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NPC Natural Product Communications

EDITOR-IN-CHIEF
HONORARY EDITOR
DR. PAWAN K AGRAWAL
PROFESSOR GERALD BLUNDEN
Natural Product Inc.
The School of Pharmacy & Biomedical Sciences,
7963, Anderson Park Lane,
University of Portsmouth,
Westerville, Ohio 43081, USA
Portsmouth, PO1 2DT U.K.
agrawal@naturalproduct.us
axuf64@dsl.pipex.com

EDITORS ADVISORY BOARD


PROFESSOR ALESSANDRA BRACA
Dipartimento di Chimica Bioorganicae Biofarmacia, Prof. Berhanu M. Abegaz Prof. Leopold Jirovetz
Universita di Pisa, Gaborone, Botswana Vienna, Austria
via Bonanno 33, 56126 Pisa, Italy
braca@farm.unipi.it Prof. Viqar Uddin Ahmad Prof. Karsten Krohn
Karachi, Pakistan Paderborn, Germany
PROFESSOR DEAN GUO Prof. Øyvind M. Andersen Prof. Hartmut Laatsch
State Key Laboratory of Natural and Biomimetic Drugs, Bergen, Norway Gottingen, Germany
School of Pharmaceutical Sciences,
Peking University, Prof. Giovanni Appendino Prof. Marie Lacaille-Dubois
Beijing 100083, China Novara, Italy Dijon, France
gda5958@163.com Prof. Yoshinori Asakawa Prof. Shoei-Sheng Lee
Tokushima, Japan Taipei, Taiwan
PROFESSOR YOSHIHIRO MIMAKI Prof. Lee Banting Prof. Francisco Macias
School of Pharmacy, Portsmouth, U.K.
Tokyo University of Pharmacy and Life Sciences, Cadiz, Spain
Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan Prof. Julie Banerji Prof. Imre Mathe
mimakiy@ps.toyaku.ac.jp Kolkata, India Szeged, Hungary
Prof. Alejandro F. Barrero Prof. Joseph Michael
PROFESSOR STEPHEN G. PYNE Granada, Spain
Department of Chemistry Johannesburg, South Africa
University of Wollongong Prof. Anna R. Bilia Prof. Ermino Murano
Wollongong, New South Wales, 2522, Australia Florence, Italy Trieste, Italy
spyne@uow.edu.au Prof. Maurizio Bruno Prof. M. Soledade C. Pedras
Palermo, Italy Saskatoon, Cnada
PROFESSOR MANFRED G. REINECKE
Department of Chemistry, Prof. César A. N. Catalán Prof. Luc Pieters
Texas Christian University, Tucumán,Argentina Antwerp, Belgium
Forts Worth, TX 76129, USA Prof. Josep Coll Prof. Peter Proksch
m.reinecke@tcu.edu Barcelona, Spain Düsseldorf, Germany
PROFESSOR WILLIAM N. SETZER Prof. Geoffrey Cordell Prof. Phila Raharivelomanana
Department of Chemistry Chicago, IL, USA Tahiti, French Plynesia
The University of Alabama in Huntsville Prof. Cristina Gracia-Viguera Prof. Monique Simmonds
Huntsville, AL 35809, USA Murcia, Spain
wsetzer@chemistry.uah.edu
Richmond, UK
Prof. Duvvuru Gunasekar Prof. Valentin Stonik
PROFESSOR YASUHIRO TEZUKA Tirupati, India Vladivostok, Russia
Institute of Natural Medicine Prof. A.A. Leslie Gunatilaka Prof. Winston F. Tinto
Institute of Natural Medicine, University of Toyama, Tucson, AZ, USA
2630-Sugitani, Toyama 930-0194, Japan
Barbados, West Indies
tezuka@inm.u-toyama.ac.jp Prof. Kurt Hostettmann Prof. Karen Valant-Vetschera
Lausanne, Switzerland Vienna, Austria
PROFESSOR DAVID E. THURSTON Prof. Martin A. Iglesias Arteaga Prof. Peter G. Waterman
Department of Pharmaceutical and Biological Chemistry, Mexico, D. F, Mexico
The School of Pharmacy,
Lismore, Australia
University of London, 29-39 Brunswick Square, Prof. Jerzy Jaroszewski
London WC1N 1AX, UK Copenhagen, Denmark
david.thurston@pharmacy.ac.uk

