmolytic components of the essential oil such as carvone.

Keywords:Aloysia

antispasmodicCa-antagonist

(-)- carvonerat intestine

Verbenaceae

Introduction

Many plants are used in folk medicine to treat gastrointestinal disorders, such as spasm or indigestion. Several plants of the genus Aloysia, Verbenaceae, are used as eupeptic in South America. In a previous work it was described for the first time the antispasmodic effect of Aloysia citriodora Ortega ex Pers., which is known as “cedrón” or “hierba luisa”. Its pharmacological mechanism of action was related to activation of the guanylil-cyclase and the potassium channels (Ragone et al., 2007). In this work, the antispasmodic properties of two other species of Aloysia were validated and the pharmacological basis of the popular use was evaluated, as well as the correlation with its chemical composition. These two species, Aloysia polystachya (Griseb.) Moldenke known as “burrito” or “té-burro”

and Aloysia gratissima (Gillies & Hook.) Tronc. known as “palo amarillo”, are also used as eupeptic in South America. The leaves are prepared as an aromatic infusion commonly used as dietary supplement or eupeptic tea; and also as decoction or alcoholic tincture for abdominal pain or against nausea and dizziness (Soraru & Bandoni, 1978; Alonso & Desmarchelier, 2005). In spite of its wide use and the local experiences for cultivation (Burdyn et al., 2006) there were not studies about its gastrointestinal properties. Nevertheless, there were described anxiolytic and antidepressant effects of A. polystachya (Mora et al., 2005; Hellion-Ibarrola et al., 2008). Also, it has been described the presence of (-)-carvone in the essential oil of A. polystachya (Werdin-González et al., 2010), which was related to antispasmodic effect of Mentha genus (de Sousa et al., 2008; Goncalves et al., 2008) as well as other terpenic

Antispasmodic effects of Aloysia polystachya and A. gratissima tinctures and extracts are due to non-competitive inhibition of intestinal contractility Alicia E. Consolini et al.


compounds. At a taxonomic level the genus Aloysia is related to the genus Lippia (Pascual et al., 2001), which is widely used in the North hemisphere and exhibits an antispasmodic activity. In this way, in this work we studied whether the aqueous extract and tinctures of A. polystachya and A. gratissima are antispasmodic on acethylcholine-induced contractions on the isolated rat intestine, and which are the mechanisms and relation to composition. Isolated rat duodenum and ileum were a useful model to study the gastrointestinal effects of the extract and drugs. They have muscarinic receptors and let to study the antispasmodic effects over the smooth muscle contractured by depolarization or cholinergic stimulation (Karamenderes & Apaydin, 2003; Emendorfer et al., 2005).


Plant material

Aloysia polystachya (Griseb.) Moldenke and Aloysia gratissima (Gillies & Hook.) Tronc., Verbenaceae, were provided by a local herboristery, and the plants were authenticated by Prof. Dra. Etile Spegazzini with the voucher numbers 1153 (22-05-06) and 1155 (18-06-07) for A. polystachya and 1154 (23-06-06) and 1156 (19-07-07) for A. gratissima (LPE). Their samples are kept in the Herbarium Museum of Botany and Pharmacognosy (LPE), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina.

Pharmacological studies

Extracts preparation

Aqueous extracts (AE) were prepared by boiling 20 g of dried leaves in 100 mL distilled water for 20 min, according to the ethomedicinal use. After filtration, the respective decoction was lyophilized, affording a 10% w/w yield. The lyophilized extract was diluted in distilled water and Tyrode solution for in vitro tests the day of each experiment. With this procedure, the essential oil is not included in the lyophilized sample. The ethanolic tinctures (T) were obtained by maceration during 8-10 h at 20% in ethanol 70º.

Animals

The research was conducted in accordance with the internationally accepted principles for the laboratory animal use and care as was established by US guidelines (NIH publication # 85-23 revised in 1985).

Biological preparation and contractile measurements

Sprague-Dawley rats (200-250 g) were subjected to a 24 h fasting with free access to water before experimentation. The animals were anaesthetized by inhalation with diethyl ether and then quickly sacrificed by the opening of torax and abdomen, in order to avoid a relaxant effect of any other anaesthetic on the intestinal tissue. Duodenums and ileums (about 2 cm long) were prepared and mounted in organ baths of 20 mL containing Tyrode solution at 37 °C constantly oxygenated with air (pH 8.2) as in other works (Emendorfer et al., 2005). The preparations were equilibrated for at least 45 min at 1 g of pre-load. Tissues were connected to either, an isotonic force transducer Letica TRO 015 (PanLab, Spain) or an isometric one Power Lab MLT0201 (Power Lab, AD Instruments). Both types of response were statistically compared in each protocol, without significant differences between results. Because of that, both types of data were mixed. The signals were acquired in a computer respectively by a National Instruments PC-516 NI-Daq SW with Virtual Bench logger and by Chart 4 of Power Lab program.

Solutions and drugs

The solutions used had the following composition: Tyrode (Tyr): 150 mM NaCl, 2.7 mM KCl, 2 mM MgCl2, 12 mM NaHCO3, 0.4 mM PO4H2Na, 1.8 mM Ca Cl2 , bubbled with air (pH 8.2). Tyrode-0Ca: by eliminating Ca Cl2. Tyrode-0Ca-40 mM K+: by adding 0.6 ml KCl 10% to 20 mL Tyrode 0Ca. The drugs employed in biological tests were: Acetylcholine bromide (ACh, Sigma, USA), tetraethylammonium chloride (TEA, Sigma, USA) and metoclopramide (Novartis) and the monoterpene (-)-carvone (Sigma, USA). For phytochemical assays there were used the flavonoids quercetin (Sigma, USA), vitexin (Extrasynthese, France), isovitexin (Extrasynthese, France) and hesperidin (Sigma, USA), and also (-)-carvone (Sigma, USA). The flavonoids were initially dissolved in dimethylsulphoxide (DMSO) as well as (-) carvone, and then diluted with water to obtain the lower concentrations. A DRC control was done with DMSO at 1% or Ethanol 1% in the Tyrode medium, which were respectively not different from the DRC without vehicle.

Protocols

Dose-response curves to acetylcholine

Antispasmodic effects of Aloysia polystachya and A. gratissima tinctures and extracts are due to non-competitive inhibition of intestinal contractility

Alicia E. Consolini et al.


Dose-response curves (DRC) to acetylcholine (ACh) were done for the rats duodenums and ileums after a stabilization of 45 min. ACh concentrations were cumulatively added to the bath (to reach from 0.01 to 10 μg/mL) in the absence and the presence of a unique concentration of either, the respective AE, the T, or a reference drug as it was shown in the respective figure. They were added 5 to 10 min before the DRC and remained during it in the bath, and control curves were done with the vehicle of the tinctures, ethanol of 70°. When several concentrations of AE or T were assed, a growing order of concentrations was used for making the DRC in each organ.

Dose-response curves to CaCl2

After an stabilization during 45 min in Tyrode, the external Ca+2 was eliminated with Tyrode-0Ca and then, the muscle was depolarized with Tyrode-0Ca-40 mM K+. The DRC of Ca2+ were obtained by cumulatively adding CaCl2 to reach concentrations from 0.0195 to 17.5 mmol/L, in the absence and presence of growing concentrations of the respective AE or T. Each concentration of AE or T was added 5 min before the depolarization with high-K+ in Tyrode-0Ca and remained in the bath during it.

Dose relaxation curves of the extracts

Tissues were contracted by depolarization with ClK 40 mM in the Tyrode until obtaining a tonic response. At this point, growing concentrations of AE (shown in the respective figure) were cumulatively added to construct a dose-relaxation curve. In another group of muscles, the tonic contracture was obtained by adding 10 µg/mL acetylcholine (ACh). Then, the relaxation curves of AE or T were obtained in the absence and in the presence of 40 mmol/L tetraethylammonium (TEA, a non-selective K+-channels blocker).

Pharmacological and statistical analysis

From the DRC there were calculated the pD2 of the agonist (as -log EC50, in molar), and the affinity (pD’2) of the pure non-competitive antagonists as pD’2= -log [B’] + log [(EA-EAB’max)/(EA-EAB’)-1], where B’ is the concentration of the non-competitive antagonist, in molar, and EA, EAB’ and EAB’max are the maximal effects of the agonist respectively in the absence (A) and in the presence of either the used concentration of antagonist (B’) and the maximal concentration of it (B’max) (Van der Brink, 1977; Kenakin, 1984). In order to evaluate whether carvone act as competitive antagonist of ACh or Ca, the Schild method was applied for the

50% effect of DRC and the linear correlation of log (concentrations ratio-1) vs. log[carvona] (in Molar) was statisticaly analyzed (Kenakin 1984; Wyllie & Chen, 2007). For the extracts, the IC50 was calculated by extrapolation to 50% the individual relaxation curves obtained by plotting the maximal effect of the agonist from the respective DRC curves vs [extract] (expressed as mg lyophilized by mL). All results are expressed as media±ESM. Regression of DRC and statistics were done by using Graph Pad Prism 4.0 program, and by applying one-way ANOVA test for multiple comparisons followed by Tukey’s a posteriori tests. Paired t-test for comparison of paired results were also done. In all tests it was considered a significance of p<0.05.

Phytochemical studies

HPLC analysis

Since the presence of vitexin and isovitexin had been demonstrated in Aloysia citriodora leaves, it was evaluated whether these flavonoids were present in the AE of A. polystachya and A. gratissima. It was applied the previously developed method of high performance liquid chromatography (HPLC) contrasted with standards of both flavonoids (Ragone et al., 2007). It was used a 322-H2 series pump, a 155/156 UV/Vis detector and a work station equipped with the UniPoint LC, 3.3 version software (Gilson SAS, Villiers-Le-Bel, France). A Rheodyne 7125 manual sample injector (Rheodyne, CA, USA) with a fixed volume of 20 µL was also used. Cromatographic conditions were: LiChrocart RP-18 (250 mm x 4 mm internal diameter, 5 µm particle size) column (Merck, Darmstadt, Germany) as stationary phase under isocratic elution mode, at room temperature (25 °C). The mobile phase was composed by 2-propanol:tetrahydrofurane:water (5:15:85) at pH 4.33, flow rate was set at 1.0 mL/min and detected at 336 nm. The samples were prepared from the lyophilized AE of both plants, in the mobile phase at 700 µg/mL of concentration, which was filtered and injected by triplicate in the HPLC system. The following compounds were used as standard: vitexin and isovitexin.

Total flavonoids content Total content of flavonoids was evaluated from the aerial parts of both, A. polystachya and A. gratissima, by adaptation of the Kostennikova method modified by Méndez (Gutiérrez-Gaitén et al., 2000). Briefly, 1 g of aerial parts were extracted at reflux during 2 h with 20 mL sol. 10% H2SO4 and 20 mL of ethanol 50°. After cooling, filtering, and washing with 30 mL ethanol 50°, it was evaporated on a water



boiling bath up to the half of volume, and cooling on an ice bath. The obtained precipitated was filtered and washing with cool water (10 °C, four times of 10 mL). Both, the precipitate and the remaining solid obtained during cooling, were dissolved again with ethanol 96° previously heated at 50 °C, transferred and completed volume to 100 mL with ethanol 96°. A volume of 5 mL of this solution was diluted to 100 mL (1:20) to read the absorbance at a wavelength of 285 nm (A285) on a Thermo spectrophotometer, Helios-beta model (Thermo Fisher Scientific, Waltham, MA, USA). As a standard it was used a 40 µg/mL solution of hesperidin (Sigma) in ethanol 50°, and this one was measured as a blank. Then, the standard and hydroalcoholic extractions of both species of Aloysia were submitted to the measure of an UV-spectra in the range of 200-400 nm in order to stablish the wavelength of maximum absorption. From those results, it was calculated the concentration of flavonoids in the sample expressed as µg/mL hesperidin (Cm) as Cm=Ch.Am/Ah, where Ch is the hesperidin concentration (in µg/mL), Am is the absorbance of the sample at 285 nm, and Ah is the absorbance of hesperidin at 285 nm. Then, it was calculated the % of total flavonoids expressed as hesperidin (g flavonoids by 100 g of aerial parts of the plant).

TLC analysis

The presence of the ketonic monoterpene (-)-carvone in both species of Aloysia was evaluated by thin-layer chromatography (TLC), on silica gel F 254 Merck 0.25 mm and three systems of mobile phase: (a) toluene/ethylacetate (7:3), (b) toluene/ethylacetate (96:4), and (c) toluene/ethylacetate (9:1). The samples were 10% hexanic extractive solutions of the aerial parts of A. pachystachya and A. gratissima, and a standard of (-)-carvone (Sigma) at 1% in dichloromethane. The presence of flavonoids was evaluated by TLC on silica gel F254 Merck 0.25 mm and several mobile phases, as follows: (d) Cl2CH2/MeOH (95:5), (e) Cl2CH2/MeOH (99:1), (f) EtOAc/MeOH/H2O (100:17:10), and (g) EtOAc/MetH/AcH/ H2O (100:11:11:26) (superior phase). As a standard it was used a 1% methanol solution of quercetin (Sigma). The detection of bands of flavonoids was performed under UV light (254 and 366 nm) with and without ammonia clouds, and with natural product spray (1% methanol solution of 2-amino ethyl-diphenil-boric acid) and it was observed under visible and UV light. The 10% methanol extractive solutions of aerial parts of both species of Aloysia and the lyophilized of 20% AE were run.

Results

Pharmacological studies

The extract of both, Aloysia polystachya (AE-A.p) and A. gratissima (AE-A.g) reduced the maximal effect of the DRC on a dose-dependent way (Figure 1A), suggesting a non-competitive antagonism over the cholinergic contraction. The extrapolated IC50 of AE-A.p was 1.8±0.27 mg lyophilized/mL, but for AE-A.g it was only possible to find an IC25 of 0.276±0.077 mg lyophilized/mL because it inhibited the DRC in a lower degree (Figure 1B). The tinctures of both plants also non-competitively inhibited the DRC of Ach in a higher degree than the AE (Figure 2), with IC50 of 3.15±0.57 mg drug/mL for T-A.p and 6.46±2.28 mg drug/mL for T-A.g.

-4 -3 -2 -1 0 1 20

20

40

60

80

100controlA. polystachya 0.1 mg/mLA. polystachya. 0.3 mg/mLA.polystachya. 1 mg/mLA.polystachya. 1.5 mg/mLA.polystachya. 2 mg/mL

A.polystachya. 3 mg/mL

**

* * *

***

*

-log [ACh] (µg/mL)

% E

-3 -2 -1 0 1 20

20

40

60

80

100

controlA. gratissima 0.1 mg/mLA. gratissima 0.3 mg/mLA. gratissima 1 mg/mL

*

**

log [ACh] (µg/mL)

% E

Figure 1. Effects of Aloysia polystachya (a) and Aloysia gratissima (b) extracts on the dose-response curves of: acetylcholine (pD2: 5.77±0.13). Results as mean±SEM. Concentrations (mg lyophilized/mL) in labels. Two-way ANOVA by treatment: F=28.77, df 6, p<0.0001, by doses: F: 161.3, p<0.0001, ,post-tests: *p<0.05 vs control.

a

b




0 50 100 150 200 2500

50

100

150

200

T 2 8 14 mg/mL

T -A.gratissima

time (s)

% to

nic

K 40

mM

2.5 5.0 7.5 10.0

-8-7-6-5-4-3-2-101

AE A.gratissimaTEA 40 mM + AE A.gratissima

[extract] (mg lyoph./mL)

AE A.polystachya

∆ to

nic

of A

Ch

(v)

0 200 400 600 800 1000 1200

0

50

100

150

200

AChACh + A. polystachya

AE .0.3--1------3 -----------T 2-----T8

time (s)

% to

nic

of A

Ch

Figure 3. Relaxant effects on the tonic contractures of: (a) A. gratissima tincture on the high-K contracture vs. time; (b) A. polystachya AE and T on the ACh-contracture vs time, and (c) AE of both Aloysia spp on the ACh-contracture vs. extract concentrations. Results as mean±SEM. Concentrations (mg/mL) are shown in the figure with arrows.

To assay whether the effects of both T on the DRC of ACh were associated to K+-channels activation, the DRC of ACh were done in the absence and the presence of 10 mM TEA. Figure 4 shows that TEA did not inhibited the non-competitive antagonism of T-A.p. and T-A.g. on DRC-ACh, but potentiated it. In order to evaluate whether that potentiation was due to the release of a relaxant neurotransmitter such as dopamine (DA) evoked by the depolarization due to TEA, the DRC of ACh was done in the presence of 10 mM TEA, 6 mg/mL T-A.p and 10 mM metoclopramide to block the effect of dopamine in D2 receptors. The comparison of Figure 4A with Figure 4C shows that this combined treatment did not reduce but increased the non-competitive blockade produced by T-A.p. and TEA. In order to elucidate whether the non-competitive

-4 -3 -2 -1 0 1 20

20

40

60

80

100

120 controlA. polystachya T 0.66 mg/mLA. polystachya T 2 mg/mLA. polystachya T 6.6 mg/mL

**

* *

**

**

* * *

**

** * * *

etanol 70%

-log [ACh] (µg/mL)

% E

-4 -3 -2 -1 0 1 20

20

40

60

80

100

120 control (n= 7)A. gratissima T 0.66 mg/mLA. gratissima T 2 mg/mLA. gratissima T 6.6mg/mLA. gratissima T 13 mg/mL

*

** * *

*

**

**

*

* *

*

* **

* * *

* **

-log [ACh] (µg/mL)

% E

Figure 2. Effects of Aloysia polystachya (a) and Aloysia gratissima (b) tinctures on the dose-response curves of: acetylcholine. Results as mean±SEM. Concentrations (mg leaves/mL) in labels. Two-way ANOVA for (a): by treatment: F=173.9, df 3, p<0.0001, by doses: F: 137.6, df 8, p<0.0001; for (b): by treatment: F=61.32, df 4, p<0.0001, by doses: F: 62.89, df 8, p<0.0001. Post-tests: *p<0.01 vs control.

On the tonic contracture induced by Tyrode-K 40 mM the EA-A.p did not produce relaxation, but EA-A.g relaxed in a concentration-dependent way (Figure 3A). A similar behavior of the EA was obtained when the tonic contracture was induced by ACh, in which only the EA-A.g relaxed in a concentration-dependent way and it was unaffected by the unspecific blocking of all types of K+-channels with 40 mM TEA (Figure 3B). Nevertheless, both T-A.p. and T-A.g. relaxed in a concentration-dependent way the ACh-induced tonic contracture (Figure 3C). The effect of T-A.p. was not reduced but potentiated by the specific blocking of Ca2+-dependent K+-channels with 1 mM TEA.

a

b

a

b

c



antagonism of both T on the ACh-DRC were associated to inhibition of Ca+2 influx to the smooth muscle, DRC of Ca+2 were done on high-K+-medium to activate the channels. Figure 5 shows that both, T-A.p and T-A.g produced a non-competitive inhibition of Ca+2 influx. The extrapolated IC50 were respectively 1.8±0.08 mg/mL for A. polystachya and 9.3±1.4 mg/mL for A. gratissima.

-4 -3 -2 -1 0 1 20

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40

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120

control

T- A. polystachya 6.6 mg/mL

TEA 10 mM

TEA 10 mM + T- A.polystachya 6.6

*

*

*

*

*

*

** * * *

^ ^ ^

-log [ACh] (µg/mL)

%E

-4 -3 -2 -1 0 1 20

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40

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control

T-A. gratissima 6.6 mg/mL

TEA 10 mM

TEA 10 mM + T-A. gratissima 6.6

** * *

*

* * **^*

*

-log [ACh] (µg/mL)

%E

-4 -3 -2 -1 0 1 20

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40

60

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120

control

metoclopramide 10 µM

metoc 10 µM+T-A. poly 6 mg/mL

metoc10 µM+TEA10mM+T-A.poly 6

*

** * * *

* * * * * * *

-log [ACh] (µg/mL)

% E

Figure 4. Effects of Aloysia polystachya (a, c) and A. gratissima (b) tinctures on the dose-response curves of: acetylcholine in the absence (control) and the presence of 10 mM TEA and 10 µM metoclopramide. Results as mean ± SEM. Concentrations (mg leaves/mL) in labels. Two-way ANOVA for (a): by treatment: F=69.5, p<0.0001; for (b): by treatment: F=42.86, p<0.0001; for (c) by treatment: F: 368, p<0.0001. Post-tests: *p<0.05 vs the respective control.

Effects of (-)carvone

Since it was described the presence of (-)-carvone in the essential oil of A. polystachya (Werdin-González et al. 2010), it could contribute to the effects of tinctures. Since its mechanism has not been studied before, in this work we evaluated its effects on the DRC of ACh and Ca2+. Figure 6 shows that (-)-carvone developed a non-competitive blockade of both DRC, since it decreased Emax. Also, the Schild method confirmed that (-)-carvone is not a competitive antagonist of ACh (slope different than 1) or Ca2+ (there is not significative linear correlation). From the DRC it was calculated the affinity of (-)-carvone on its own cellular site, which was estimated by the pD´2. It resulted 4.002±0.117 (n=20) from the DRC of ACh and 3.86±0.19 (n=18) from the DRC of Ca2+.

-5 -4 -3 -20

25

50

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125

Control

T- A. polystachia 0.66 mg/mL

T- A. polystachia 2 mg/mL

**

*

*

*

*

*

T-A.polystachya 6 mg/mL

* *

*

*

*

log [Ca] (M)

% E

-5 -4 -3 -20

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125

ControlT- A. gratissima 6.6 mg/mL

T- A. gratissima 20 mg/mL

***

*

*

***

log [Ca] (M)

% E

Figure 5. Effects of Aloysia polystachya (a) and A. gratissima (b) tinctures on the dose-response curves of calcium in the high-K Tyrode. Two-way ANOVA for (a): by treatment: F=258.9, df 3, p<0.0001, by doses: F: 577, df 5, p<0.0001; for (b): by treatment: F=449.3, df 2, p<0.0001, by doses: F: 175.4, df 5, p<0.0001 Post-tests: *p<0.05 vs control.

Phytochemical assays

HPLC determinations

a

b

a

b

c




Contrarily to what was described for Aloysia citriodora (Ragone et al., 2007) the HPLC chromatograms showed no presence of vitexin or isovitexin in the AE of Aloysia polystachya and Aloysia gratissima (Figure 7). Retention times of the standard drugs were Rt 11.51 min for vitexin and 16.85 min for isovitexin, with a good separation profile comparable to that previously described.

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120controlcarvone 3 uMcarv 10 uM carv 30 uM

carv 100 uM

**

*

pD´2 carv 3.86 ± 0.19 (n=18)

car 300 uM

log [Ca] (M)

% E

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125controlcarvone 3 µM carv 10 µM carv 30 µMcarv 100 µM carv 300 µM

pD´2 carvone: 4.002 ± 0.117 (n= 20)

*

* * * ** *

log [ACh] (µg/mL)

% E

-5 -4 -3-0.05

0.05

0.15

0.25

0.35 slope: 0.081±0.019r2: 0.903, p=0.0497

+log [carvone] (M)

+log

(A2/

A1-

1)

4 5 6

-0.001

0.000

0.001

0.002r2: 0.3378p=0.30

log[carvone] (M)

+log

(A2/

A1-

1)

Figure 6. Effects of (-)-carvone on the dose-response curves of: (a) ACh (n=4, see pD’2 of carvone); and (b) Ca2+ (n=4, see pD´2 of carvone). Two-way ANOVA for (a): by treatment: F: 43.23, df 5, p<0.0001, by doses: F: 52.58, df 7, p<0.0001; for (b): by treatment: F: 4.702, df 5, p=0.0006, by doses: F: 195.1, df 7, p<0.0001. Post-tests: *p<0.05 vs. control. In (c) are shown the Schild graphs obtained from the DRC of ACh (at left) and Ca2+ (at right) and the linear correlation results, which demonstrate the absence of competitive antagonism.

Total content of flavonoids

Figure 8 shows that hesperidin exhibited a maximum absorption at 285 nm with a lower peak absorption at 230 nm. The extractives from A.polystachya and A.gratissima exhibited also two peaks at 285 nm and 330 nm, by which it was chosen the wavelength of 285 nm for measuring. Table 1 shows the results of absorbance as well as the calculated % of flavonoids total content.

Table 1. Results of absorbance and total content of flavonoids expressed as hesperidin.

Hesperidin (40 µg/mL)

Extractive solutions

A. polystachya A. gratissima

Absorbance at 285 nm 0.627 0.330 0.277

Concentration in the 1:20 dilution (µg/mL) --- 21 17.67

% total flavonoids in the plant (g flavonoids/100 g aerial parts)

--- 4.2% 3.53%

Table 2. Chromatographic TLC results of the methanol extracts of Aloysia spp (EtOAc/MeOH/H2O 100:17:10). Aloysia pachystachya Aloysia gratissima Quercetin

--- 0,40

Blue-clear fluorescence (UV 366)

0.50 Yellow-orange

0.50 Yellow-orange

0.60 Brown

0.60 Brown

0.64 Brown

0.64 Brown

0.68 Brown

0.68 Brown

0.71 Brown

0.71 Brown

0.78 Brown

0.78 Brown

0.80 Yellow-orange

0.80 Yellow-orange

0.85 Yellow-orange

0.85 Yellow-orange

0.90 Yellow-orange

0.90 Yellow-orange

0.90 Yellow-orange

1.00 1.00

TLC results

Table 2 shows the results obtained with TLC for detecting the presence of flavonoids in the systems of higher resolution. The methanol extracts of both plants

Results show Rf values, colour at visible with natural products reactive and/or fluorescent aspects at UV 366.



Table 3. Chromatographic TLC results of the hexane extracts of Aloysia spp.Toluene/EtOAc 96:4 Toluene/EtOAc 9:1

Aloysia pachystachya Aloysia gratissima Carvone Aloysia pachystachya Aloysia gratissima Carvone

0 Violet-blue

0 Violet-blue

0.00 Violet-blue

0.00 Violet-blue

0.13 Violet-blue

0.13 Violet-blue

0.17 Violet-blue

0.17 Violet-blue

0.17 Violet-blue

0.17 Violet-blue

0.25 Violet-blue

0.25 Violet-blue

0.25 Blue-clear fluorescence

(UV366)


(UV366)


(UV366)


(UV366)

0.30 Violet-blue

0.30 Violet-blue

0.38 Violet-blue

0.38 Violet-blue

0.40 Violet

0.40 Violet

0.40 Violet

0.45 Violet (very low)

0.45 Violet (very low)

0.60 Violet-blue

0.60 Violet-blue

0.50 Violet

0.50 Violet

0.50 Violet

1.00 Violet-blue

1.00 Violet-blue

0.58 Violet-blue

0.58 Violet-blue

1.00 Violet-blue

1.00 Violet-blue

Table shows Rf values, colour at visible in the presence of sulphuric anisaldehyde and/or fluorescence aspect at UV 366.

Figure 7. HPLC traces of the standard solutions of vitexin (first peak) and isovitexin (second peak) (a) in comparison with the HPLC traces of the aqueous extract of Aloysia polystachya (b) A. gratissima (c) and A. citriodora (d). Retention time scale in minutes are detailed in the text.

a b

c d




glycosides, the stronger at Rf 0.50. The band at Rf 0.90 is similar to that of the standard, the flavonol quercetin. On this system, the aglycons flavonoids run with the front of mobile phase (Rf 0.80 a 1.00) while the glycosides run in the middle. The AE of A. polystachya developed three bands of very low intensity (Rf 0.45; 0.60 and 0.80) which correspond with those of the methanolic extract. The AE of A. gratissima only showed two bands (Rf 0.45 and 0.80). Table 3 shows the results of TLC done with the hexane extractives of both Aloysia spp. The ketonic monoterpene (-)-carvone was used as a standard. The cromatographic profiles are very similar for both species. At 254nm there were found 6 bands with Rf between 0 and 0.60. One of them (Rf 0.40) has the same Rf and aspect than that of the standard (-)-carvone. At 366 nm it appears a band with blue clear fluorescence (Rf 0.25), which does not react with sulphuric anisaldehyde. With this compound were seen the same seven bands, from which it is stronger the band of Rf 0.30 in violet. In system (c) there were found similar profile of bands for both Aloysia spp., except a low-intensity band at 0.45 in A.polystachya. In both, a band at Rf 0.30 with blue clear fluorescence appears at 366, which does not react with sulphuric anisaldehyde. Also, there is a band at Rf 0.50 which became red violet after sulphuric anisaldehyde, with Rf and aspect similar to that of (-)-carvone. Then, TLC results suggest that both species of Aloysia have similar content in monoterpenic compounds, such as carvone.

Discussion

This work gives support to the popular use of Aloysia polystachya (“burrito”) and A. gratissima (“palo amarillo”) as antispasmodic, since both directly relaxed the intestinal smooth muscle and non-competitively inhibited the DRC of ACh. There were assessed two phytotherapic preparations, the tinctures obtained by maceration (T) and the aqueous extract obtained by decoction (AE). The T induced a higher antispasmodic effect than the AE, as it was evidenced by the higher reduction in the effects of ACh (see the Emax of the DRC). These differences suggest either that the ethanolic maceration could extract more active principles or that these ones are thermo-sensitives. The non-competitive inhibition of the ACh effect suggests that one or more active principles would be interacting with a cellular site different from the muscarinic receptor, as it is classically interpreted from the DRC (Van der Brink, 1977; Kenakin, 1984). Several antispasmodic plants demonstrated to be non-competitive antagonists of ACh on duodenal or ileal smooth muscles (Sánchez de Rojas et al., 1995; Karamenderes & Apaydin, 2003; Camara et al., 2003; Emendorfer et al., 2005). This behaviour was also found in flavonoids, such as quercetin (Di Carlo et al., 1999). The presence of flavonoids as quercetin was evidenced in the AE by TLC, which could explain

Figure 8. UV Spectrum of hesperidin standard (a), and the extractive solutions of A. polystachya or “burrito” (b) and A. gratíssima or “palo amarillo” (c).

yielded until 10 common bands with Rf between 0.50 and 1.00. The extract of A. gratissima has another extra band at Rf 0.40 with strong blue clear fluorescence. According to Wagner & Bladt (1995) this is typical of extracts with flavonoids containing phenol-carboxylic acids, such as caffeic and chlorogenic acids. After boric acid, they appear until four bands yellow-orange (Rf 0.50; 0.80; 0.85 and 0.90) characteristics of flavonols and flavones and their



part of the effects of both Aloysia spp. It was reported previously that quercetin relaxed the high-K+ contracture on rat intestine (Ragone et al., 2007) and guinea-pig ileum (Di Carlo et al., 1999). Also, the total content of flavonoids was estimated by a method which compares the UV-absorption spectrum of the extracts with that of hesperidin. This compound could be present among other flavonoids, according to the characterization done by TLC. The flavonoids total content of A. pachystachya was slightly higher than that of A. gratissima. This difference could be the origin of the pharmacological differences: A. pachystachya induced higher reduction of the ACh effect than A. gratissima in the extracts, and higher potency in the tinctures (IC50 of 3.14±0.57 vs 6.46±2.28 mg/mL, respectively). Nevertheless, the flavonoids vitexin and isovitexin were not found by HPLC in A. polystachya or A. gratissima. These flavonoids have been identified in the AE of Aloysia polystachya in which explain part of the antispasmodic effect (Ragone et al., 2007), and there were also described in other plants as the genus Passiflora (Muller et al., 2005). Then, the antispasmodic effects of the species of Aloysia could be related to several types of flavonoids which differ among them. The first hypothesis underlying the non-competitive inhibition of ACh contractions was that the plants could activate K+-channels, as Aloysia citriodora does. A K+-channel opener hyperpolarizes and relaxes the smooth muscles exposed to agonists but not those exposed to high-K+ depolarization, and this is used as a tool to evidence this mechanism (Karaki et al., 1997). Then, results of the AE and T on the tonic contractures induced by high [K+] and ACh suggested that A. polystachya could have that mechanism. Nevertheless, the relaxation was not inhibited by TEA at 10-40 mM (concentrations at which it non-selectively blocks all types of K+-channels), or TEA at 1 mM (which selectively block the Ca-dependent K+-channels KCa without affecting the voltage-dependent ones, Kv) (Zhao et al., 2009). Contrarily, the non-competitive inhibition of the T on the ACh dose-response curve was potentiated by TEA, suggesting that the depolarization induced by TEA could stimulate the release of a relaxant neurotransmitter such as dopamine (DA). Also, the depolarization could be contributing to activate and inactivate the Ca2+ channels and then, it could be the origin of the potentiated blockade of T on the smooth muscle. It would suggest that T may contain Ca-blockers among the active principles. The pre-treatment with the competitive antagonist of D2 receptor, metoclopramide, did not reduce the effect of T-A. polystachya. Then, the relaxant effect of T-A. polystachya with or without TEA was not due to release of dopamine acting on D2 receptors. The potentiation of metoclopramide on the non-competitive blockade of the T suggests also interaction with other constituents of the plant. The following hypothesis was that the AE and

especially the T have active principles which could act as antagonists of Ca+2 influx. This hypothesis was supported by the relaxation of the tonic contracture induced by high-K+ and ACh, and by the potentiation evoked by depolarization with TEA. The dose-response curves of CaCl2 were also non-competitively inhibited by the T of A. polystachya and A. gratissima. The IC50 on the Ca2+-DRC were similar to (for A. gratissima) or lower than (for A. polystachya) the IC50 on the ACh-DRC. These comparisons suggest that the blockade of Ca2+ influx would be responsible for the non-competitive inhibition on the ACh-induced contractions. It is known that the interference with the Ca2+ channels may result competitive for drugs as dihydropiridines, but some other typical drugs as diltiazem or verapamil interfere in a non-competitive way with the internal sites of the channel (Karaki et al., 1997). Alternatively, other cellular sites could be affected by these plants, such as inhibition on calmoduline or sensitivity of actomyosin myofilaments (Karaki et al., 1997). The AE of A. citriodora also inhibited the DRC of Ca2+ in a non-competitive way, but the effect was associated to the activation of guanylil-ciclase and K+ channels (Ragone et al., 2007). The presence of flavonoids could also explain the Ca2+-blockade, since these compounds induce relaxation of smooth muscle and blockade of calcium influx (Gharzouli & Holzer, 2004). The taxonomically related genus Lippia also contains flavonoids (Skaltsa & Shammas, 1988) and exhibits antispasmodic properties. Nevertheless, the specific flavonoids seems to differ among the different species of Aloysia, especially A. citriodora. Furthermore, the presence of the ketonic monoterpene (-)-carvone was described in about an 85% in the essential oil of A. polystachya (Werdin-González et al., 2010). We found by TLC a similar band when there were assessed together the standard of this drug and the hexane extract of the aerial parts. The effect of (-)-carvone on smooth muscle was also assessed, finding that this compound non-competitively blocked the DRC of ACh and Ca2+, both with the same pD´2 (about 4). This parameter estimates the affinity of a pure substance for its own receptor site, which differs from the agonist receptor. It is defined as the -log [antagonist] (in Molar) that reduces to the half the effect of the agonist (Kenakin et al., 1984). Also, the Schild analysis confirmed that (-)-carvone is not a competitive antagonist of ACh or Ca2+, since there was obtained either absence of correlation (for ACh) or a slope different than one (for Ca2+) according to Kenakin et al. (1984) and Wyllie & Chen (2007). Neither the pD´2 nor the Schild graphs could be calculated for the extracts or tinctures because they are not pure substances, but the DRC showed non-competitive antagonism because of the fall in Emax (Van der Brink, 1977). In spite of the




different parameters estimated (pD´2 vs IC50), either for the tinctures and for (-)-carvone assays, the parameters of inhibition on the DRC of ACh were similar to that of the DRC of Ca2+. It suggests that in tinctures as well as in (-)-carvone the Ca2+-influx blockade may be the origin of the interference with the ACh effect. Then, if considering that at least a fraction of the essential oil could be extracted in the tinctures, and that the pharmacological test is more sensitive than the TLC, it is possible to suggest that carvone could contribute to the effect of A. polystachya and A. gratissima tinctures. In summary, the results support the popular use of A. polystachya and A. gratissima as intestinal antispasmodics. Also, they are mostly explained by the non-competitive blockade of the Ca2+ influx. The effect was higher with the tinctures than with the acqueous extract, by which the maceration with ethanol of 70º must be extracting the active principles which cause the effect, such as some flavonoids and essential components as carvone. Since the flavonoids are known relaxants of the smooth muscle, their presence suggest that they would contribute to the antispasmodic effect of these plants. Here it is shown that also the monoterpene carvone could be in part responsible for the effect of the tinctures, since it non-competitively inhibits the effect of ACh and Ca2+ influx, at the same concentration range, as well as the tinctures.