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Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

NPC-SILAE: Special Issue

I am very grateful to Professor Luca Rastrelli, Dipartimento di Scienze Farmaceutiche e Biomediche, University of Salerno,
84084 Fisciano (SA), Italy, for organizing this issue, originating from the XIX SILAE (Società Italo-Latinoamericana di
Etnomedicina) Congress, which was held at Cagliari, Italy, from September 6th-10th, 2010, and attended by a large number of
participants from Latin America and the European Union. The present issue highlights some significant aspects of
ethnomedicine. The editors join me in thanking Professor Rastrelli, the authors and the reviewers for their efforts that have
made this issue possible, and to the production department for putting it into print.

Pawan K. Agrawal
Editor-in-Chief
Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

Editorial
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
scientific newsletters, books and reports, and spearheads programs that raise the bar of understanding for science worldwide.

Natural Product Communications 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 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 60 submissions were proposed for publication in the special issue of Natural Product Communications in
October 2010, each of which was reviewed by at least two anonymous referees. Following the review, 31 papers from different
universities of Argentina (7), Brazil (8), Colombia (2), Cuba (1), Honduras (1), Ireland and Serbia (1), Italy (6), Mexico (2) and
Venezuela (3) were selected for publication in this Special Issue. They are original papers on all aspects of natural products
including isolation, characterization, spectroscopic properties, biological activities, synthesis, analytical methods and tissue
culture; several are collaborative works between two or more countries.

Ten papers present the compositions of essential oils from different aromatic plants using analytical techniques such as GC,
GC/MS, GC/MS-LRI, esGC, GC-C-IRMS (1, 2, 4, 7, 8, 11, 14, 16, 22, 28) and also NMR spectroscopy (22). Although
essential oils have been used therapeutically for centuries, there is little published research on many of them. Bonaccorsi et al.
(1) report in their article on samples of Egyptian nerolì oils, obtained from the flowers of bitter orange (Citrus aurantium,
Rutaceace). For all the samples the composition was determined by GC/FID and by GC/MS-LRI; the samples were also
analyzed by esGC to determine the enantiomeric distribution of twelve volatiles and by GC-C-IRMS for the determination of
the 13CVPDB values of some mono and sesquiterpene hydrocarbons, alcohols and esters. The analytical procedures allowed the
quantitative determination of 86 components (1). Radulović et al. (7) identify 109 constituents from an essential oil sample
obtained from dry leaves of Nepeta × faassenii Bergmans, a hybrid species produced by crossbreeding N. mussinii Spreng. with
N. nepetella L. The chemical composition of the oil was compared, using multivariate statistical analyses (MVA), with those of
the oils of other Nepeta taxa, in particular N. mussinii and N. nepetella. The authors also report the chemical composition
dissimilarity relationships of 36 Nepeta essential oil samples. Some authors report the in vitro activity of the essential oil
against bacteria (2, 11, 16, 28). Bruno et al. (2) record the results obtained with the oil from the aerial parts of Salvia verbenaca
(Labiatae) aerial parts against Bacillus subtilis, Staphylococcus aureus, S. epidermidis, Streptococcus faecalis, Escherichia coli,
Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas aeruginosa; Perez et al. (11), the essential oil of Chrysactinia
mexicana (Astaraceae) roots against Streptococcus pneumonia; Rios Tesch et al. (16), Lantana camara var. moritziana
(Verbenaceae) leaf essential oil against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella
pneumoniae, Salmonella typhi, and Pseudomonas aeruginosa; and Mora et al. (28), Phthirusa adunca (Loranthaceae) aerial
parts essential oil against Salmonella typhi, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Klebsiella
pneumonia. Results confirm that many essential oils possess in vitro antimicrobial activity against pathogens when compared
with reference drugs. Martini et al. (8) report the chemical composition of two Lamiaceae, Ocimum selloi and Hesperozygis
myrtoides, widely used in Brazilian traditional medicine. The authors report, for the first time, the chemical composition of the
essential oil from H. myrtoides, a very aromatic small bush found in the region of Aiuruoca (Minas Gerais State, Brazil); this
plant is also used in the preparation of a drink with “cachaça”, the Brazilian sugar cane spirit, where the plant is soaked in the
bottle’s spirit and buried for one year before being consumed. The essential oils showed activity against Candida albicans C.
glabrata, C. krusei, C. parapsilosis and C. tropicalis. Oliva et al. (14) also report the activity against Candida yeasts (C.
albicans, C. dubliniensis, C. glabrata, C. krusei, C. guillermondii, C. parapsilosis and C. tropicalis) of an essential oil obtained
from Aloysia triphylla (Verbenaceae), a promising alternative from the Argentinean flora for the treatment of candidiasis.
Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

Veloza et al. report the application of dioxirane chemistry to essential oils in order to generate modified compounds with
potential uses in several areas of medicine and industry (22). Polyphenols are among the most widespread class of metabolites
in nature, and their distribution is almost ubiquitous.

Nine papers describe the isolation and structure elucidation, as well as the bioactivity, of phenolic compounds. Interesting
biological properties are reported, such as antioxidant (17, 18, 26, 29), antifungal (13), antimicrobial (17), antiplatelet (20),
antiangiogenic (25), chemopreventive and apoptotic activities (21). Arevalo et al. (3) report a new phenolic apiosyl derivative,
two uncommon apiosyl derivatives, and known phenyl propanoids and flavonoids from Martinella obovata (Bignoniaceae)
collected in Honduras, and used by indigenous peoples to treat various eye ailments, including inflammation and conjunctivitis.
It is well known that the consumption of polyphenol-rich products, mainly due to their antioxidant properties, is beneficial for
human health, Salvador et al. (18), as well as Aislan et al. (29), report the antioxidant capacity and phenolic organic acids and
flavonoids content of Myrtaceae plants of the south of Brazil. The article by Mendiondo et al. [17] presents the antioxidant and
antimicrobial activity of the methanolic extract of Chuquiraga straminea (Asteraceae). The extract was also active against ten
methicillin resistant and sensitive S. aureus strains isolated from nosocomial infection; kaempferol and quercetin glycoside
derivatives seem to be responsible for some of the observed biological activity of the extracts. Derita and Zacchino (13)
dedicate their article to the bio-guided fractionation of the active dichloromethane extract of Polygonum persicaria L.
(Polygonaceae). This genus is represented in Argentina by 21 species and some of them have been used in traditional medicine
of that country to treat complaints related to fungal infections, such as skin ailments and vaginal disease. Isolated sesquiterpene
dialdehydes and flavonoids showed activity against yeasts, Aspergillus spp. and dermatophytes with MICs between 3.9 - 250
µg/mL. The results validate the popular use of this plant.

Data from the literature show that some flavonoids and other phenolic substances have the property to interfere with the
platelet system. A diet rich in phenolic compounds may favorably contribute to reducing the risks of cardiovascular diseases
through several mechanisms. Douglas et al. (20) assessed the inhibitory activity toward clotting formation and platelet
aggregation of an aqueous extract of leaves from Petroselinum crispum, an aromatic herb from the Apiaceae family that has
been employed in the food, pharmaceutical, perfume and cosmetic industries. The active principles, cosmosiin (apigenin 7-O-
glucoside) and apigenin, showed in vitro antiplatelet aggregation activity. Angiogenesis is a crucial step in many pathological
conditions like cancer, inflammation and metastasis formation; on this basis, the search for antiangiogenic agents has widened.
In order to identify new compounds able to interfere with the Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1),
Lepore et al. (25) investigated the extract of Calycolpus moritzianus (Myrtaceae) leaves by a competitive ELISA-based assay.
Phytochemical and pharmacological investigation of the active fractions led to the isolation of flavonoids and terpenes. The
authors hypothesized that the inhibitory activity of PlGF and VEGF interaction with Flt-1 receptor by the C. moritzianus
CHCl3 extracts and fractions may be due to the presence of a combination of compounds acting synergistically or as vehicles
enhancing the biological activity. These results suggest that the use of C. moritzianus extract is preferable to that of a purified
single compound. Torrenegra et al. (21) evaluated the activity of 3,5-dihydroxy-7-methoxy-flavanone, 3,5-dihydroxy-7-
methoxyflavone and 3,5,7-trihydroxy-6-methoxyflavone present in Chromolaena leivensis (Asteraceae) on cell viability, cell
cycle distribution, mitochondrial membrane depolarization and viability of peripheral blood mononuclear cells and fibroblasts.
3,5-Dihydroxy-7-methoxyflavone showed activity on mitochondrial membrane, whereas both 3,5-dihydroxy-7-methoxy-
flavanone and 3,5-dihydroxy-7-methoxyflavone slightly increased the proliferation of peripheral blood mononuclear cells
either with phytohemagglutinin or without it, and the proliferation of fibroblasts.