Acknowledgments

We kindly thanks to Lic. Esperanza Ruiz for her technical assistance on the HPLC experiments, and to Dra Etile Spegazzini for the identification of the plants. This work was supported by the grants X-408 (2005-2008) and X-513 (2009-2012) from the Universidad Nacional de La Plata (UNLP), and was presented as a thesis of Andrea Berardi for obtaining the Magister en Plantas Medicinales from Facultad de Ciencias Exactas, UNLP.

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*Correspondence

Alicia E. ConsoliniDepartamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata47 y 115 (1900), La Plata, [email protected] Tel.: 54 221 423 5333 int.42

901

Article


695X2011005000142

Received 28 Dec 2010Accepted 17 May 2011



21(5): 901-907, Sep./Oct. 2011Inhibition of Saccharomyces cerevisiae Pdr5p by a natural compound extracted from Brazilian Red Propolis

Cinzia Lotti,1 Gabriellen M. Migliani de Castro,2,3 Leandro F. Reis de Sá,2 Beatriz dos Anjos Fonseca Sampaio da Silva,2,3 Ana Claudia Tessis,3 Anna L. Piccinelli,1 Luca Rastrelli,1 Antonio Ferreira-Pereira*,2

1Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Italy,2Laboratório de Bioquímica Microbiana, Departamento de Microbiologia Geral/IMPPG/CCS, Universidade Federal do Rio de Janeiro, Brazil,3Instituto Federal do Rio de Janeiro, Brazil.

Abstract: Multidrug resistance of cancer cells and pathogenic microorganisms leading to the treatment failure of some forms of cancer or life-threatening bacterial or fungal infections is often caused by the overexpression of multidrug efflux pumps belonging to the ATP-binding cassette transporters superfamily. The multidrug resistance of fungal cells often involves the overexpression of efflux pumps belonging to the pleiotropic drug resistance (PDR) family of ABC transporters. Possibly the best-studied fungal PDR transporter is the multidrug resistance transporter Pdr5p of Saccharomyces cerevisiae. Some research groups have been searching for new inhibitors of these efflux pumps in order to alleviate resistance. Natural products are a great source for the discovery of new compounds with biological activity. Propolis is a complex resinous material collected by honeybees from exudates and buds of certain plant sources and this material is thought to serve as a defense substance for bee hives. Propolis is widely used in traditional medicine and is reported to have a broad spectrum of pharmacological properties. Literature reported some biological functionalities of propolis, such as antibacterial, antiviral, fungicidal, anti-inflammatory and anti-carcinogenic activities. The chemical composition of propolis is qualitatively and quantitatively variable. Components isolated from methanolic extract of red Brazilian propolis (Alagoas, Northeast of Brazil) are isoflavonoids (including pterocarpans, isoflavans, isoflavones), flavanones and polyprenylated benzophenones. In this work we demonstrated the effects of five different isolated compounds on the ATPase activity of Pdr5p. Out of all five substances tested, only BRP-1 was able to completely abolish the enzymatic activity while others worked as positive modulators of the enzyme activity. BRP-1also inhibited the efflux of Rhodamine 6G from yeast cells overexpressing Pdr5p. Taken together, these results demonstrate that Brazilian propolis could be a source of promising compounds that can alleviate the MDR phenomenon, particularly in some fungi, where it could be used as an adjuvant for the treatment with azoles.

Keywords:Brazilian Red Propolis

multidrug resistancePdr5p

R6Gyeast

Introduction

The overexpression of pleiotropic drug resistance (PDR) efflux pumps of the ATP-binding cassette (ABC) transporter superfamily, similar to the human multidrug resistance protein P-glycoprotein (ABCB1), frequently correlates with multidrug resistance of fungal cells. Multidrug resistance leads to the resistance to several structurally and functionally unrelated drugs and can be the cause of treatment failure of anticancer, antimicrobial and antifungal

chemotherapy (Lamping et al., 2010). The pleotropic drug resistant protein Pdr5p is a plasma membrane associated ATP-binding cassette (ABC) transporter and the major multidrug efflux pump of Saccharomyces cerevisiae. The overexpression of multidrug efflux pumps has been reported to cause multidrug resistance in several pathogenic microorganisms such as fungi (Morschhäuser, 2010; Ernst et al., 2005), parasites (Pradines et al., 2005), as well many pathogenic bacteria (Hawkey & Jones, 2009). The emergence and spread of multidrug

Inhibition of Saccharomyces cerevisiae Pdr5p by a natural compound extracted from Brazilian Red PropolisCinzia Lotti et al.


resistance (MDR) poses a severe and increasing public health threat, since new pharmaceutical drugs, especially drugs, show moderate to severe toxicity and/or side effects (Shukla et al., 2008). Efforts to identify natural products as inhibitors of MDR exporters have the potential to provide a large number of novel drug leads since many of these products have been used for centuries without harmful side effects (Wang et al., 2010). Propolis is a complex resinous material collected by honeybees (Apis mellifera) from exudates and buds of certain plant sources and this material is thought to serve as defense substance for bee hives (Pietta et al., 2002). It is used to block holes and cracks, to repair combs, to strengthen the thin borders of the comb and for making the entrance of the hive weathertight or easier to defend. Propolis is also used as an “embalming” substance to cover hive invaders which bees have killed but cannot transport out of the hive (Ghisalberti, 1978). The action against micro-organisms is an essential characteristic of propolis and it has been used by human beings since ancient times for its pharmaceutical properties. For all types of propolis a broad spectrum of pharmacological properties is reported. Literature reported some biological functionalities of propolis, such as antibacterial and antiviral properties and many other beneficial biological activities including antiinflammatory, antiulcer, local anaesthetic, hepatoprotective, antitumor and immunostimulating properties (Sforcin et al., 2000; Kujumgiev & Popov, 1996; Banskota et al., 2002; Awale et al., 2008; Marcucci, 1995; Burdok, 1998; Popova et al., 2005). For this reason propolis is widely used as a popular remedy in folk medicine, as a constituent of biocosmetics, health food and today represents an important raw material for many health products and constitute a new interdisciplinary area for research (Matsuda, 1994). Propolis chemical composition is qualitatively and quantitatively variable, depending on the season, the species of bee, vegetation and the area of collection; in tropical regions, for example, because of the difference in vegetation, the chemical composition of propolis is very different (Lotti et al., 2010). Its colour also varies depending on its botanical source, the most common being dark brown, but red propolis has also been observed in tropical countries such as Brazil and Cuba (Park et al., 2002; Cuesta-Rubio et al., 2007). The major components isolated from methanolic extract of red Brazilian propolis (Alagoas, Northeast of Brazil) are isoflavonoids (including pterocarpans, isoflavans, isoflavones), flavanones and polyprenylated benzophenones (Trusheva, 2006; Awale et al., 2008). This type of Brazilian Red Propolis (BRP) demonstrated a notable antimicrobial activity

against Staphylococcus aureus and mutans (Alencar et al., 2007) and cytotoxicity against human pancreatic PANC-1 cancer cell line (Awale et al., 2008). In this work we show that BRP is also able to inhibit the enzymatic activity and the rhodamine 6G transport of Pdr5p from Saccharomyces cerevisiae. Thus BRP could be a new tool for the study of the mechanism of multidrug resistance and work as a natural compound to reverse this phenotype in yeast.


Plant material

Samples of raw red propolis produced by Apis mellifera bees were collected in a Northeast region of Brazil (Alagoas State) in the mangrove area. Methanol extract of BRP was obtained by maceration of ground propolis sample with methanol and stored at 5 °C in the dark.

Standard and samples

Compounds BRP-1-5 were isolated from Brazilian red propolis sample and identified by spectroscopic methods.

General experimental procedure

A Bruker DRX-600 NMR spectrometer, operating at 599.19 MHz for 1H and at 150.86 MHz for 13C, using the UXNMR software package was used for NMR experiments; chemical shifts are expressed in δ (parts per million) referring to the solvent peaks δ H 3.34 and δ C 49.0 for CD3OD, δ H 7.27 and δ C 77.0 for CDCl3; coupling constants, J, are in hertz. Preparative HPLC was performed on a Agilent 1100 series system consisting of a G-1312 binary pump, a G-1328A Rheodyne injector, a G-1322A degasser and a G-1315A photodiode array detector (PDA). TLC analyses were performed with Macherey-Nagel precoated silica gel 60 F254 plates.

Isolation of compounds

A portion of dry extract was fractionated over Sephadex LH-20 column (100 cm x 5.0 cm) using methanol as elution solvent. Major fractions were purified by HPLC-RI (Waters 590 series pumping system equipped with a Waters R401 refractive index detector) in isocratic elution mode using Luna C8 column (250 x 10 mm, 10 µm) (methanol-water 55:45 v/v), both from Phenomenex (Torrance, CA, USA), to yield pure compounds, which were identified as isomeric mixture of guttiferone E and xanthocymol (1) (Gustafson et al.,

Inhibition of Saccharomyces cerevisiae Pdr5p by a natural compound extracted from Brazilian Red Propolis

Cinzia Lotti et al.


1992), 7-O-methylvestitol (2) (Piccinelli et al., 2005), vestitol (3) (Piccinelli, 2005), biochanin A (4) (Chang et al., 1994), and formononetin (Chang et al., 1994) (5), respectively.

O O

OHO

OHHO

1

O O

OHO

OHHO

OOH

OCH3

R

2 R = OCH33 R = OH

OOH

OCH3

HO

4 R = H5 R = OH

OR

Preparation of plasma membrane

Plasma membrane fractions were obtained from yeast S. cerevisiae mutants AD124567 and AD 1234567 (Decottignies et al., 1998) as previously reported by Rangel et al. (2008). The plasma membranes highly enriched in Pdr5p were stored in liquid nitrogen.

ATPase activity assay

ATP hydrolysis was determined by incubating Pdr5p enriched membranes (0.013 mg/mL final concentration) in 96-well plate at 37 °C for 60 min in a reaction medium containing 100 mM Tris-HCl pH 7.5, 4 mM MgCl2, 75 mM KNO3, 7.5 mM NaN3 and 0.3 mM ammonium molybdate and 3 mM ATP). The compounds BRP-1-5 were added from stock solution in dimethylsulfoxide up to 5% v/v final concentration. After the incubation period the reaction was stopped with 1 % SDS as described previously (Dulley, 1965). The released inorganic phosphate (Pi) was measured as described previously (Fiske & Subbarrow, 1925). As control plasma membranes prepared from the PDR5 deleted strain AD1234567 were used. The difference between the ATPase activity of AD124567 membranes and the activity of AD1234567 corresponds to the Pdr5p mediated ATPase activity.

R6G efflux by intact cells

Rhodamine efflux assays were performed as described previously by Niimi et al. (2004) with minor changes. Briefly, log-phase AD124567 or AD1234567 cells were washed four times with distilled water. The pellet was resuspended in water and incubated

for 15 min at 4 °C. Soon after, the cell suspension was centrifuged and the pellet was resuspended in 50 mM 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES)-NaOH, pH 7.0. To deplete intracellular energy, the cells were incubated for 30 min a 30 °C with 2-deoxyglucose (final concentration 5 mM). Then R6G was added at a final concentration of 15 µM and incubated for 30 min at 30 °C. R6G equilibrated cells were then washed twice with distilled water and resuspended with HEPES buffer to an OD of 10. Approximately 1x108 cells were incubated at 30 °C with or without BRP-1 and glucose at a final concentration of 0.2% was added to start the reaction. Glucose free controls were included in all experiments. After 13 min the cells were pelleted and the R6G present in the supernatant was spectrophotometrically quantified using a spectrophotometer (P 582-Micronal- Brazil) at a wavelength of 527 nm.

BRP-1 MIC as a measure of toxicity against S. cerevisiae

The MIC of BRP-1 for Pdr5p-hyperexpressing strain AD124567 and isogenic null mutant AD1234567 was determined by a microdilution method according to Clinical and Laboratory Standards Institute (CLSI; former NCCLS M27-A) with slight modifications using 96-well microtiter plates. Cells in YPD medium were inoculated at 1x104 cells per well and incubated at 30 °C for 48 h with shaking (150 rpm) in the presence of 50 µM of BRP-1. Cell growth was monitored at 600 nm with a microplate reader (Fluostar Optima, BMG Labtech, Offenburg, Germany).

Intracellular accumulation of R6G

This essay was performed as previously described by Sharma et al. (2009) with slight modifications. Yeast cells in the logarithmic phase of growth were washed three times with phosphate-buffered saline (PBS) pH 7,0. The pellet was resuspended in PBS and then de-energized by incubating for 45 min at 30 °C with 2-deoxyglucose (final concentration 5mM) and dinitrophenol (final concentration 5 mM). The de-energized cells were pelleted, washed twice (9000 rpm 2 min) with PBS without glucose, and then resuspended in PBS (with glucose 2%). The cell suspension was incubated with de BRP-1 (final concentration 100µM) at 30 °C by 5 min. Then R6G was added at a final concentration of 2.2 µM and incubated for 30 min at 30 °C. After washing three times with PBS without glucose (9000 rpm, 2 min) the cells were resuspended with the same buffer and their fluorescence visualized using a Nikon Eclipse E400 (Nikon, Japan).




All experiments were carried out at least three times and the results are expressed as mean±SD. Standard deviation and IC50 values were calculated using the computer program Sigma Plot version 10 (SPSS Science Marketing).

Results

ATPase activity assay The effects of BRP-1-5 on the Pdr5p ATPase activity are shown in (Figure 1). It was observed that only BRP-1 was able to inhibit the enzyme activity in a final concentration of 100 µM. In contrast, BRP-2, 3 and 4 stimulate the enzyme activity while BRP-5 was ineffective. BRP-1 was chosen for the following experiments. The determination of the IC50, value of the Pdr5p ATPase activity for BRP-1 is shown in (Figure 2). The results show that BRP-1 is a very strong inhibitor of the Pdr5p ATPase activity with an apparent IC50 of 1.35 µM.

ATP

ase Activity (µm

olPi.m

in-1

.mg

-1)

0,00

0,05

0,10

0,15

0,20

0,25

0,30

Control BRP-1 BRP-2 BRP-3 BRP-4 BRP-5

*ND

Figure 1. Modulatory effects of BRP compounds on the Pdr5p ATPase activity. Pdr5p-enriched plasma membranes were incubated in the presence of 100 µM of BRP-1, BRP-2, BRP-3, BRP-4 and BRP-5. The ATPase activity was measured as described in Materials and Methods. The control bar represents the enzymatic activity in the absence compounds. The data presents means±SD of three independent experiments. *ND=not detected.

Determination of BRP-1 toxicity for S. cerevisiae

It is known that propolis can be very toxic for bacterial (Alencar et al., 2007) and human cells (Awale et al., 2008). We therefore investigated the toxic effects BRP-1 against S. cerevisiae cells. Figure 3 shows that even in the presence of high concentrations of BRP-1 (almost 50 times more than the IC50 value), no toxicity of BRP-1 against yeast cells could be detected (without compound).

[BRP-1] (µM)

0 5 10 15 20

ATPase Activity (% of control)

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20

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60

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Figure 2. Inhibition of Pdr5p ATPase activity by BRP-1. The enzymatic activity was measured as described in Materials and Methods in the presence of different concentrations of BRP-1 (0.1 to 20 µM). Data represent means±SD of three independent experiments. The control represents 100% of enzymatic activity in the absence of compound.

[BRP-1] (µM)

CONTROL 50

Relative G

rowth (%

of control)

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20

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60

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Figure 3. Toxicity of the BRP-1 against AD 124567 strain. The cells were treated with BRP-1 at a final concentration of 50 µM, as described in Materials and Methods using a 96-wells microtiter plate . The control bar corresponds to the 100% of growth without compound.

Effect of BRP-1 on the intracellular accumulation and the efflux of R6G

The effects of BRP-1 on the accumulation and retention of R6G was first evaluated by fluorescence microscopy (Figure 4). The Pdr5p-hyperexpressing strain AD124567 treated with BRP-1 (final concentration 100 µM) showed a strong fluorescence, indicating an intracellular accumulation of R6G (Figure 4H), compared to the control in the absence of BRP-1 (Figure 4G). In the presence of 100 µM BRP-1, AD124567 cells accumulated R6G at levels comparable with those of cells treated with FK-506 (Figure 4F), a known inhibitor of Pdr5p, similar to AD1234567 cells that do not express Pdr5p at all (Figure 4E).

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Cinzia Lotti et al.


by prokaryotic or eukaryotic cells. This problem is currently of growing significance with increasing numbers of HIV patients, who are highly susceptible to opportunistic fungal infections due to their severely compromised immune system. MDR caused by the overexpression of multidrug efflux pumps frequently develops in such patients due to their long-lasting prophylactic treatment regimes, but similar MDR can also be observed in some plant and animal pathogens and even in human tumour cells. Therefore, it is necessary to develop strategies that can reverse this drug resistance and natural compounds could be a valuable source material to find potent inhibitors of these multidrug efflux pumps. In this work, we investigated five different compounds isolated from Brazilian red propolis type, which were purified, identified and named as: guttiferone E/xanthocymol (1); 7-O-methylvestitol (2); vestitol (3); biochanin A (4) and formononetin (5). From those five compounds 1 showed a strong inhibition (Figure 1) of the enzymatic activity of the multidrug efflux transporter Pdr5p of S. cerevisiae. Its IC50 value was lower than those reported for other natural compounds such as enniatin (Ivnitski-Steele et al., 2009) or curcumin (Sharma et al., 2009) two well-known inhibitors of multidrug efflux transporters. BRP-1 was not toxic to the yeast cells used in this work even at high concentrations (50 µM). Purified BRP-1 (100 µM), effectively inhibited the efflux of R6G of Pdr5p overexpressing cells and was able to inhibit the Pdr5p ATPase activity at similar concentrations in vitro (Figure 4H). These results suggest that BRP-1 acts as an inhibitor of the Pdr5p ATPase activity by

In order to quantify the inhibition of energy-dependent R6G efflux by BRP-1 the R6G efflux of the strain AD124567 was measured at different concentrations of BRP-1 (Figure 5). BRP-1 caused a dose-dependent inhibition of the glucose-dependent R6G efflux, with 80% of inhibition achieved at 100 µM. This inhibition by BRP-1 was very strong with an IC50 value similar to the IC50 value measured for the inhibition of the (8.14 µM).