The chemical composition of naturally grown herbs may vary according to climatic conditions, harvest time, storage condition,
and so on. As such, the same type of herb can vary in its composition and concentrations of chemical constituents from batch to
batch. These variabilities can result in significant differences in pharmacological activity. Therefore, the identification and
extraction of active ingredients from a medicinal plant represent a new approach to the development of natural product based
drugs. Picerno et al. (26) report on the evaluation of polyphenol components and antioxidant properties of fresh bergamot juice
(Citrus bergamia, Rutaceae), as well as on the production and characterization of powders obtained by loading the fresh juice
onto maltodextrins as a carrier (BMP) for spray-drying. Moreover, a formulation study to develop tablets containing BMP for
oral administration has been performed. The characteristics of the tablets were evaluated in terms of disintegration time and the
release of the active compounds into water and simulated biological fluids.

Five papers deal with the evaluation of pharmacological activity of crude extracts from plants used in Mexican (6),
Argentinean (9, 10 and 31) and Cuban (12) traditional medicine. Pazos et al. (6) investigated the effect of Morinda citrifolia
(Rubiaceae) seed (noni oil) on serum lipid levels in normolipidemic and hyperlipidemic induced mice. They found that
administration of noni oil causes a reduction in total cholesterol and triglyceride levels in both models. GC-MS analysis of the
fatty acid methyl esters indicated the presence of five major fatty acids. The mean linoleic acid content of crude noni seed oil
was 67.8%; these results indicate that noni seeds may be a useful new source of vegetable oil. Few medicinal plants have been
Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

scientifically evaluated for their safety, efficacy and potential benefits, despite the great public interest in these herbs. Sabini et
al. (10) evaluated the cytotoxic and genotoxic activities of a cold aqueous extract obtained from Achyrocline satureioides
(Asteraceae) using the Allium cepa test, whereas Escobar et al. (31) assessed the genotoxic and cytotoxic activities of a
methanolic extract of Verbascum thapsus (Scrophulariaceae) using a micronucleus test in mouse bone marrow. Numerous
investigations have reported bioactive properties for both medicinal plants and the results obtained in these present studies
allow the conclusion to be made that the aqueous extract of A. satureioides and the methanolic extract of V. thapsus do not
contain genotoxic and cytotoxic compounds. Cytotoxicity, antiviral and virucidal activities of aqueous extracts of Baccharis
articulata were also evaluated by Cristina Vanesa Torres et al. (9). Extracts exhibited more than 95% virucidal activity against
Herpes suis virus type 1. These findings support the potential application of these extracts as a disinfectant or antiseptic
consistent with ancient ethnopharmacological thinking. In Cuba, alcoholic extracts of propolis are popular as a homemade
remedy. Three main types of Cuban propolis directly related to their secondary metabolite classes were described: brown
Cuban propolis (BCP), rich in polyisoprenylated benzophenones, red Cuban propolis (RCP), containing isoflavonoids as the
main constituents, and yellow Cuban propolis (YCP) with a variety of triterpenoids as the major chemical components.
Monzote et al. (12) assessed the activity of Cuban propolis extracts (brown, red and yellow type) on Leishmania amazonensis
and Trichomonas vaginalis. All propolis samples caused inhibition of growth of the Leishmania parasite. RCP was the most
active and the most cytotoxic. Only five propolis extracts showed activity against T. vaginalis and in this case YCP samples
were the most active.

According to the World Health Organization (WHO), more than 80% of the world's people, mostly in poor and less-developed
countries, depend on traditional medicine for their primary healthcare requirements. They use medicinal plants not only for
themselves but also for their domestic animals. Traditionally, people collected the ingredients for their medicines from forests.
However, due to rapid and extensive deforestation, accompanied by uncontrolled over-exploitation, the wild populations of
medicinal plants are disappearing very fast.

Three papers deal with biodiversity and nature protection. The cerrado, a vast tropical savanna ecoregion of Brazil,
particularly in the states of Goiás and Minas Gerais, is characterized by an enormous range of plant and animal biodiversity.
The cerrado is one of the world's threatened biodiversity hotspots. About 60% of its vegetation has already been removed and
the remaining areas are isolated in forest fragments. Due to the devastation, many natural compounds with potential biological
activities have been lost. Soares et al. (23) compared the basal cytotoxicity of active compounds extracted from plants of the
Brazilian “cerrado”. Those with low toxicity were subjected to further anti-inflammatory assays as natural products with low
cytotoxicity constitute an excellent alternative source for complementary treatments for inflammatory disease. The viability
was assayed using the neutral red uptake assay in Mac Coy cells after 24 h of exposure. The dose evaluated was 50µg/µL. The
test substances were: cinnamic acid, p-coumaric acid, chlorogenic acid, syringic acid, vanilic acid, homogentisis acid,
scandenin, palustric acid, diosgenin, and cabraleone. From 1975 until the beginning of the 1980s, many governmental
programs have been launched with the intent of stimulating the development of the "cerrado" region, through subsidies for
agriculture. As a result, there has been a significant increase in agricultural and cattle production. On the other hand, urban
pressure and rapid establishment of agricultural activities in the region have rapidly reduced the biodiversity of the ecosystems.
Camargo et al. (30) tried to diagnose the current public programs focused on herbal medicines in Brazil from 1985 to 2006.

Sharry et al. (19) dedicated their article to the establishment of vegetative propagation systems for three native forest species
widely used in Argentinean folk medicine: Erythrina crista-galli (Fabaceae), Acacia caven (Mimosaceae) and Salix
humboldtiana (Salicaceae). In the last few years the use of in vitro culture techniques for trees has facilitated the cloning of
selected phenotypes, leading to the preservation and manipulation of vegetal material. The authors are able to support the
conservation of native forest resources for medicinal use by means of vegetative propagation techniques: macro and
micropropagation and somatic embryogenesis.

The last four papers are quite different from the others, each one dealing with its own subject of great importance. Marques
et al. (5) report for the first time the isolation of six aristolactams from Ottonia anisum (Piperaceae). Aristolactams belong to a
large and important group of naturally occurring alkaloids that possess a phenanthrene lactam skeleton with a
phenolic hydroxy function. They constitute an important alkaloid group due to their unique structural features and potent
biological activities, such as anti-inflammatory, anti-arthritis, anti-PAF, anti-mycobacterial, and neuro-protective.
Aristolactams have been reported from plants of the Annonaceae, Monimiaceae, Menispermaceae, Piperaceae and Saururaceae
families. Nicoletti (15) analyzed, first by HPTLC and later by isolation and analysis of spectroscopic data, the presence of a
sildenafil derivative (thiosildenafil) in herbal products. Quality assurance has enabled health professionals to prescribe safely
herbal medicines that the population has been taking for quite a long time.The presence of synthetic drugs in the formulation of
herbal products in order to improve the efficacy has been reported in several cases.
Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

Viegi et al. (24) revised the use of toxic or potentially toxic plants for the treatment of ailments in livestock and pets in
ethnoveterinary practice in Italy. More than 250 of the entities used (81% for curative purposes) can be toxic unless dosed
appropriately. The species belong to 71 families, among which the Fabaceae predominates. Drugs derived from natural
sources are usually produced by harvesting the natural source or through semi-synthetic methods: semisynthesis is usually used
when the precursor molecule is too structurally complex, too costly or too inefficient to be produced by total synthesis. It is
also possible that the semisynthetic derivative outperforms the original biomolecule itself with respect to potency, stability and
safety. Usubillaga et al. (27) undertook the isomerization of kaurenic acid to obtain ent-kaurenic acid, a tetracyclic diterpene
that has been reported to have antimicrobial, antiparasitic and cytotoxic activity. The occurrence of kaurenic acid, which has an
exocyclic double bond at Δ16, is widespread in the plant kingdom, while the occurrence of its isomer ent-kaur-15-en-19-oic
acid is rare.