[BRP-1] (µM)

0 20 40 60 80 100

Rhodam

ine 6G efflux ( %

of control)

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Figure 5. Inhibition of R6G efflux in strain AD124567 by BRP-1. The efflux of R6G was measured after incubation of the cells in the presence of different concentrations of BRP-1 as described in the Material and Methods. Data represent means±SD of three independent experiments.

Discussion

Resistance to chemotherapy is a clinical problem in patients with infectious diseases caused

Figure 4. Fluorescence Microscopy of R6G intracellular accumulation of R6G induced by BRP-1. Upper panels (A-D) show the phase contrast microscopy and lower panels (E-H) depict fluorescence of the cells. (A) and (E) show the strain AD1234567 (negative control); (B) and (F) show the Pdr5p-overepressing strain (AD124567); (C) and (G) show the strain AD124567 incubated 2 h with FK506 (10 µM); (D) and (H) AD124567 incubated 2 h with 100µM BRP-1.

Rho

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blocking its transport mechanism as the R6G efflux of whole cells was effectively inhibited by BRP-1 at low concentrations. Thus BRP-1 is a very promising natural compound that can effectively inhibit multidrug efflux transporters such as Pdr5p and can be used to reverse MDR of cells overexpressing these efflux pumps function. We expect these results to aid the understanding of Pdr5p and possibly many other multidrug efflux pumps opening new perspectives to improve the treatment of infectious diseases in which these transporters cause treatment failure of traditional synthetic drugs.

Acknowledgments

We thank Dr. Erwin Lamping (Dental School, University of Otago/Dunedin, New Zealand) for a critical review of this manuscript. The present work was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Procad), Conselho Nacional de Desenvolvimento Científico e Tecnológico and Fundação Universitária José Bonifácio.

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Lamping E, Baret PV, Holmes AR, Monk BC, Goffeau A, Cannon RD 2010. Fungal PDR transporters: phylogeny, topology, motifs and function. Fungal Genet Biol 47: 127-142.

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*Correspondence

Antonio Ferreira-PereiraLaboratório de Bioquímica Microbiana, Departamento de Microbiologia Geral/IMPPG/CCS, Universidade Federal do Rio de Janeiro21941902 Rio de Janeiro-RJ, [email protected] Tel.: 5521 2562 6494Fax: 5521 2560 8344 #108

908

Article


Received 14 Jan 2011Accepted 23 Mar 2011Available online 2 Sep 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 908-914, Sep./Oct. 2011 Antileishmanial activity of nerolidol-rich

essential oil from Piper claussenianum

André Mesquita Marques,1 Anna Léa Silva Barreto,2 José Alexandre da R. Curvelo,2 Maria Teresa Villela Romanos,2

Rosangela M. de A. Soares,*,2 Maria Auxiliadora C. Kaplan1

1Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Brazil,2Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo Góes, Universidade Federal do Rio de Janeiro, Brazil.

Abstract: Leishmaniasis is one of the most neglected tropical diseases, representing a group of parasitic diseases worldwide spread, occurring in 88 tropical and subtropical countries. Approximately 350 million people live in areas of active transmission of leishmaniasis, with about 1-2 million estimated new cases occurring every year. More than 90% of the cutaneous cases appear in developing countries. Efforts to improve the therapeutic arsenal against leishmaniasis have led to the search for new and cheap range of drugs. In this study, the nerolidol-rich essential oil from Piper claussenianum (Miq.) C. DC., Piperaceae, was assayed on arginase activity of Leishmania amazonensis. The effect of this essential oil on arginase activity levels showed an enzyme inhibition of 62.2%. This result stimulates the scientific interest about the potential value of this plant species on neglected diseases as potential new natural product source of pharmacological interest for the treatment of leishmaniasis.

Keywords:essential oilLeishmania amazonensis nerolidolPiper claussenianum

Introduction

Leishmaniasis is one of the most neglected tropical diseases, due to few tools available for controlling and also the lack of clear criteria for the disease management. Leishmaniasis represents a group of parasitic diseases worldwide spread, occurring in 88 tropical and subtropical countries. Approximately 350 million people live in areas of active transmission of Leishmania, as many as 12 million people are believed to be currently infected, with about 1-2 million estimated new cases occurring every year throughout Africa, Asia, Europe, and the Americas directly affected by leishmaniasis. More than 90% of the cutaneous cases appear in developing countries (Desjeux et al., 2004; WHO 2007). This illness can be promoted by more than twenty species of trypanosomatids from the genus Leishmania. The parasite is transmitted by insect vectors of the genera Lutzomyia and Phlebotomus (Diptera: Psychodidae: Phlebotominae), and is able to affect animals and humans (Ashford et al., 2000). The cutaneous leishmaniasis (CL) has affl icted humankind for centuries, and represent an infection characterized by a heteroxenic cycle affecting mainly skin or mucous membranes and is distinguished by the presence of ulcerative lesions. CL may cause the impairment and destruction of mucosal

and cutaneous structures, frequently producing facial mutilation (Murray et al., 2004). Since the discovery of the fi rst class of drugs able to treat leishmaniasis (i.e., pentavalent antimonials), the search for substances with antileishmanial activity, without toxic effects, and able to overcome the emergence of drug resistant strains still remains a current goal (Santos et al., 2008). Efforts to improve therapeutic arsenal against leishmaniasis have led to the search for new and cheap range of drugs. The searching for new drugs with antileishmanial activities, including natural product compounds, have been undertaken worldwide, although problems with the side effects of the chemotherapies currently used have not yet been solved (Santin et al., 2009). Natural products have been widely employed in folk medicine to treat infectious diseases, mainly in communities with inadequate conditions of health and sanitation. Many studies have shown plants of many regions of the world containing compounds with anti-protozoal activity. It has been reported that various essential oils (EO) show inhibitory action against diverse human parasites related to neglected tropical diseases such as L. amazonensis (Marques et al., 2010; Silva et al., 2009; Ueda-Nakamura et al., 2006). In this work a nerolidol-rich essential oil from leaves of Piper claussenianum (Miq.) C. DC., Piperaceae,

Antileishmanial activity of nerolidol-rich essential oil from Piper claussenianum

André Mesquita Marques et al.


was tested against L. amazonensis enzymatic activity of arginase, in order to understand the growth inhibition mechanism of this EO over this specific parasite, which has been previously reported elsewhere (Marques et al., 2010).


Plant material and essential oil extraction

Leaves (100 g) of Piper claussenianum (Miq.) C. DC., Piperaceae, were collected in Castelo-ES in January 2010. The botanical vouchers were identified by Dr. Elsie Franklin Guimarães and kept at the Herbarium (HB) of the Rio de Janeiro Botanical Garden (JBRJ), registered under number RB 489043. The fresh and dried plant materials were submitted to hydrodistillation for 2 h in a modified Clevenger-type apparatus. The obtained essential oils (EO) were dried over anhydrous sodium sulphate, yielding 1% w/v in both studied specimens.

GC-FID analysis

The gas chromatography analyses were carried out on a GC 2010 Shimadzu with a ZB-1MS fused silica capillary column (30 m x 0.25 mm x 0.25 μm film thickness). The operating temperatures used were as follows: injector 260 oC, detector 290 oC and column oven 60 °C up to 290 oC (10 °C/min). Hydrogen at 1.0 mL min-1 was used as a carrier gas. The percentages of the compounds were obtained by GC-FID analysis.

GC-MS analysis

Qualitative analyses were carried out on a GC-QP2010 PLUS Shimadzu with a ZB-5MS fused silica capillary column (30 m x 0.25 mm x 0.25 μm film thickness) under the experimental conditions reported for GC-FID analysis. The essential oil components were identified by comparing their retention indices and mass spectra to published data and computer matching with WILEY 275 and the National Institute of Standards and Technology (NIST 3.0) libraries provided by a computer-controlled GC-MS system. The results were also confirmed by comparing the compounds’ elution order with their relative retention indices reported in the literature (Adams, 2001). The retention indices were calculated for all the volatile constituents using the retention data of linear n-alkanes C8-C24.

Nuclear magnetic resonance spectroscopy

The EO obtained was analysed by 1H and 13C NMR and recorded on a Brüker DRX 400 spectrometer. The chemical shifts were determined in CDCl3, using

TMS as the internal standard. The spectrometric data for the 1H NMR spectra are organised in agreement with the convention: chemical shift (number of protons and multiplicity), and the data for 13C NMR spectra are organised by chemical shift. The signals of the NMR analyses were compared to the literature. (Marques et al., 2010).

Biological assays

Parasite

One L. amazonensis strain (MHOM/BR/75/Josefa) originally isolated from a human CL, kindly provided by Dr Elvira Saraiva (UFRJ, Brazil) was used in this study. Infective promastigote forms were maintained by weekly transfers in 25-cm2 culture flasks with Schneider’s insect medium (SIM) (Sigma Aldrich® St. Louis/ USA), pH 7.2, supplemented with 10% fetal bovine serum (FBS) and Gentamicine (80 µg/mL) at 26 °C. By this method, a five passage strain was used in all experiments carried out.

Protozoal arginase activity

Arginase enzyme activity was measured as previously described by Kropf et al. (2005), with slight modifications. Promastigotes were incubated for 72 h at 26 °C in presence or absence of the P. claussenianum leaves essential oil IC50 (Marques et al., 2010). Inhibition concentrations previously tested (Marques et al., 2010) was used in all experiments, since the EO stock solution was obtained by a single isolation and extraction. After incubation, 108 cells were mixed with 100 µL of 0.1% Triton X-100 and underwent five cycles of 3 min in the vortex and 1 min on ice bath. Thereafter, 100 µL of 25 mM Tris-HCl pH 8.3 were added to the sample. For arginase activity measurements, 10 µL of 10 mM MnCl2 were added to each 100 µL of the lysate. The arginase was then activated by heating the sample for 10 min at 56 °C, and arginine hydrolysis was conducted by incubated the sample with 100 µL of 0.5 M L-arginine, pH 9.7 at 37 °C for 20 min. The reaction was stopped by adding 800 µL of H2SO4 (96%), H3PO4 (85%) and H2O (1/3/7, v/v/v) to the sample and mixing with 40 µL of 4% of α-isonitrosopropiophenone (dissolved in ethanol). After heating (95 °C for 30 min), urea concentration was determined by spectrometry at 540 nm wavelenght.

Interaction Leishmania-mammal host cells

To assess the influence of IC50 P. claussenianum leaves essential oil on parasite host cell interactions, parasites were treated or not for 1 h with the EO, centrifuged at 4500 rpm, at 4 °C for 3 min, washed twice

Antileishmanial activity of nerolidol-rich essential oil from Piper claussenianumAndré Mesquita Marques et al.


in 10 mM PBS pH 7.2 and ressuspended in cold sterile Dulbecco's modified Eagle's medium (DMEM) (GIBCO®) to adjust the inoculum to 5 x 106 cells. Coverslips adhered macrophages, obtained from BALB/c mice peritoneal washing, were incubated with the parasites, in a ratio of 10 parasites per host cell, in 300 μL of DMEM supplemented with 2% fetal calf serum at 37 °C 5% CO2 for an hour. After interaction, the material was fixed in methanol for 5 min, washed and stained with Giemsa 36% for 40 min. Thereafter, coverslips were dehydrated in acetone and xylene, in concentrations 90:10, 70:30, 30:70 and 0:100 respectively, and stuck to a blade 26 x 76 with resin Entellan®.

Nitrite determination

Nitrite is a stable oxidation product of nitric oxide (NO), that can be measured spectrophotometricaly by adding Griess reagent (0.1% sulfanilamide and 0.1% N-1 naphtylethylenediamine in 2.5% phosphoric acid) at the same volume of infected macrophages cultures supernatants. After 20 min at room temperature and in the dark, the absorbance of the chromophore was measured at 540 nm wavelenght. Nitrite concentrations were estimated by comparison with a standard curve prepared with sodium nitrite in DMEM supplemented with 10% fetal bovine serum (v/v). Positive control of NO production, were macrophages treated with INFγ (50 ng/mL) or treated with INFγ and the EO.

Cytotoxicity assay

To evaluate the possible essential oil’s toxicity to mammal cells, a cytotoxicity assay was performed using L929 fibroblast cells (mouse) and Raw cells (mouse macrophages). The cells were grown in Eagle’s minimum essential medium (Eagle-MEM) supplemented with 10% (v/v) fetal bovine serum, glutamine (0.03 mg/mL), garamycin (50 μg/mL), fungizone (amphotericin B) (2.5 mg/mL), NaHCO3 (0.25%) and HEPES (10 mM) (Schmidt, 1979). Cell cultures were prepared in 96-well microtiter plates (Falcon Plastics, Oxnard, CA, USA) and incubated at 37 oC in a 5% CO2 atmosphere. Cytotoxicity was evaluated by the neutral red dye-uptake method (Neyndorff et al., 1990). To determine the cytotoxicity of the P. claussenianum essential oil extracted from leaves, several concentrations of each sample (range from 40 to 0.56 mg/mL) were placed in contact with confluent L929 fibroblast cell monolayers and incubated at 37 oC in a 5% CO2 atmosphere for 24 h. After incubation, cells were incubated in the presence of 0.01% neutral red solution for 3 h at 37 oC in a 5% CO2 atmosphere. Then, the medium was removed and the cells were fixed with 4% formalin in phosphate-buffered saline, pH 7.2. The dye incorporated by the viable cells

was eluted using a mixture of methanol: acetic acid: water (50:1:49), and the dye uptake was determined by measuring the optical density (OD) of the eluate at 490 nm in an automatic spectrophotometer (ELx800TM-Bio-TeK Instruments, Inc.). The 50% cytotoxic concentration (CC50) was defined as the concentration that caused a 50% reduction in dye uptake. All experiments were performed in triplicate, and the statistical analysis of the differences between mean values obtained was done by Student’s t test. p values of 0.05 or less were considered significant.


The essential oils obtained by hydrodistillation from leaves of P. claussenianum in fresh and in dried material yielded about 1% (w/v) to both cases. Results for the qualitative and quantitative analysis of the essential oils are shown in Table 1 for the leaf volatile constituents. In total, sixteen different compounds were identified, ranging from 92.48% and 97.72% of the oils for fresh and dried leaves respectively. The oils showed a rich sesquiterpene content. Sesquiterpenes were identified as the main volatile fraction of the leaves, being Nerolidol the major constituent, ranging of up to 81.0% in EO obtained by hydrodistillation from fresh and dried samples. The structure of nerolidol was confirmed by GC-MS, 1H NMR and 13C NMR spectra (Marques et al., 2010). The oil from leaves of P. claussenianum was characterized by their high content in (E)-nerolidol (81.41; 83.29%) as well as linalool (5.20; 2.15%), γ-muurolene (1.09; 3.22%), (E)-caryophyllene (0.64; 1.40%) and γ-elemene (0.54; 0.78%) fresh and dried material, respectively. The high percentage of nerolidol in EO from leaves of P. claussenianum allowed us to perform biological assays against L. amazonensis to understand the mechanism of growth inhibition of this parasite in cutaneous leishmaniasis. The fresh leaf EO with 81.41% of nerolidol was used to perform the biological assays on arginase inhibition. Arginase is a dependent manganese metalloenzyme able to hydrolyze L-arginine to L-ornithine and urea, and its activity has been related to the synthesis of nitric oxide (NO), and therefore associated to cytotoxic process and immunological mechanisms (Kanyo et al., 1996; Da Silva et al, 2002). The arginase activity in the Tripanossomatidae family has been shown in some specific genre, and it has been used for identification and classification purposes. The genus Leishmania has a detectable arginase activity which is considered to have a role in the production of ornithine reported by Da Silva et al. (2002) and therefore essential for the growth of the parasite, since previous studies revealed that arginase null protozoans are unable to generate polyamines (Vincendeau et al.,




2003; Roberts et al., 2004). The effect of the leaf essential oil of P. claussenianum showed 62.17% inhibition in the levels of arginase activity (Figure 1). Regarding to the Leishmania-macrophage interaction mechanisms, when parasites were pretreated for one hour with the IC50 of the EO from leaves of P. claussenium, the infected macrophages percentage was reduced in 42.7% (p=0,01). When the EO was used on infected macrophages the infected macrophages percentage was reduced in 31.25% (p=0,03) (Figure 2). The production of NO from infected macrophages treated for 24 h with the IC50 of the EO was measured by the nitrite concentration in the culture supernatants, using Griess reagent. In this case there was an increase of 17.2% in NO production, an increase greater than that observed when infected cells were treated with INF-γ. Interestingly an increase of 20,5% in NO production could be seen when EO and INF-γ were used together (Figure 3).

Table 1. Identified compounds in the essential oil from leaves of P. claussenianum.