The congresses of SILAE are international events whose organizations are submitted to an International Organizer Committee
composed of professors from Italian and Latin America Universities. The Italo-Latin American Congress of Ethnomedicine
arose from the necessity to evaluate the important potentialities of little known medicinal and alimentary plants, typical and
traditional plants of the Latin American continent and to provide connections between Italian and other European and Latin-
American researchers, with common objectives of research in the areas in which the projects will be articulated. Traditional
medicine is used by 85% of the World’s population and is of great importance in developing countries. In accordance with the
requirements of the World Health Organization, a scientific basis and proof for the use of medicinal plants is required and so
the organization of such a Congress provides an important exchange of such information and coordination of scientific activity.
This Natural Product Communications special issue provided an opportunity for publication of original, peer-reviewed, full-
length 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 Editor 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. I am also grateful to my staff who lent their considerable talents to the project: Annalisa
Piccinelli, Florence Somma, Luca Campone and the webmaster of SILAE, Vincenzo Barbarulo. I thank all of them for their
commitment, continued support and friendship.

I am also very grateful to the Editorial Board of Natural Product Communications 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. Finally, I would like to thank the Editor-in-Chief, Pawan Agrawal, for his valuable input and
for careful supervision. Thank you Pawan!
Luca Rastrelli
Dipartimento di Scienze Farmaceutiche e Biomediche,
University of Salerno,
Via Ponte don Melillo,
84084 Fisciano (SA), Italy
E-mail: rastrelli@unisa.it

References

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Product Communications, 6, 1009-1014
2. Canzoneri M, Bruno M, Rosselli S, Russo A, Cardile V, Formisano C, Rigano D, Senatore F. (2011) Chemical composition and
biological activity of Salvia verbenaca essential oil. Natural Product Communications, 6, 1023-1026
3. Arevalo C, Ruiz I, Piccinelli AL, Rastrelli L. (2011) Phenolic derivatives from the leaves of Martinella obovata (Bignoniaceae).
Natural Product Communications, 6, 957-960
4. Pimentel Victório C, Moreira CB, da Costa Souza M, Sato A, do Carmo de Oliveira Arruda R. (2011) Secretory cavities and
volatiles of Myrrhinium atropurpureum Schott var. atropurpureum (Myrtaceae): an endemic species collected in the restingas of
Rio de Janeiro, Brazil. Natural Product Communications, 6, 1045-1050
5. Marques AM, Velozo LSM, de L. Moreira D, Guimarães EF, Kaplan MAC. (2011) Aristolactams from roots of Ottonia anisum
Sprengel (Piperaceae). Natural Product Communications, 6, 939-942
6. Pazos DC, Jiménez FE, Garduño L, López VE, Cruz MC. (2011) Hypolipidemic effect of seed oil of noni (Morinda citrifolia).
Natural Product Communications, 6, 1005-1008
7. Radulović N, Polina D., Blagojević, KR, de Sousa Menezes F. (2011) Essential oil of Nepeta x faassenii Bergmans ex Stearn (N.
mussinii Spreng. x N. nepetella L.): a comparison study. Natural Product Communications, 6, 1015-1022
Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