Compounds aRILit bRI Fresh H.D %

Dry H.D % Identification

α-pinene 939 936 0.17 - RI, GCMS

β-pinene 979 976 0.18 0.15 RI, GCMS

β-myrcene 991 987 - 0.18 RI, GCMS

(Z)-β-ocimene 1037 1033 0.62 - RI, GCMS

(E)-β-ocimene 1050 1043 0.85 - RI, GCMS

cis-linalool oxide 1087 1083 0.57 - RI, GCMS

linalool 1097 1101 5.20 2.15 RI, GCMS

γ-elemene 1338 1340 0.12 1.79 RI, GCMS

β-elemene 1391 1387 0.54 0.78 RI, GCMS

(E)-caryophyllene 1419 1415 0.64 1.40 RI, GCMS

α-humulene 1455 1451 0.59 1.09 RI, GCMS

γ-muurolene 1480 1477 1.09 3.22 RI, GCMS

α-selinene 1498 1497 0.35 - RI, GCMS

δ-cadinene 1523 1520 0.38 0.76 RI, GCMS

(E)-nerolidol* 1563 1563 81.41 83.29 RI, GCMS

α-eudesmol 1654 1654 0.50 2.91 RI, GCMS

% Peak sum of identified compounds 92.48 97.72

The effect of essential oil of P. claussenianum on arginase activity levels showed a strong and significant inhibition of the enzyme activity. The ability to survive and multiply within macrophages is a particular feature of several microorganisms including Trypanossoma cruzi and Leishmania. In order to sustain a chronic infection, parasites must subvert macrophage-accessory cell activities and ablate the development of protective immunity

(Alexander et al., 2002). The increase in NO production by macrophages infected and treated with the EO suggests that it may be considered a useful strategy in controlling the infection by inhibiting parasite arginase. The most important mechanism for the killing of Leishmania and for the control of leishmaniasis is the production of nitric oxide by macrophages (Holzmuller et al., 2006). These immune cells can control Leishmania infection when Th1 response is mounted and pro-inflammatory cytokines are released. This process leads to the induction of inducible nitric oxide sinthase (iNOS) and NO production. Arginine, a substrate of NO production, is modulated by both Th1 and Th2 responses in a manner that Th1 response increases conversion of arginine to NO, whereas the Th2 response promotes arginase induction (Wanasen & Soong, 2008). The inhibition of the enzymatic activity may be able to modulate cellular infection in Leishmania major and Leishmania infantum infected macrophages, while the increasing parasite arginase activity by IL-4 induction promotes Leishmania growth (Iniesta & Gomes-Nieto, 2001). Futhermore, Iniesta and colleagues in 2005 demonstrated that L. major infection leads to arginase I induction in macrophages, and that host arginase I induction supports Leishmania growth. Previous studies have demonstrated that the arginase activity in Leishmania spp. is able to modulate the nitric oxide synthase activity (Boucher et al., 1999; Wanasen & Soong, 2008). Besides, studies using models of visceral leishmaniasis by L. donovani showed that the parasite is able to modulate the activity of arginase in spleen cells of infected animals, increasing the enzyme expression and favoring the maintenance of infection (Osorio et al., 2008). Strikingly, the active role played by Leishmania arginase in diverting arginine away from the iNOS pathway was demonstrated by the increased host NO response to L. mexicana knockout for arginase infection in mice. The L. mexicana knockout for arginase infection led to an enhanced Th1 response associated to an INF-γ upregulation as well. Taken together, these responses led to a significant growth attenuation of the parasite in mice (Gaur et al., 2007). Considering the importance of the cellular signaling pathways involving the host cells and parasites of the Leishmania genus there are few studies in the literature connecting the importance of the parasite arginase inhibition and macrophage NO production. Therefore, arginase-specific inhibitors may be considered a potential alternative among new substances with pharmacological interest for the treatment of leishmaniasis. The interference in Leishmania - mammalian host cell interaction suggests that the essential oil of P. claussenianum may action on early steps of this process of interaction. Therefore this EO may be able to modulate the virulence factors not yet identified, or to block specific

aRI Lit: Literature Retention Indices; bRI: Experimental Retention Indices; H.D: Hydrodistillation; *The structure of (E)-nerolidol was also confirmed by NMR and Internal Standard FID analysis.



-0,5 0,5 1,5 2,5

Control

IC50

Con

cent

ratio

n es

sent

ial o

il

Urea concentration (µg)

Figure 1. Essential oil from leaves of P. claussenianum in arginase activity of promastigotes of L. amazonensis. The cells were incubated in the absence or presence of IC50 for 72 h at 26 °C. The enzyme activity was measured by the concentration of urea in the system (p=0.03).

A

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

% In

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ed m

acro

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0%

5%

10%

15%

20%

25%

30%

35%

40%%

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Figure 2. A. Percentage of macrophages infected with L. amazonensis pretreated or not with the IC50 of essential oil from leaves of P. claussenianum after one hour of interaction. Reduction of 42.7% infection (p=0.01). B. Percentage of macrophages infected with L. amazonensis treated or not with the IC50 of essential oil from leaves of P. claussenianum for 24 h. Reduction of 31.25% of infection (p=0.03).

0

5

10

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20

25

Sodi

um N

itrite

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)

MØ MØ+LA MØ+LA+INFγ

MØ+LA+EO MØ+LA+INFγ+EO

Figure 3. Nitrite dosages of culture supernatants of macrophages (MØ) infected with L. amazonensis and treated with the IC50 of the EO for 24 h. As controls were used uninfected and infected MØ treated with INF-γ.




receptors. This experiment was carried out with sub-inhibitory concentration of the EO in order to avoid compromising the viability of parasites. Santos et al. (2009) observed similar results when L. amazonensis promastigotes were pre-treated with protease inhibitors for HIV. The citotoxicity assay revealed that the EO extracted from leaves of P. claussenianum has no toxicity effect to fibroblasts nor macrophages cell lines in any concentration tested (ranging from 40 to 0.56 mg/mL). Macrophages and fibroblasts generate from mice were challenged with the essential oil extracted from the leaves of P. claussenianum (range 40 to 0.56 mg/mL), whose major component is (E)-nerolidol, demonstrating no cytotoxic effect on either cell lines in any concentration. This finding agrees with literature data, in fact toxic effects were not observed in vivo after nerolidol intraperitonial or topic administration in mouse (Arruda at al., 2005). In vivo studies have demonstrated that animals treated with nerolidol in concentrations up to 200 mg/kg showed no clinical signs or morbidity, and in addition, peripheral blood cells and liver taken from these animals showed no DNA damage (Pículo et al., 2010). Thus, the absence of citotoxicity in high concentrations may be considered a relevant issue pointing to the possible administration of this compound as an alternative for the management of leishmaniasis, since it is able to control protozoa growth, as well as modulate some crucial virulence factors, such as the arginase activity and the interaction mechanisms between parasites and mammal host cells.

Conclusion

The essential oil extracted from the leaves of P. claussenianum is a natural product rich in (E)-nerolidol that has a surprising biological activity, showing efficient growth inhibition of promastigote forms of L. amazonensis, and a significant inhibition on its arginase activity as well as an important enhancement in the NO levels in infected macrophages induced by this EO. Moreover, this essential oil is able to interfere with the parasite-host cell interaction, reducing the percentage of infected cells. Nevertheless, no cytotoxic effect was evidenced against any of the mammalian cells lines tested. The results presented in this study points to the P. claussenianum leaves essential oil as an important source in the searching for new alternatives for the treatment and management of cutaneous leishmaniasis.

Acknowledgments

The authors would like to thank to the National Council for Scientific and Technological Development for financial support.

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*Correspondence

Rosangela Maria de Araújo SoaresInstituto de Microbiologia Prof. Paulo de Góes, Centro de Ciências da Saúde, Bloco I, Universidade Federal do Rio de Janeiro21941-590 Rio de Janeiro-RJ, Brazil [email protected].: +55 21 2562 6711/ 2562 6513Fax: +55 21 2562 6512

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695X2011005000159

Received 6 Jan 2011Accepted 31 Jul 2011

Available online 2 Sep 2011


21(5): 915-920, Sep./Oct. 2011Antiviral activity of Ageratina havanensis and major chemical compounds from the most active fraction

Gloria del Barrio,*,1 Iraida Spengler,2 Trina García,2 Annele Roque,1 Ángel L. Álvarez,1,3 José S. Calderón,4 Francisco Parra3

1Departamento de Microbiología y Virología, Facultad de Biología, Universidad de la Habana, Cuba,2Centro de Estudios de Productos Naturales, Facultad de Química, Universidad de La Habana, Cuba, 3Instituto Universitario de Biotecnología de Asturias, Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, España, 4Instituto de Química, Universidad Nacional Autónoma de México, México.

Abstract: The antiviral activity of extracts obtained from Ageratina havanensis (Kunth) R.M.King & H.Rob., Asteraceae, against rabbit vesivirus (RaV) (Caliciviridae) and human herpes simplex viruses type 1 and 2 (HSV-1, HSV-2) (Herpesviridae) were analyzed, and the main metabolites from the most active extract were isolated and characterized. The antiviral properties were investigated by measuring the inhibition of viral-induced cytopathic effect in Vero cells. The strongest inhibitory effects were found for ethyl acetate extract from leaves (SI=5 for RaV and SI=5.4 for HSV-1). The crude ethyl acetate extract was further fractionated by chromatographic methods and the structures of isolated compounds were established through comprehensive spectroscopic analyses, including IR, 2D NMR and MS. Four flavonoids were identified: 5,4’-dihydroxy-7-methoxyflavanone (sakuranetin), 3,5,4’-trihydroxy-7-methoxyflavanone (7-methoxyaromadendrin), 4’-O-β-D-glucosyl-5,3’-dihydroxy-7-methoxyflavanone (4’-O-β-D-glucosyl-7-methoxy-eriodictyol) and 4’-O-β-D-glucosyl-5-hydroxy-7-methoxyflavanone (4’-O-β-D-glucosylsakuranetin). This is the first report on antiviral activity for Ageratina havanensis.

Keywords:Ageratina havanensis

antiviralHSVRaV

sakuranetin

Introduction

The herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) stand out because of the wide variety of clinical symptoms they cause, varying from mild self-limiting skin lesions to severe illnesses such as encephalitis and pneumonia, frequent among immune compromised patients. The relatively high toxicity of commercially-available synthetic anti herpes compounds, and the continuous spread of drug-resistant viral strains emphasize the need for new herpes inhibitors. Moreover, genital HSV lesions are associated to an increased risk of HIV acquisition (Freeman et al., 2006). On the other hand, the Caliciviridae includes many relevant human and animal pathogens, such as the human noroviruses which are the main causes of viral acute gastroenteritis outbreaks worldwide, and the rabbit hemorrhagic disease virus (RHDV), which is responsible for great

economical losses due to devastation of entire domestic rabbit populations. Therefore, the search for novel anti-caliciviral drugs also merits great attention. In addition, many caliciviruses do not replicate in cell cultures, thus making diffi cult their study. The rabbit vesivirus (RaV) is one of the few in vitro replicating caliciviruses and, for this reason, it is an excellent model to search for anti-caliciviral agents (Martin-Alonso et al., 2005). Ageratina (snakeroot) is a plant genus comprising about 250-290 perennials and rounded shrubs in the sunfl ower family, Asteraceae. Some species possess different biological properties, such as wound healing, cell proliferation stimulation and antimycotic action (Romero-Cerecero et al., 2008; Romero-Cerecero et al., 2011). Ageratina havanensis (Kunth) R. M. King & H. Robinson (common name: Havana snakeroot) is a species of fl owering shrub in this family, native of the Caribbean and Texas (Diggs et al., 1999).

Antiviral activity of Ageratina havanensis and major chemical compounds from the most active fractionGloria del Barrio et al.


Since ancient times, plants have been widely used in traditional medicine by human populations. Chromones and flavonoids highlight among the most important naturally-occurring antiviral compounds (Torres et al., 2002). Flavones, flavonols and flavonoid glucosides were previously isolated from Ageratina havanensis (Yu et al., 1987). Although there is no evidence for antiviral use of this plant, other species in the Asteraceae have been used in folk medicine to treat gastrointestinal disorders, kidney affections and microbial infections (Romero-Cerecero et al., 2008). The present work reports for the first time the antiviral activity of A. havanensis against HSV-1, HSV-2 and RaV, as well as the isolation and characterization of major compounds from the most active extract.


Plant material

Ageratina havanensis (Kunth) R.M.King & H.Rob., Asteraceae (leaves and stems), was collected in Alamar (Havana) in November 2009 and identified by Prof. Iralys Ventosa from Instituto de Ecología y Sistemática (Havana, Cuba), where a voucher specimen was deposited (Ref. HAC-42498). The fresh leaves and stems were separated and oven-dried at 40 ºC, ground to fine powder (yielding 145 g and 284 g, respectively) and stored in airtight bottles.

Extraction and isolation

The powdered oven-dried plant samples (from separate leaves and stems) were soaked in n-hexane and extracted with ethanol (EtOH). The EtOH extracts were concentrated by reduced pressure to yield syrup, further dissolved in water and extracted with ethyl acetate (EtOAc) and n-butanol (n-BuOH) in a separating funnel. The resulting extracts were concentrated under reduced pressure in order to obtain the final EtOAc extracts (leaves yielded 11.12 g and stems yielded 1.55 g) and n-BuOH extracts (leaves yielded 28.35 g and stems yielded 4.80 g). The EtOAc extracts were further fractionated with an n-hexane/chloroform/methanol (2:1:1) mixture through Sephadex LH-20-packed column to give eleven fractions (A-K). Fractions I and J were purified by preparative thin-layer chromatography with a dichloromethane/methanol (9.7:0.3) mixture to give the compounds 1 and 2. Fraction E was chromatographed on silica gel using dichloromethane/methanol mixtures of increasing polarities. Compound 3 was obtained with 10% dichloromethane/methanol. The G fraction was further fractionated by column chromatography on silica gel with n-hexane/chloroform/methanol (2:3:0.5) to yield compound 4.

Compound 1: 5,4’-dihydroxy-7-methoxyflavanone (sakuranetin): white solid, m.p. 180-181 ºC, IR (KBr, cm-1): 3450; 2990; 1640; 1605, 1450, 1370; 1280, 1260; 1090. MS 70eV (m/z): 302 (M+); 273 (M+-29); 167 (A1 + H)+; 136 (B1 + H2O) +.

Compound 2: 3,5,4’-trihydroxy-7-methoxyflavanone (7-methoxyaromadendrine): white solid, m.p. 154-156 ºC, IR (KBr, cm-1): 3530; 3452; 3356; 3144; 1622; 1512; 1447; 1206; 1071. MS 70eV (m/z): 286 (M+); 285 (M+-1); 167 (A1 + H)+; 120 (B1 + 2H) +.

Compound 3: 4’-O-β-D-glucosyl-5,3’-dihydroxy-7-methoxyflavanone: white solid, m.p.206-207 ºC, IR (cm-1): 3200, 2840, 1668, 1580, 1370.

Compound 4: 4’-O-β-D-glucosyl-5-hydroxy-7-methoxyflavanone: white solid, m.p. 201- 204 ºC, IR (cm-1): 3420, 2940, 1625, 1490, 1380, 1260, 1200, 1140. HR-MS (FAB+) (m/z): [M+H]+: 449, C22H24O10.

Structure elucidation procedures

The melting points (m.p.) were determined on a Reichert-Thermovar apparatus. The optical rotations were determined with a Perkin–Elmer 341 polarimeter. IR spectra were recorded on a Mattson Genesis spectrophotometer, series FT-IR. 1H and 13C NMR measurements were obtained on Varian Inova 400 and 600 MHz NMR spectrometers with TMS as the internal reference. Mass spectra were recorded on Brucker Apex 70 eV. TLC was performed on 0.2 mm-thick Kiesegel 60 F254 layers (Merck). Sephadex LH-20 and Silicagel (Merck) were used for column chromatography.

Cytotoxic assays

Vero cells (ATCC, CCL81) were seeded in 96-well plates at a density of 2x104 cells/well, and incubated at 37 ºC in a 5% CO2 atmosphere for 48-72 h, until 90% or greater confluence of the monolayers was reached. The cells were then incubated with increasing concentrations of the extracts at 37 ºC in a 5% CO2 atmosphere, for 72 h. Afterwards, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added (final concentration 0.5 mg/mL) and the plates were further incubated for 4 h to allow formazan production. The solid purple precipitate was dissolved with dimethylsulphoxide (Sigma) and the absorbance at 570 nm was measured using a µQUANT Spectrophotometer (Bio-Tek Instruments) with a reference wavelength of 620 nm. The concentration reducing cell viability by 50% (cytotoxic mean concentration, CC50) was calculated by regression analyses, as previously described (Mosmann, 1983).

Antiviral activity of Ageratina havanensis and major chemical compounds from the most active fraction

Gloria del Barrio et al.


Antiviral assays

Ninety-six well plates containing confluent cell monolayers were pre-incubated for 1 h with increasing non-cytotoxic concentrations of the plant extracts, with a replicate number of six. Afterwards, the cells were infected with HSV-1, HSV-2 or RaV (10 TCID50). The plates were incubated at 37 ºC in a 5% CO2 atmosphere and observed daily for viral-induced cytopathic effect (CPE) using a light microscope. The extract was present until the end of the experiment. When CPE was observed in all virus control wells, the percentage of wells with CPE was determined for each treatment concentration, as previously reported (Al Jabri et al., 1996). Acyclovir (ACV) at concentrations varying from 0.1-10 µg/mL served as positive control during HSV evaluations. A positive control for RaV is not yet available.

Statistics

Statistical analyses were performed using the Statistica 6.1 software. Differences among antiviral activity of extracts were determined using Student unpaired t-tests. Values of p<0.05 were considered indicative of statistical differences. The concentration reducing cell viability by 50% (mean cytotoxic concentration, CC50) and that reducing viral-induced CPE by 50% (antiviral effective mean concentration, EC50) were calculated by regression analyses using the dose-response curves (not shown) generated from the experimental data. A selectivity index (SI) was calculated for each test sample by dividing its CC50 by the corresponding EC50 value.


Cytotoxic effects and antiviral activity of Ageratina havanensis

The cytotoxic effects of samples on Vero cells

were determined by the MTT colorimetric assay, as previously described (Mosmann, 1983). The influence of A. havanensis extracts on cell viability was indirectly measured as a function of the ability of mitochondrial enzymes (active in viable cells) to convert MTT in a purple solid formazan precipitate. All extracts exhibited concentration-dependent cytotoxic effects on Vero cells to different extents. Regarding the source of extracts, those obtained from leaves showed a higher toxicity when compared to their homologues from stems. On the other hand, when comparing extracts from the same plant organ, it should be mentioned that the n-BuOH extract was the most toxic, the EtOH the most innocuous, and the EtOAc exerted intermediate toxic effects (Table 1). The CC50 values found for A. havanensis extracts on Vero cells correlated well with the morphological changes recorded during microscopic examination of treated cultures. These alterations were gradual, concentration-dependent and comprised cell rounding, presence of cytoplasmic inclusions and loss of monolayer confluency. The antiviral activity of extracts at non-cytotoxic concentrations was assessed by measuring their protective effects on infected Vero cells (Al Jabri et al., 1996; Cann, 1999; del Barrio & Parra 2000; Álvarez et al., 2011). SI values greater than 2 were considered indicative of specific antiviral activity (Tsuchiya et al., 1985; De Clercq, 1993). The first samples to be evaluated were the EtOH extracts from leaves and stems, since these were first generated during the extraction methodology. In view of the results obtained in this antiviral screening, we proceeded the fractionation by means of liquid-liquid partition chromatography with EtOAc and n-BuOH. The SI values increased with fractionation, as we should expect if the purification of antiviral compounds succeeded. The strongest antiviral activity was recorded for the EtOAc extract from leaves against HSV-1 (SI=5.4) and RaV (SI=5). The EtOAc extract from stems was also active against RaV (SI=3.3). The anti-herpetic activity of this extract remains to be investigated since the maximum

Table 1. Cytotoxic effects and antiviral activity of extracts from Ageratina havanensis leaves and stems.

ExtractCytotoxicity Antiviral activity

CC50aHSV-1 HSV-2 RaV

EC50a SI EC50 SI EC50 SIEtOHl 2834±448 809.9±59.6 3.5 1050±42.9 2.7 1153±49.3 2.5EtOAcl 1670±0.2 311.6±10.1 5.4 >450 <4 334.1b 5.0

n-BuOHl 404.9±43.5 >225 <2 128.1±22.8 3.2 174.2±14.5 2.3EtOHs 5685±117 2628±103 2.2 2614±158 2.2 1307±206 4.3EtOAcs 2270±99.9 >450 <5 >450 <5 684.0b 3.3

n-BuOHs 457.4±28.1 240.9±5.7 1.9 145.4±22.1 2.8 147.9±3.67 3.1ACV >1000 0.9±0.2 >1000 0.7±0.1 >1000 - -

Data are presented as the mean value of two independent experiments and their standard deviations. aCC50 and EC50 data are expressed as µg/mL. bValues from a single experiment. lExtract from leaves, sextract from stems.



concentration tested against HSV-1 and HSV-2 did not reach the EC50 value (SI<5). On the other hand, no significant antiviral activity was observed for the n-BuOH extracts against HSV-1 (SI<2). However these extracts were indeed active against the rest of viruses, with SI ranging from 2.3 to 3.2 (Table 1). Several authors consider that the antiviral activity of compounds occurring in plants take place via a direct inactivating (virucidal) effect over the extracellular virus particle (Sydiskis et al., 1991). This may be especially true for enveloped viruses (e.g. HSV) which are more sensitive to physic and chemical disrupting agents. However, being RaV a non-enveloped and small virus, and consequently more resistant to external environmental agents, the antiviral activity found here is more attractive because A. havanensis compounds could hamper specific targets within the virus replication cycle, thus deserving further research. Based on its high SI values, the leaves EtOAc extract was selected for further purification of metabolites, as described below.

Structural data of compounds isolated from A. havanensis leaves ethyl acetate extracts

In order to isolate and characterize the major compounds in A. havanensis, a combination of column chromatographic techniques (Sephadex LH-20, silica gel and preparative thin-layer chromatography) was used. The structures of the isolated compounds were elucidated by physical and spectroscopic data measurements (m.p., IR, 1H NMR, 13C NMR, 2D NMR and MS) and by comparing the obtained data with previously published values.

The 1H and 13C-NMR spectroscopic data obtained for these compounds (not shown) are consistent with previously reported values: Compound 1 was identified as 5,4’-dihydroxy-7-methoxyflavanone (sakuranetin) (Domínguez & De la Fuente, 1973); compound 2 corresponded to 3,5,4’-trihydroxy-7-methoxyflavanone (7-methoxyaromadendrine) (Agrawal, 1989); compound 3 was identified as 4’-O-β-D-glucosyl-5,3’-dihydroxy-7-methoxyflavanone (4’-O-β-D-glucosyl-7-methoxy-eriodictiol), previously isolated from the plant Aerva persica (Ahmed et al., 2006), but this is the first report for this glucoside in the Asteraceae; and compound 4 matched data for 4’-O-β-D-glucosyl-5-hydroxy-7-methoxyflavanone (4’-O-β-D-glucosyl-7-methoxysakuranetin) (Zhang et al., 2006), which is a new flavonoid in the Ageratina genus. Antiviral activities have been previously reported for several naturally-occurring flavonoids (Kaul et al., 1985; Kwon et al., 2010; He et al., 2011). It is known that some of them such as epigallocatechin gallate, epigallocatechin and quercetin, inhibited virus-induced cytopathic effects and reduced virus-plaque yields in Vero cells infected with HSV-1 and HSV-2 (Lyu et al., 2005). The antiherpetic activity of several flavonoid rich extracts from different plants has also been shown (Goncalves et al., 2001; Fernandez-Romero et al., 2003; Silva et al., 2010). Regarding their mechanism of action against herpes virus, it has been reported that flavonoids could have virucidal effects (Kaul et al., 1985; Hayashi et al., 1997; Goncalves et al., 2001) or act as inhibitors of the viral attachment and penetration (Isaacs et al., 2000; Silva et al., 2010).

O

O

OH

OH

H3CO

1

O

O

OH

OH

H3CO

2

OH

O

O

O

OH

H3CO

3 R = OH4 R = H

O

OH

OHOH

HOR

Antiviral activity of Ageratina havanensis and major chemical compounds from the most active fraction

Gloria del Barrio et al.


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Goncalves JL, Leitao SG, Monache FD, Miranda MM, Santos MG, Romanos MT, Wigg MD 2001. In vitro antiviral effect of flavonoid-rich extracts of Vitex polygama (Verbenaceae) against acyclovir-resistant herpes simplex virus type 1. Phytomedicine 8: 477-480.

Hayashi K, Hayashi T, Otsuka H, Takeda Y 1997. Antiviral activity of 5,6,7-trimethoxyflavone and its potentiation of the antiherpes activity of acyclovir. J Antimicrob Chemother 39: 821-824.

He W, Li LX, Liao QJ, Liu CL, Chen XL 2011. Epigallocatechin gallate inhibits HBV DNA synthesis in a viral replication - inducible cell line. World J Gastroenterol 17: 1507-1514.

Isaacs CE, Xu W, Pullarkat RK, Kascsak R 2000. Retinoic acid reduces the yield of herpes simplex virus in Vero cells and alters the N-glycosylation of viral envelope proteins. Antiviral Res 47: 29-40.

Kaul TN, Middleton E Jr, Ogra PL 1985. Antiviral effect of flavonoids on human viruses. J Med Virol 15: 71-79.

Kwon HJ, Kim HH, Ryu YB, Kim JH, Jeong HJ, Lee SW, Chang JS, Cho KO, Rho MC, Park SJ, Lee WS 2010. In vitro anti-rotavirus activity of polyphenol compounds isolated from the roots of Glycyrrhiza uralensis. Bioorg Med.Chem., 18, 7668-7674.

Lyu SY, Rhim JY, Park WB 2005. Antiherpetic activities of flavonoids against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro. Arch Pharm Res 28: 1293-1301.

Martin-Alonso JM, Skilling DE, Gonzalez-Molleda L, del BG, Machin A, Keefer NK, Matson DO, Iversen PL, Smith AW, Parra F 2005. Isolation and characterization of a new Vesivirus from rabbits. Virology 337: 373-383.

Mosmann T 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63.

Romero-Cerecero O, Zamilpa A, Jimenez-Ferrer JE, Rojas-Bribiesca G, Roman-Ramos R, Tortoriello J 2008.

Further antiviral evaluations and mechanistic experiments are needed in order to elucidate the mechanism of action of metabolites isolated from A. havanensis.

Concluding remarks

The search for antiviral compounds among medicinal plants is actually a retrospective study aiming to establish the rationale for the use of products commonly employed by folk. The bioactivity-guided purification approach, partially followed in this work, has demonstrated to be an effective strategy to look for novel drugs. The outlined anti-HSV activities found for Ageratina havanensis suggest that EtOAc extract or derived compounds can be considered as phytopharmaceutical candidates for the treatment of herpetic infections. The results found using the in vitro anti-RaV model may also contribute to gain knowledge on calicivirus antiviral strategies. This is the first report on Ageratina havanensis antiviral activity. Further experiments are required to investigate the mechanism of action and the molecules responsible for the inhibition of specific viral targets.

Acknowledgements

This study was partially funded at F. Parra laboratory by AECID PCI grant D/030639/10. Financial support (grant: UNOV-10-BECDOC) given to Á. L. Álvarez by the University of Oviedo, is acknowledged.

References

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Al Jabri AA, Wigg MD, Oxford JS 1996. Initial in vitro screening of drug candidates for their potential antiviral activities. In: Mahy BWJ, Kangro HO (Eds.), Virology Methods Manual. Academic Press, p. 303-305.

Álvarez AL, Habtemariam S, Juan-Badaturuge M, Jackson C, Parra F 2011. In vitro anti HSV-1 and HSV-2 activity of Tanacetum vulgare extracts and isolated compounds: an approach to their mechanisms of action. Phytother Res 25: 296-301.

Cann A 1999. Antiviral testing. In Cann A (Ed.) Virus culture: a practical approach, New York: Oxford University Press, p. 201-219.

De Clercq E 1993. Antivirals for the treatment of herpesvirus infections. J Antimicrob Chemother 32 Suppl A: 121-132.

del Barrio G, Parra F 2000. Evaluation of the antiviral activity



Double-blind clinical trial for evaluating the effectiveness and tolerability of Ageratina pichinchensis extract on patients with mild to moderate onychomycosis. A comparative study with ciclopirox. Planta Med 74: 1430-1435.

Romero-Cerecero O, Zamilpa-Alvarez A, Ramos-Mora A, Alonso-Cortes D, Jimenez-Ferrer JE, Huerta-Reyes ME, Tortoriello J 2011. Effect on the wound healing process and in vitro cell proliferation by the medicinal mexican plant Ageratina pichinchensis. Planta Med 77: 979-983.

Silva IT, Costa GM, Stoco PH, Schenkel EP, Reginatto FH, Simoes CM 2010. In vitro antiherpes effects of a C-glycosylflavonoid-enriched fraction of Cecropia glaziovii Sneth. Lett Appl Microbiol 51: 143-148.

Sydiskis RJ, Owen DG, Lohr JL, Rosler KH, Blomster RN 1991. Inactivation of enveloped viruses by anthraquinones extracted from plants. Antimicrob Agents Chemother 35: 2463-2466.

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filifolium. Bol Soc Chil Quím 47: 259-263.Tsuchiya Y, Shimizu M, Hiyama Y, Itoh K, Hashimoto Y,

Nakayama M, Horie T, Morita N 1985. Antiviral activity of natural occurring flavonoids in vitro. Chem Pharm Bull 33: 3881-3886.

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Zhang X, Hung TM, Phuong PT, Ngoc TM, Min BS, Song KS, Seong YH, Bae K 2006. Anti-inflammatory activity of flavonoids from Populus davidiana. Arch Pharm Res 29: 1102-1108.

*Correspondence

Gloria del BarrioDepartamento de Microbiología y Virología, Facultad de Biología, Universidad de la HabanaC/ 25, 455 e/ J e I, Vedado, 1040. La Habana, [email protected]. +53 7 836 7941Fax: +53 7 832 1321

921

Article


695X2011005000148

Received 20 Dec 2010Accepted 19 Jun 2011



21(5): 921-927, Sep./Oct. 2011Assessment of estrogenic, mutagenic and antimutagenic activity of nemorosone

Mariana S. Camargo,1 Soraya D. Varela,1 Ana Paula S. de Oliveira,1 Flávia Ap. Resende,1 Osmay Cuesta-Rubio,2 Wagner Vilegas,3 Eliana A. Varanda*,1

1Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Brazil,2Instituto de Farmacia y Alimentos, Universidad de La Habana, Cuba,3Instituto de Química, Universidade Estadual Paulista, Brazil.

Abstract: Currently, a wide range of research involving natural products is focused on the discovery of new drugs in many different therapeutic areas. A great number of the synthetic compounds on the market were derived from natural products, especially plants. Nemorosone is the major constituent of the floral resin of Clusia rosea Jacq., Clusiaceae, and in Cuban propolis. In vitro studies have shown cytotoxic activity in this substance against various tumor cell lines, including those resistant to various cytotoxic drugs, whereas it has low cytotoxicity to non-tumoral cells. Therefore, in order to characterize the biological activity of nemorosone, a substance with potential antitumor activity, and in view of preclinical testing of the toxicity of drug candidate compounds, the main aim of this study was to determine the mutagenic and antimutagenic activity of nemorosone by the Ames test, using the strains TA97a, TA98, TA100 and TA102 of Salmonella typhimurium. Secondly, to characterize the estrogenic activity in an experimental recombinant yeast model (Recombinant Yeast Assay) mutagenic activity was observed at in any of the concentrations in any of the test strains. To evaluate the antimutagenic potential, direct and indirect mutagenic agents were used: 4 nitro-o-phenylenediamine (NPD), mitomycin C (MMC) and aflatoxin B1 (AFL). Nemorosone showed moderate antimutagenic activity (inhibition level 31%), in strain TA100 in the presence of AFL, and strong antimutagenic activity in TA102 against MMC (inhibition level 53%). Estrogenic activity was observed, with an EEq of 0.41±0.16 nM at various tested concentrations.

Keywords:Ames test

antimutagenic activityestrogenic activity

nemorosone

Introduction

Nemorosone is found in the resin and latex of plants of Clusia (fam. Clusiaceae, syn. Guttiferae) species (Lokvam et al., 2000) and it is the major constituent of Clusia rosea Jacq. floral resins (Cuesta-Rubio et al., 2001; Popolo et al., 2011) and brown Cuban propolis (Cuesta-Rubio et al., 2002; Cuesta-Rubio et al., 2007; Pardo-Andreu et al., 2011). This compound belongs to the class of polycyclic polyisoprenylated benzophenones, a group of acylphloroglucinol derivates wich are characteristic secondary metabolites of the Guttiferae family (Pagano et al., 2008). The cytotoxicity of nemorosone in several human cancer cell lines has been reported in the past years (Cuesta-Rubio et al., 2002; Diaz-Carballo et al., 2003; Popolo et al., 2011). This compound showed cytotoxic activity in vitro against a range of tumor cell lines, such as breast, colon, ovary, liver and lung

carcinoma (including both wild type and chemotherapy-refractory) and it presents low cytotoxicity against normal cell lines (Diaz-Carballo, et al. 2003; Diaz-Carballo et al.,2008; Popolo et al., 2011). The National Cancer Institute (USA), which considers that a pure compound is active when its IC50 is lower than 4 µg/mL (Cordell et. al., 1993); nemorosone showed IC50 values lower than 3.6 µg/mL against several cancer cell lines (Cuesta-Rubio et al. 2001). Antineoplastic agents of plant origin, such as vinblastine, vinorelbine, etoposide, teniposide and others, are currently used as parts of anticancer treatment protocols (Bulanas & Kinghorn, 2005). Phytochemicals have very good prospects as anticancer agents on account of their great chemical diversity and unlimited potential for rational modification (Diaz-Carballo et al., 2008). Compounds capable of provoking mutations are found in our daily diet, drinks and medication

Assessment of estrogenic, mutagenic and antimutagenic activity of nemorosoneMariana S. Camargo et al.


as well as in polluted air and water. It is known that mutations are a major factor for the onset of cancer. However, chemoprevention, that is the prevention of cancer development by chemical substances that act as antimutagenic agents, with the capacity to interact with mutagenic compounds or their metabolites and reduce their effects, is one possible way to prevent cancer (Nogueira et al., 2006; Lira et al., 2008; Chen et al., 2011). In recent years, endocrine disrupting chemicals (EDC) have become a major issue in the field of environmental science, owing to their ability to interact with human estrogen receptors, thus interfering with the endocrine system (Crews et al., 2000; Brix et al., 2010). Epidemiological studies and animal experiments have shown that estrogen can have carcinogenic properties. Studies to clarify the molecular mechanisms of carcinogenesis by estrogen suggest that estrogen causes carcinogenic effects by combining genotoxicity and stimulation of cell proliferation (Bhat et al., 2003; Cavalieri et al., 2000). The main aim of this study was to assess the mutagenic and antimutagenic activity of nemorosone by the Salmonella reversion assay, which is widely used for the detection of mutagenic and antimutagenic agents, especially those present in plant extracts. To elucidate further the biological activity of this compound, its estrogenic activity was also tested by recombinant yeast assay.


Chemicals

Dimethylsulfoxide (DMSO), nicotinamide adenine dinucleotide phosphate sodium salt (NADP), D-glucose-6-phosphate disodium salt, magnesium chloride, L-histidine monohydrate, D-biotin, sodium azide (SAZ), 2-anthramine (2-ANTR), 4-nitro-o-phenylenediamine (NPD), mitomycin C (MMC), aflatoxin B1 (AFL), 17ß-estradiol, Triton X-100, SDS 10%, 2-mercaptoethanol, 4-methylumbelliferyl ß-D-galactoside were purchased from Sigma Chemical Co (St. Louis, USA). Oxoid Nutrient Broth No. 2 (Oxoid, England) and Difco Bacto Agar (Difco, USA) were used as bacterial media.

Plant material

Flowers of Clusia rosea Jacq., Clusiaceae, were collected in Havana (Cuba) in September 2009 and identified by Dr. Victor Fuentes Fiallo. A voucher specimen (No. 9576) was deposited in the Herbarium of La Estacion Experimental de Plantas Medicinales de Guira de Melena.