8. Martini MG, Bizzo HR, de L. Moreira D, Neufeld PM, Miranda SN, Alviano CS, Alviano DS, Leitão G. (2011) Analysis of the
chemical composition and antimicrobial activities of the essential oils from Ocimum selloi Benth. and Hesperozygis myrtoides (A.
St.-Hil.) Epling (Lamiaceae) . Natural Product Communications, 6, 1027-1030
9. Torres CV, Domínguez MJ, Carbonari JL, Sabini MC, Sabini LI, Zanon SM. (2011) Study of antiviral and virucidal activities of
aqueous extract of Baccharis articulata against Herpes suis virus. Natural Product Communications, 6, 993-994
10. Sabini MC, Cariddi LN, Escobar FM,, Bachetti RA, Sutil SB, Contigiani MS, Zanon SM, Sabini LI. (2011) Evaluation of
cytogenotoxic effects of cold aqueous extract from Achyrocline satureioides by Allium cepa L test. Natural Product
Communications, 6, 995-998
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(2011) Activity against Streptococcus pneumoniae of the essential oil and 5-(3-buten-1-ynyl)-2, 2'-bithienyl isolated from
Chrysactinia mexicana roots. Natural Product Communications, 6, 1035-1038
12. Monzote Fidalgo L, Sariego Ramos I, García Parra M, Cuesta-Rubio O, Márquez Hernández I, Campo Fernández M, Piccinelli AL,
Rastrelli L. (2011) Activity of Cuba propolis extracts on Leishmania amazonensis and Trichomonas vaginalis. Natural Product
Communications, 6, 973-976
13. Derita M, Zacchino S. (2011) Validation of the ethnopharmacological use of Polygonum persicaria for its antifungal properties.
Natural Product Communications, 6, 931-933
14. Oliva M, Carezzano E, Gallucci N, Casero C, Demo M. (2011) Antimycotic effect of the essential oil of Aloysia triphylla against
Candida species obtained from human pathologies. Natural Product Communications, 6, 1039-1043
15. Nicoletti M. (2011) Isolation and identification of thiosildenafil in a health supplement. Natural Product Communications, 6,
1003-1004
16. Rios Tesch N, Mora F, Rojas L, Díaz T, Velasco J, Yánez C, Rios N, Carmona J, Pasquale S. (2011) Chemical composition and
antibacterial activity of the essential oil of Lantana camara var. moritziana (Otto & Dietr.) López-Palacios. Natural Product
Communications, 6, 1031-1034
17. Mendiondo ME, Juárez BE, Zampini C, Isla MI, Ordoñez R. (2011) Bioactivies of Chuquiraga straminea Sandwith, subfamily
Barnadesioideae (Asteraceae) . Natural Product Communications, 6, 965-968
18. Salvador MJ, Andreazza NL, de Lourenço CC, Aislan Pascoal CRF, Alves Stefanello ME. (2011) Antioxidant capacity and
phenolic content of four Myrtaceae plants of the South of Brazil. Natural Product Communications, 6, 977-982
19. Sharry S, Adema M, Basiglio Cordal MA, Villarreal B, Nikoloff N, Briones V, Abedini W. (2011) Propagation and conservation of
native forest genetic resources of medicinal use by means of in vitro and ex vitro techniques. Natural Product Communications, 6,
985-988
20. Chaves DSA, Frattani F, Assafim M, de Almeida AP, Zingali RB, Costa SS. (2011) Phenolic chemical composition of
Petroselinum crispum extract and its effect on haemostasis. Natural Product Communications, 6, 961-964
21. Torrenegra RDG, Rodriguez AO. (2011) Chemical and biological activity of leaves extracts of Chromolaena leivensis. Natural
Product Communications, 6, 947-950
22. Veloza LA, Orozco LM, Sepúlveda-Arias JC. (2011) Use of dimethyldioxirane in the epoxidation of the main constituents of the
essential oils obtained from Tagetes lucida, Cymbopogon citratus, Lippia alba and Eucalyptus citriodora. Natural Product
Communications, 6, 925-930
23. Soares VCG, Bonacorsi C, Andrela ALB, Bortoloti LV, Campos SC, Fagundes FHR, Piovani M, Cotrim CA, Vilegas W, Toyama
MH. (2011) Cytotoxicity of active ingredients extracted from plants of the Brazilian “Cerrado”. Natural Product Communications,
6, 983-984
24. Viegi L, Vangelisti R. (2011) Toxic plants used in ethnoveterinary medicine in Italy. Natural Product Communications, 6, 999-
1000
25. Lepore L, Gualtieri MJ, Malafronte N, Dal Piaz F, Ambrosio L, De Falco S, De Tommasi N. (2011) Anti-angiogenic activity
evaluation of secondary metabolites from Calycolpus moritzianus (O. Berg) Burret leaves. Natural Product Communications, 6,
943-946
26. Picerno P, Sansone F, Mencherini T, Prota L, Aquino RP, Rastrelli L, Lauro MR. (2011) Citrus bergamia juice: phytochemical and
technological studies Natural Product Communications, 6, 951-955
27. Rojas J, Aparicio R, Villasmil T, Peña A, Usubillaga A. (2011) On the isomerization of ent-kaurenic acid. Natural Product
Communications, 6, 935-938
28. Mora FD, Ríos N, Rojas LB, Díaz T, Velasco J, Carmona JA, Silva B. (2011) Chemical composition and in vitro antibacterial
activity of the essential oil of Phthirusa adunca (Meyer) from Venezuelan Andes Natural Product Communications, 6, 1051-1053
29. Pascoal ACRF, Ehrenfried CA, Eberlin MN, Alves Stefanello ME, Salvador MJ. (2011) Free-radical scavenging activity,
determination of phenolic compounds and HPLC-UV/DAD/ESI-MS profile of Campomanesia adamantium leaves. Natural
Product Communications, 6, 969-972
30. Saranz Camargo EE, Medeiros Bandeira MA, Gomes de Oliveira A. (2011) Diagnosis of public programs focused on herbal
medicines in Brazil. Natural Product Communications, 6, 1001-1002
31. Escobar FM, Sabini MC, Zanon SM, Cariddi LN, Tonn CE, Sabini LI. (2011) Genotoxic evaluation of a methanolic extract of
Verbascum thapsus using micronucleus test in mouse bone marrow Natural Product Communications, 6, 989-991
Natural Product Communications
2011
Volume 6, Number 7
Contents
Original Paper Page