Nemorosone was extracted from the floral resins of C. rosea and isolated as previously reported (Cuesta-Rubio et al., 2001) In brief, nemorosone was crystallized from floral resins of Clusia rosea employing a mixture of EtOH-H2O. The product was subject to a VLC on silica gel with C6H12-EtOAc mixtures to purify it to homogeneity. It was identified through 1-D and 2-D NMR experiments, and its purity was verified by HPLC-DAD and HPLC-MS. Ames mutagenicity assay

The Salmonella mutagenicity assay was performed by the pre-incubation method, for 20-30 min with Salmonella typhimurium strains, TA97a, TA98, TA100 and TA102, with and without metabolic activation (Maron & Ames, 1983). The metabolic activation mixture (S9 mix) was freshly prepared before each test from an Aroclor-1254-induced rat liver fraction purchased (lyophilized) from Moltox Molecular Toxicology Inc. (Boone, NC). S. typhimurium strains were kindly provided by Dr. B. Ames, University of California, Berkeley, CA, USA. The benzophenone was diluted in dimethylsulfoxide and tested at concentrations of 5, 10, 20 and 30 µg/plate. The chosen sublethal concentrations were based on the toxicity of the benzophenone to the strains. Toxicity was apparent either as a reduction in the number of his+ revertants, or as an alteration in the auxotrophic background. The various concentrations of nemorosone to be tested were added to 500 μL of buffer (pH 7.4) and 100 µL of S. typhimurium inoculum. After that, 2 mL of top agar was added to the mixture and poured on to a plate containing minimal agar. The plates were incubated at 37 °C for 48 h and the his+ revertant colonies were counted manually. The influence of metabolic activation was tested by replacing the buffer with 500 µL of S9 mixture (4%). All experiments were performed in triplicate. The standard mutagens used as positive controls in experiments without S9 mix were 4-nitro-o-phenylenediamine (NPD-10 µg/plate) for TA98 and TA97a, sodium azide (2.5 µg/plate) for TA100 and daunomycin (3 µg/plate) for TA102, while 2-anthramine (0.63 µg/plate) was used in the experiments with metabolic activation for TA98, TA100 and TA97a and 2-aminofluorene (10 µg/plate) for TA102. The statistical analysis was performed with the Salanal computer program using the Bernstein model (Bernstein et al., 1982). The data (revertants/plate) were assessed by analysis of variance (ANOVA), followed by linear regression. The mutagenic index (MI) was also calculated for each dose, as the average number of revertants per plate divided by average number of revertants per plate of the negative (solvent) control. A sample was considered positive when MI≥2 for at least



one of the tested doses and if the response was dose dependent (Varella et al., 2004; Santos et al., 2010).

Ames antimutagenicity assay

In accordance with the pre-incubation method, developed by Maron & Ames (1983), various concentrations of nemorosone were mixed with 100 µL of S. typhimurium inoculum and the mutagenic agent (NPD, AFL or MMC), and incubated at 37 °C for 20-30 min. After this, 2 mL of top agar was added, supplemented with traces of histidine and biotin, and the content of each tube was lightly homogenized and poured onto a plate of minimal glucose agar. After the top agar solidification, the plates were incubated for 48 h at 37 °C, and the number of revertant colonies per plate was counted. The entire assay was performed in triplicate. The percentage of mutagenicity inhibition was calculated as in Tachino et al. (1994), where:

The antimutagenic effect was considered negligible when a value lower than 25% was obtained, moderate when a value between 25% and 40% was obtained and strong at values greater than 40% (Neigi et al., 2003). Cell viability was also determined in each antimutagenesis experiment to assess the bactericidal potential of the mutagens. The responses were considered toxic when sample survival was less than 60% of the total observed for the negative control (Vargas et al., 1993).

Recombinant yeast assay (RYA)

Yeast strain BY4741 (MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0) from Euroscarf (Frankfurt, Germany) was transformed with plasmids pH5HE0 and pVitBX2, as described elsewhere (Garcia-Reyero et al., 2001). Expression plasmid pH5HE0 contains the human estrogen hormone receptor HE0 (Green & Chambon, 1991) cloned into the constitutive yeast expression vector pAAH5. The reporter plasmid pVITB2x contains two copies of the pseudopalindromic estrogen responsive element ERE2 from X. laevis vitellogenin B1gene (5’AGTCACTGTGACC-3’) inserted into the unique KpnI site of pSFLΔ-178k (Garcia-Reyero et al., 2005). In brief, transformed yeast cells were first grown overnight in non-selective medium (YPD) at 30 °C.

Next, they were grown overnight in minimal medium (6.7 g/L yeast nitrogen base without amino acids, Difco, Basel, Switzerland; 20 g/L glucose, supplemented with 0.1 g/L of prototrophic markers as required). The final culture was adjusted to an optical density (OD) of 0.1 and distributed in a siliconized 96-well polypropylene microtiter plate. Aliquots of 10 μL nemorosone solution at a final concentration giving 15 µg/well were added to 90 μL of yeast culture and these initial inoculum were used for subsequent serial dilutions (1:10, 1:30, 1:90, 1:270 and 1:810). Positive controls were made by adding 17ß-estradiol at a final concentration of 10 nM. For a toxicity control, 10 nM 17ß-estradiol was added to a sample with a dilution factor of 1:30. Plates were incubated for 6 h at 30 °C under mild shaking. After incubation, 50 μL of the yeast cells lysis reagent Y-PERTM (PierceTM, Rockford, IL, USA) was added to each well and further incubated at 30 °C for 30 min. Finally, 50 μL of assay buffer was added to the lysed cells. The assay buffer was prepared by mixing 100 mL Z-buffer, 1 mL Triton X-100, 1 mL 10% SDS, 70 μL 2-mercaptoethanol and 21 mg 4-methyl umbelliferyl-ß-D-galactoside. Z-Buffer is a mix of: 60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl and 1 mM MgSO4, pH 7.0. After brief centrifugation, plates were read in a spectrofluorometer (Tecan SpectraFluor Plus), set at 355 nm excitation and 460 nm emission. Fluorescence was recorded for 20 min (one measurement per min); ß-galactosidase activity was calculated as rate of the increase in arbitrary fluorescence units per min, using standard linear regression methods. Estrogenicity values are reported as nM concentration (in the sample) equivalent to 17ß-estradiol (EEQ). These values were calculated by adjusting ß-galactosidase values from serial dilutions of each sample to the Hill equation by nonlinear methods (Noguerol et al., 2006). The entire assay was performed on four replicates.


The explosive growth of phytotherapy has created a new awareness of the potential value of natural products as anticancer agents (Diaz-Carballo et al., 2003). It may be hoped that, if nemorosone can be developed into a clinical drug in the future, it would prove valuable in both first and second-line therapies (Diaz-Carballo et al., 2008). Tumor cells possess the characteristic of proliferating much faster than normal cells, so cell proliferation is used as one of the targets for the development of chemotherapeutic agents (Green et al., 2011). These drugs however, when administered to patients, may kill normal cells and thus result in debilitating side effects (Keyomarsi & Pardee, 2003;

100x

inhibitor)(without plate

revertants inducedinhibitor)(with plate

revertants induced

1Inhibition % −=



Brumlik et al., 2008). Most anti-tumoral agents are designed to action cell proliferation (Green et al., 2011) and apoptosis induction (Peng et al., 2003). Drugs inhibit DNA synthesis by two mechanisms that are generally associated: the drug either binds to DNA by intercalation and stops the replication (Liu et al., 2011); or interferes directly with molecules required for DNA polymerization and/or initiation of replication (Pommier et al., 1998; Bruning & Mylonas et al., 2011). The ability of such drugs to intercalate into DNA can induce mutations in normal cells (Hoffmann et al., 2003). The accumulation of several random mutations can lead to aberrations in the cells and conversion of non-carcinogenic cells into carcinogenic cells (Ferguson & Denny, 1995; Raynaud et al., 2008). Thus it is essential that chemotherapeutic drugs be carefully tested not only for anticancer or antitumor activity, but also for their potential mutagenicity (Narayan et al., 2005). The Ames test is used worldwide as an initial screen to determine the mutagenic potential of new chemicals and drugs. The test is also used to furnish data for submission to regulatory agencies, for the registration or acceptance of many chemicals, including drugs and biocides. International guidelines have been developed for use by corporations and testing laboratories, to ensure uniformity of testing procedures (Mortelmans & Zeiger, 2000). In this study, the mutagenicity of nemorosone was assessed by the Ames test, using four different concentrations of the compound and four bacterial strains (Salmonella typhimurium TA97a, TA98, TA100 and TA102), each strain carrying different mutations in various genes in the histidine operon. A metabolic activation system (S9 mix) was added to S. typhimurium during the assay to metabolize the nemorosone by the cytocrome P450, enzymes extracted from rat liver. The results show that nemorosone did not induce any increase in the number of revertant colonies, indicating

the absence of mutagenic activity. The results for mutagenic activity of nemorosone are presented in Table 1, showing the number of revertants/plate, the standard deviation and the mutagenic index (MI) after treatment with this benzophenone. The mutagenic index (MI) was not higher than 2 at any tested concentration. Given that oxidative stress is strongly implicated in the toxicity of chemotherapy, much effort has been focused on the research of diverse antioxidants as potential chemoprotective agents. Tzanova et al. (2009) demonstred the low toxicity of benzophenones to three cell lines and potent destruction of reactive oxygen species generated by tert-butyl hydroperoxide (tBHP). Interestingly, one of the investigated benzophenones was shown to protect non-cancerous cells against tBHP-induced death. The antimutagenic activity of nemorosone was also assessed by the Ames test and the results are displayed in Table 2. The benzophenone was tested in association with direct (NPD and MMC) and indirect mutagens (AFL) using strains TA98, TA102 and TA100, respectively. When strain TA98 was used in association with NPD was used, no reduction in the number of revertant colonies was observed. For strain TA100, a moderate protective effect was observed when tested with AFL (31% inhibition). However, in strain TA102, nemorosone showed a strong protective effect when tested with MMC (53% inhibition). Mitomycin C is a well known anti-tumor drug whose genotoxic effects in non-tumor cells are of special significance, owing to the possibility that it may induce secondary tumors in cancer patients (Aydemir et al., 2005). It is quite possible that plants and their components may modulate the genotoxicity of anticancer drugs and thus may reduce the chances of cancer patients developing secondary tumors (Siddique et al., 2009). The present results show that nemorosone can reduce the mutagenic damage of one anti-cancer drug therapy (mitomycin C) reducing the probability of secondary tumors being induced in the cancerous patients treated with this drug.

Table 1. Mutagenic activity expressed as the mean and standard deviation of the number of revertants and mutagenic index (in brackets) in strains TA98, TA100, TA102 and TA97a exposed to nemorosone at various doses, with (+S9) or without (-S9) metabolic activation.

Treatment µg/plate

Revertants/plate in S. typhimurium strains

TA98 TA100 TA102 TA97a

-S9a +S9b -S9c +S9b -S9d +S9e -S9a +S9b

DMSO 32.3±3 33.7±2.3 214.7±14.3 166±10 196.67±11.5 285±67 123.2±6 221.3±2.3

5 42±2(1.2) 37.7±3.5(1.1) 183.7±14.1(0.8) 163±9.5(1) 176±15.7(0.9) 291±35(1) 112.4±18(0.9) 243.7±3.5(1.1)

10 35.7±8(1.1) 35.7±3.2(1.1) 226±11.5(1.2) 200±4(1.2) 199.67±5(1) 324±8(1.1) 158±1(1.3) 260.7±3.2(1.1)

20 44.3±13(1.3) 31.8±6.4(0.9) 184.7±8(0.8) 176±14(1.1) 173.3±25.7(0.9) 295±29(1) 139.4±12(1.1) 236.7±6.4(0.9)

30 26.08±16(0.8) 26.3±2(0.8) 219.7±23.3(1) 159±20(1) 203±29.3(1) 255±33(0.9) 153.4±22(1.2) 170.1±2(0.8)

Control + 2150±720 1107±23 2734±53 1241±53 1496±190 1338±33a 782±72 1277±33

DMSO: 75 µL/plate(negative control); Control+Positive control: a4-Nitro-o-phenylenediamine(NPD-10 µg/plate); b2-anthramine (0,63.g/plate); cSodium azide, (2.5 µg/plate); dDaunomycin (3.0 µg/plate); e2-aminofluorene (10 µg/plate).



Figure 1. Plots of the increase in fluorescence (in fluorescence units) with time for various nemorosone concentrations and negative control in the RYA system. Concentrations of nemorosone are indicated on the right margin. ß-galactosidase activity was calculated from the slopes of lines fitted to the data by standard linear regression methods. Values are means of four experiments performed in triplicate.

These results shows that nemorosone, at various concentrations, has no estrogenic activity detectable by this method but does possesses a protective activity against some kinds of induced mutation. Further studies should be performed to assess the biological activity of this benzophenone and clarify its mechanism of action.

Figure 2. Dose/response plot for various nemorosone dilutions, 17ß-estradiol and negative control (methanol) in the RYA system. The graph shows mean the ß-galactosidase activity in fluorescence units for each nemorosone dilution, from four experiments performed in triplicate.

References

Agradi E, Fico G, Cillo F, Francisci C, Tomè F 2002. Estrogenic activity of Nigella damascena extracts, evaluated using a recombinant yeast screen. Phytother Res 16: 414-416.

Aydemir N, Celikler S, Bilalouglu R 2005. In vitro genotoxic effects of the anti-cancer drug gemcitabine in human lymphocytes. Mutat Res 582: 35-41.

Bhat HK, Calaf G, Hei TK, Loya T, Vadgama JV 2003. Critical role of oxidative stress in carcinogenesis. Proc Natl Acad Sci 100: 3913-3918.

Bernstein L, Kaldor J, McCann J, Pike MC 1982. An empirical approach to the statistical analysis of mutagenesis data from the Salmonella test. Mutat Res 97: 267-281.

Brix R, Noguerol TN, Piña B, Balaam J, Nilsen AJ, Tollefsen KE, Levy W, Schramm KW, Barceló D 2010. Evaluation of the suitability of recombinant yeast-

Table 2. Antimutagenic activity expressed as the mean and standard deviation of the number of revertants and percent inhibition (in brackets) by nemorosone of direct (-S9) and indirect (+S9) mutagens, in strains TA98, TA100 and TA102 of S. typhimurium.

Treatment μg/plate TA98 TA100 TA102

NPD Aflatoxin Mitomycin C

Control+ 972±20 1376.3±30 2105.7±42.3

2.5 936.7±27(3.6) 1255.7±37(8.7) 2072.7±34.5(1.6)

5 808.7±10(16.8) 1278.7±24(7) 2013.3±24.2(4.4)

10 808.3±7.6(16.8) 1307.3±21(5) 2063.8±28.4(2)

20 816.7±19(14) 1045±19(24) 1803.4±9.9(14.3)

30 815±24(16.2) 940±20(31) 990.6±11.1(53)

The possible health risks and benefits associated with the consumption of plant estrogens underline the need to characterize the estrogenic potency of vegetables and seeds commonly present in the diet or used as medicinal plants (Agradi at al., 2002). The estrogenic activity of nemorosone, evaluated by the recombinant yeast assay (RYA) based on vertebrate estrogen receptors, is a convenient measure of the potential for endocrine disruption of a substance or an environmental sample (Garcia-Reyero et al., 2005; Brix et al., 2010). This assay makes use of an engineered yeast strain that harbors two foreign genetic elements: a vertebrate receptor, in this case, a human estrogen receptor, ER, and a reporter gene whose expression is made dependent on the presence of estrogens and whose final product concentration is easy to measure. This is a simplified version of the mechanism by which natural estrogens operate in vertebrates. The fundamental similarity of the transcriptional machinery in all eukaryotes ensures that it also works in yeast, in a similar way. In breast carcinoma, the estrogen receptor (Erα) is present in 75% of cases (Popolo et al., 2011). The phytoestrogens interact with these receptors inducing cancerous cell proliferation (Karayiannakis et al., 1996). The estrogenicity of nemorosone was assessed at various concentrations (0.19-15 µg/well) by RYA and the compound exhibited 0.41nM±0.16 EEq. Figure 1 shows plots of the increase in fluorescence at in different concentrations of the benzophenone and methanol (negative control) against time (for 20 min). There are no significant differences between slopes of the nemorosone linear plots and the negative control. Figure 2 shows the mean β-galactosidase activity, in fluorescence units, induced by nemorosone and the negative control, plotted against dilution factor.

Control+ - Positive Control: NPD - 4-nitro-o-phenylenediamine (10μg/plate)(-S9); Aflatoxin B1 (0.5 μg/plate) (+S9); Mitomycin C (0.5 μg/plate)(-S9).

200250300350400450500550600650700

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (minutes)

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ence

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ts

15μg5μg1.66μg0.56μg0.19μgControl

500 485 458 384 370 343

13448

0

2000

4000

6000

8000

10000

12000

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*Correspondence

Eliana Aparecida VarandaFaculdade de Ciências Farmacêuticas, UNESP, Campus AraraquaraRodovia Araraquara-Jaú, km 1, 14801-902 Araraquara-SP, [email protected].: +55 16 3301 6951Fax: +55 16 3301 6940

928

Article


Received 20 Dec 2010Accepted 20 Jun 2011Available online 2 Sep 2011

Revista Brasileira de FarmacognosiaBrazilian Journal of Pharmacognosy21(5): 928-935, Sep./Oct. 2011 Analgesic effect of leaf extract from Ageratina

glabrata in the hot plate test

Guadalupe García P,1 Edgar García S,3 Isabel Martínez G,1 Thomas R. F. Scior,2 José L Salvador,3 Mauro M Martínez P,3

Rosa E. del Río*,3

1Laboratorio de Neuroquímica, Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, Puebla, Mexico,2Laboratorio de Farmacomodelaje, Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, Puebla, Mexico,3Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Mexico.

Abstract: Ageratina glabrata (Kunth) R.M. King & H. Rob., Asteraceae (syn. Eupatorium glabratum Kunth) is widely distributed throughout Mexico and popularly known as “chamizo blanco” and “hierba del golpe” for its traditional use as external analgesic remedy. Though glabrata species has been chemically studied, there are no experimentally asserted reports about possible analgesic effects which can be inferred from its genus Ageratina. To fill the gap, we evaluated A. glabrata extracts in an animal model of nociception exploiting thermal stimuli. NMR and mass analyses identified a new thymol derivative, 10-benzoiloxy-6,8,9-trihydroxy-thymol isobutyrate (1), which was computationally converted into a ring-closed structure to explain interaction with the COX-2 enzyme in a ligand-receptor docking study. The resulting docked pose is in line with reported crystal complexes of COX-2 with chromene ligands. Based on the present results of dichloromethane extracts from its dried leaves, it is safe to utter that the plant possesses analgesic effects in animal tests which are mediated through inhibition of COX-2 enzyme.

Keywords:Ageratina glabrata 10-benzoiloxy-6,8,9-trihydroxy-thymol isobutyrateanalgesiaratsextractpainnociception

Introduction

Pain is a subjective experience that is the result of the perception of an injurious stimulus and includes an emotional component that requires that the individual is conscious so that this happens. In 1986, the International Association for the Study of Pain (IASP) defi ned pain as an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage (Merskey & Bogduk, 1994). But, the pain has a physiological component, which is called nociception. That is the process by which intense thermal, mechanical or chemical stimuli are detected by a subpopulation of peripheral nerve fi bers, called nociceptors (Basbaum & Jessell, 2000). The information is processed via specialized central nervous system pathways (Danneman, 1997). The animal models for the study of the pain measure the latency time and/or the threshold of the escape responses evaluating the effect of several drugs in the nociceptive responses that are those associated with the injurious stimulation perceived by the nociceptors (Danneman, 1997). The models of acute pain by application of intense stimuli of short duration induce

changes in motor refl exes or behavior such as retirement refl exes, vasomotor changes and vocalization, besides not requiring the accomplishment of a previous injury in the animal (González-Darder, 2000). Therefore, nociception is susceptible to a pharmacological blockade for the treatment of the pain and the common analgesic drugs are opioids and non-opioids drugs. But these drugs have disadvantages in the dosage, gastrointestinal problems, dependence and others. Different species of plants are used in folk medicine as an alternative for treatment of diseases and the traditional uses of plants as herbal remedies have played a vital role in the discovery of new molecules as therapeutic agents. In general, species of the genus Ageratina possess therapeutic properties, such as the concerned analgesic effect which in turn was reported by Mandal et al. (2005) and Chakravarty et al. (2010). The species belongs to the well-known plant family of Asteraceae, and Ageratina is a member of the tribe Eupatorieae. Ageratina constitutes a medium-sized shrub growing from 1 to 2 m with dense branches. The leaves are opposite, lamina narrowly deltoid to elliptical and the capitula densely corymbose. The genus comprises nearly 146 species according to King &

Analgesic effect of leaf extract from Ageratina glabrata in the hot plate test Guadalupe García P et al.


Robinson (1970). It is found in the tropical and temperate regions of Europe and America, especially Mexico. The plant under investigation, Ageratina glabrata (Kunth) R.M. King & H. Rob. (syn. Eupatorium glabratum Kunth) (King & Robinson, 1970), is popularly known as “chamizo blanco” or “hierba del golpe” which means “white greasewood” and “contusion herb“. The rural population recognizes its pain - relief effects in external remedies. Reports from the literature describe flavones, thymol derivatives and other phenolic terpenoids (Romo de Vivar et al., 1971; Bohlmann et al., 1977; Guerrero et al., 1978). Though A. glabrata has been chemically studied, there are no scientific reports about its analgesic effects. Therefore, we studied this Mexican plant extract in a hot plate model of nociception exploiting distinct thermal stimuli.


General

NMR measurements, including DEPT experiments, were performed at 400 MHz for 1H and 100 MHz for 13C on a Varian Mercury Plus 400 spectrometer, using CDCl3 as the solvent and TMS as internal reference. Mass spectra were recorded at 20 eV on a Hewlett-Packard 5989B Series II Plus spectrometer adapted to a HP 5890 gas chromatography. Column chromatography was carried out on Merck silica gel grade 60 (70-230 mesh).

Plant material

Specimens of Ageratina glabrata (Kunth) R.M. King & H. Rob., Asteraceae, were collected on January 4, 2006 near km 191 of México-Morelia state road No.15, in the municipality of San José de la Cumbre, State of Michoacán, México (N 19°40’859’’, W 100°50’423’’ and 2,234 meters above sea level). A voucher specimen (No. 188319) is deposited at the Herbarium of the Instituto de Ecología, A.C., Centro Regional del Bajío, Pátzcuaro, Michoacán, Mexico, where Prof. Jerzy Rzedowski kindly identified the plant material (Figure 1).

Figure 1. Photos of A. glabrata by Dr. Rosa E. del Río. The rhomboid-shaped leaves and flower grouping are clearly visible.

Preparation of plant extract

Air-dried leaves of A. glabrata (200 g) were extracted with CH2Cl2 (1.5 L) at room temperature for three days, three consecutive times. Filtration and evaporation of the extract afforded green viscous oil (19 g).

Isolation of the thymol derivative (1) from dichloromethane extract

The dichloromethane extract (0.5 g) was chromatographed on silica gel column using n-hexane and ethyl acetate mixture of increasing polarity, and finally pure ethyl acetate. Elution with hexane:EtOAc (9:1) afforded a mixture of thymol derivatives. The sub-fraction 30-32 (100 mg) was subjected to rechromatography on silica gel (5 g). Elution with n-hexane-AcOEt (8:2) afforded pure 10-benzoiloxy-6,8,9-trihydroxy-thymol isobutyrate (1) (20 mg). Identification was supported by spectroscopic analyses.

Animals

Female Sprague Dawley rats (180-200 g) were used for the study. The animals were housed in rooms maintained at 24±2 °C, twelve-hours-light-darkness cycle and maintained with ad libitum access to Harlan Tekland Global Diets® and bottled water. The animals were handled 3 min a day for four weeks until the experiment day. All animal experiments complied with recommendations of the Regulation of the Committee for the Care and Use of Laboratory Animals of the Benemérita Universidad Autónoma de Puebla in 2007, which provides for compliance with Mexican Official Norm (NOM-062-ZOO-1999) appendix A, about the “Specifications for the production, care and use of laboratory animals” (Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación; 2001).

Hot plate test

The extract was dissolved in sesame oil and administered to animals intraperitoneally at doses of 100 mg/Kg (n=9) and 150 mg/kg (n=10). The control group (n=8) was intraperitoneally exposed to a vehicle (0.7 mL) of pure sesame oil. In addition, independent groups of rats received, meloxicam intraperitoneally (n=8) at a dose of 4 mg/kg and nalbuphine (n=8) at a dose of 5 mg/kg. The control group (n=7) received isotonic saline solution (SSI, 0.4 mL). In the hot plate test, all animals were placed on a hot plate maintained at a temperature of 40 °C. The antinociceptive effect was quantified by measuring latency times for kicking or licking of hind paw. In addition, prior to the aforementioned verum and



control test, all animals had just been exposed to the hot plate in order to observe the so-called pre-injection-latency times, of which three measurements were taken. Each animal was repeatedly placed on the hot plate in time intervals of 10, 20, 30, 40, 50, 60, 70, 80 and 90 min after injection. The observation series of each animal was continued in order to collect data for every other hour until reaching the defined limit of 12 h evaluation time.

Docking simulations

The chromene derivative computationally modified and the control structure meloxicam (Engelhardt et al., 1995) were docked manually into the COX-2 catalytic site. This study was performed with the Sybyl®X software suite (Tripos International, 2010).

Vaginal smears

The vaginal smears were used to detect the phase of the estrous cycle during the hot plate test. We used this method as a control to determine whether the estrous cycle has influence on the latency times in the hot plate test.

Autopsy

The rats were observed for ten days after the nociception test in order to determine possible changes in the intake of food and water. On day eleven, animals were randomized and sacrificed using an overdose of sodium pentobarbital (55 mg/kg intraperitoneally). At autopsy the organs (heart, liver, kidneys, lungs, spleen, intestine and adjacent tissue to the administration area of A. glabrata) were given a macroscopic examination about their size, shape, color and texture.


Data were analyzed by one way ANOVA and were expressed as the means and respective standard deviation. This analysis allows checking as to whether there are any statistically significant differences (p<0.05) between the mean latency times of the groups. To determine which groups are different, the data were analyzed by Tukey test. In addition, Dunnett test was used to compare the experimental groups versus respective control group using Statistica program.


The dichloromethane extract of A. glabrata leaves was fractionated by column chromatography affording a pure compound 10-benzoiloxy-6,8,9-trihydroxy-thymol isobutyrate (1). The mass spectrum

indicated a molecular ion at m/z 388 in agreement with the molecular formula C21H24O7, m/z 370 (12%) [M-H2O]+, 178 (46%), 165 (43%), 150 (15%), 137 (5 %), 122 (23%), 105 (100%), 91 (5%), 71 (40%). The 1H NMR spectrum (Figure 2) indicates the presence of a benzoate group at 7.98, 7.55 and 7.40 ppm; singlets at 6.65 and 6.58 ppm for 2 and 5 aromatic hydrogens, respectively. The signals for the isopropyl group at 2.52 and 1.09 ppm. A singlet at 4.64 ppm for two hydrogens of methylene group in the 10-position of the molecule and the hydrogens of methylene group in C-9 were observed as a doublet each at 4.56 and 4.49 ppm. The 13C NMR spectrum (Table 1) was in agreement with the proposed structure. Eleven thymol derivatives have been obtained by Bohlmann et al 1977 from A. glabrata, but all of them differ from the new thymol derivative 1.

Table 1. NMR data for compound 1 in CDCl3.Position 13C DEPT 1H

1 149.2 C -

2 120.0 CH 6.65 (s)

3 129.2 C -

4 146.9 C -

5 112.8 CH 6.58 (s)

6 126.0 C -

7 15.7 CH3 2.16 (s)

8 78.2 C -

9 67.9 CH2 4.64 (s)

10 67.3 CH2 4.56 (d), J=12.0 Hz 4.49 (d), J=12.0 Hz

1’ 177.7 C -

2’ 33.9 CH 2.52 (sep), J=7.0Hz

3’ 18.8 CH3 1.09 (d), J=7.0 Hz

4’ 18.8 CH3 1.09 (d), J=7.0 Hz

1” 166.9 C -

2” 130.12 C -

3” 129.8 CH 7.98 (dd), J=7.0 and 1.3 Hz

4” 128.5 CH 7.40 (dd), J=7.9 and 7.7 Hz

5” 133.5 CH 7.55 (dddd), J=7.9, 7.7, 1.3, 1.3 Hz

6” 128.5 CH 7.40 (dd), J=7.9 and 7.7 Hz

7” 129.8 CH 7.98 (dd), J=7.9 and 1.3 Hz

The results obtained from this study reflect an analgesic effect for A. glabrata (100 mg/kg), meloxicam (4 mg/kg) and nalbuphine (5 mg/kg). The analysis of the data has revealed that a significant difference exists between control (vehicle and SSI) and experimental (verum) groups at doses of 100 mg/kg (p<0.05). The injected extract produced a significant increase in reaction times in all verum animals upon exposure to the thermal stimuli (Figure 3). The apex of this increase was found



during the eighth hour after injection at a dose level of 100 mg/kg. Intriguingly, dose augmentation did not enhance protective effects (a higher level of 150 mg/kg).

Figure 2. 400 MHz 1H-NMR spectrum of 10-benzoiloxy-6,8,9-trihydroxy-thymol isobutyrate (1).

Figure 3. The latency times of the substances and analgesics during the hot plate test for A. the first 90 minutes and B. continuous hourly sampling until reaching 12 h evaluation time. Each point represents the mean values of the latency time group and respective standard deviation. One way ANOVA tests show statistically significant differences (p<0.05) between all groups. On the other hand, the Tukey test finds no statistically significant difference between the verum group of A. glabrata (100 mg/kg), and both positive controls (meloxicam and nalbuphine) but in contrast to the A. glabrata 150 mg/kg group which does show a statistically significant difference with both controls (logically, vehicle and SSI do show). The basal first sample point in A. represents the latency time means for all groups before the intraperitoneally administration. In addition, Dunnett's test was applied for each time interval, comparing vehicle versus A. glabrata 100 and 150 mg/kg and SSI versus meloxicam and nalbuphine (*p<0.05).



Based on theoretical grounds and known literature the thymol derivative 1 is converted into a bicyclical system via a ring closure reaction of condensation type (Paredes & Clemente, 2005; Tenorio et al., 2006; Chemler et al., 2009). This occurs on the original thymol derivative 1 which was identified through NMR by our group. We postulate that it is chemically converted into a compound similar to positive control meloxicam which itself is a known COX-2 inhibitor (Engelhardt et al., 1995, Gris et al., 2009). To determine whether this plausible compound (Figure 4) could also block the enzyme COX-2 a docking study was conducted by Sybyl® X software suite (Tripos International, 2010) to position the chromene derivative into the binding pocket of meloxicam, exploring the interacting amino acids and complementary shape information (Wang et al., 2007). The target structure is a human COX-2 enzyme, also called prostaglandin synthase-2 (Padhye et al., 2009). The target receptor was downloaded from the Protein Data Bank site (Berman et al., 2000). Its entry code is 3LN0 (Wang et al., 2010). As a result, both the chromene derivative and the meloxicam ligands were successfully docked into the same orientation and position as seen on the experimental chromene-COX-2 complex. The crystal chromene ligand, meloxicam and our ring-closed chromene derivative have an analogous bicyclical system in common (Figure 4). Hence, the orientation and binding mode of our computed models are in good agreement with the related crystallographic structure (Figure 5).

The analgesia of the non steroidal anti-inflammatory drugs (NSAID) like meloxicam which was used as positive control, is longer than that seen under opioid analgesic drugs application (positive control nalbuphine). NSAID inhibit the synthesis of prostaglandins by cyclooxygenase (COX) enzymes. Certain prostaglandins mediate pain processes (Vanegas & Schaible, 2001). Therefore, the observed analgesic effect of the leaf extract from A. glabrata suggests that the underlying molecular mechanism is mediated via COX because of its duration and pain-suppressing character. The COX enzyme is divided into two isoforms. COX-1 is expressed constitutively throughout the body while the expression of COX-2 is inducible. However in spinal cord and brain, particularly in hippocampus, cortex and amygdala, both isoforms are expressed constitutively. This finding supports the idea that NSAID also act in the spinal cord by inhibiting prostaglandin synthesis and their effect is demonstrated in models of supraspinal analgesia as the hot plate test (Vanegas & Schaible, 2001). Contemporary research and development for potential new analgesics focus on selective COX-2 inhibition, in order to lower the incidence of gastrointestinal and renal side effects ascribed to COX-1 inhibition (Gajraj, 2003). Hence a docking study was conducted to dock meloxicam and the proposed chromene derivative into the active site of COX-2 target. The chromene structure was hypothetically designed based on the extracted thymol derivative 1 which was identified through NMR analyses.

Figure 4. The drawing show structures of chromene derivative and meloxicam in plane molecules (above) and in 3D (below) display. Although both molecules present different functional groups, there is a spatial and structural similarity.



On the other hand, there is controversy concerning the use of female rats in models of pain with regard to missing data about the hormonal action mechanism (Gordon & Soliman, 1996; Vinogradova et al., 2003; Kuba et al. 2006). The estrous cycle lasts on average four days. It consists of four phases which are proestrus, estrus, metaestrus and diestrus. In each stage variable hormone concentrations are found such as prolactin, LH, FSH, estradiol, or progesterone. Each phase of the estrous cycle is determined by a ratio of three types of cells obtained by vaginal smears (Marcondes, 2002): epithelial cells, cornified cells and leukocytes (Figure 6A-D). The influence of the phases of estrous cycle on the latency times in hot plate test, vaginal smears was performed in rats of the negative controls (vehicle and

SSI). During the hot plate test vaginal smears were performed, one at ninety minutes and again at seven hours. The statistical analysis shows no significant difference between the latency times and the phases of the estrous cycle (proestrus, estrus, and diestrus metaestrus). There is no correlation or trend between values of latency times and the phases of the estrous cycle of female rats. Hence, the latency time values of four groups with either A. glabrata 100 mg/kg, 150 mg/kg, meloxicam or nalbuphine treatments are not influenced by hormonal stages but only due to full drug effects either through the extract itself or the positive controls (Figure 6E, F). Our study design allows similar interpretation about “no hormonal influence” as earlier reported by Ratnasooriya et al. (2002) and Kiasalari et al. (2010). At autopsy there were no injuries during the

Figure 5. Computational model of chromene derivative (left) and meloxicam (right) docked into the active site of prostaglandin synthase-2 (COX-2). The parallel view allows the comparison and conclusion that the ligands’ orientation and binding mode is the same because of the highly similar overall shape and electronic properties in space. The target enzyme is displayed in ribbon mode with its helical parts and beta strands. The entry to the ligand cavity is from the right toward center in each protein model. The entire enzyme is depicted above and the detail of the cavity is displayed below.



macroscopic examination in the heart, liver, kidneys, lungs, spleen, intestine and adjacent tissue to the administration area of leaves extract of A. glabrata or vehicle. This reflects that the extract formulation is well-tolerated and administration is well performed. In addition, during the hot plate test, all animals did not show specific signs of pain such as piloerection, aggression, hunched posture, abdominal writhing, squeals on handling or pressure on administration area. They are exhaustively described by the Committee on Recognition and Alleviation of Distress in Laboratory Animals and Institute for Laboratory Animal Research (2008). Moreover, no animal died during or after treatment.

Acknowledgment

The authors thank Dr. Carlos Escamilla Weinmann, director of the Bioterium Claude Bernard of the Benemérita Universidad Autónoma de Puebla and his staff for the animals provided. Thanks are given to Dr. Jerzy Rzedowski for identifying of A. glabrata. Also thanks for Prof. Dr. U. Zaehringer, Forschungszentrum

Borstel, Germany, for the Sybyl licencing at our site. Partial financial support from CIC-UMSNH and CONACYT (118287), Mexico, is acknowledged Edgar García S. thank CONACYT for fellowship No. 205612.

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Figure 6. The phases of the estrous cycle: A. proestrus (n=4); B. estrus (n=4); C. metaestrus (n=4) and D. diestrus (n=3). Samples were prepared with hematoxylin and eosin stain under 40-fold microscopic magnification. The histogram bars show the mean and respective standard deviation values of the latency times for the control groups (vehicle and SSI) according to the phase of the estrus cycle at 90 minutes (E) and 7 h (F). Statistical analysis (one way ANOVA) does not show statistically significant difference between the groups (p<0.05).



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*Correspondence Rosa E. del RíoInstituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo. Ed. B-1, C.U. Morelia, Mich.,58030, [email protected]. +52 443 3265790 ext. 102; 3265790 ext. 103.

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