Use of Dimethyldioxirane in the Epoxidation of the Main Constituents of the Essential Oils Obtained from
Tagetes lucida, Cymbopogon citratus, Lippia alba and Eucalyptus citriodora
Luz A. Veloza, Lina M. Orozco and Juan C. Sepúlveda-Arias 925
Validation of the Ethnopharmacological Use of Polygonum persicaria for its Antifungal Properties
Marcos Derita and Susana Zacchino 931
On the Isomerization of ent-Kaurenic Acid
Julio Rojas, Rosa Aparicio, Thayded Villasmil, Alexis Peña and Alfredo Usubillaga 935
Aristolactams from Roots of Ottonia anisum (Piperaceae)
André M. Marques, Leosvaldo S. M. Velozo, Davyson de L. Moreira, Elsie F. Guimarães and
Maria Auxiliadora C. Kaplan 939
Anti-angiogenic Activity Evaluation of Secondary Metabolites from Calycolpus moritzianus Leaves
Laura Lepore, Maria J. Gualtieri, Nicola Malafronte, Roberta Cotugno, Fabrizio Dal Piaz, Letizia Ambrosio,
Sandro De Falco and Nunziatina De Tommasi 943
Chemical and Biological Activity of Leaf Extracts of Chromolaena leivensis
Ruben D. Torrenegra G. and Oscar E. Rodríguez A. 947
Citrus bergamia Juice: Phytochemical and Technological Studies
Patrizia Picerno, Francesca Sansone, Teresa Mencherini, Lucia Prota, Rita Patrizia Aquino, Luca Rastrelli and
Maria Rosaria Lauro 951
Phenolic Derivatives from the Leaves of Martinella obovata (Bignoniaceae)
Carolina Arevalo, Ines Ruiz, Anna Lisa Piccinelli, Luca Campone and Luca Rastrelli 957
Phenolic Chemical Composition of Petroselinum crispum Extract and Its Effect on Haemostasis
Douglas S. A. Chaves, Flávia S. Frattani, Mariane Assafim, Ana Paula de Almeida, Russolina B. Zingali and
Sônia S. Costa 961
Bioactivities of Chuquiraga straminea Sandwith
María Elena Mendiondo, Berta E. Juárez, Catiana Zampini, María Inés Isla and Roxana Ordoñez 965
Free Radical Scavenging Activity, Determination of Phenolic Compounds and HPLC-DAD/ESI-MS Profile of
Campomanesia adamantium Leaves
Aislan C.R.F. Pascoal, Carlos Augusto Ehrenfried, Marcos N. Eberlin, Maria Élida Alves Stefanello and
Marcos José Salvador 969
Activity of Cuban Propolis Extracts on Leishmania amazonensis and Trichomonas vaginalis
Lianet Monzote Fidalgo, Idalia Sariego Ramos, Marley García Parra, Osmany Cuesta-Rubio, Ingrid Márquez Hernández,
Mercedes Campo Fernández, Anna Lisa Piccinelli and Luca Rastrelli 973
Antioxidant Capacity and Phenolic Content of four Myrtaceae Plants of the South of Brazil
Marcos José Salvador, Caroline C. de Lourenço, Nathalia Luiza Andreazza, Aislan C.R.F. Pascoal and
Maria Élida Alves Stefanello 977
Cytotoxicity of Active Ingredients Extracted from Plants of the Brazilian “Cerrado”
Veronica CG Soares, Cibele Bonacorsi, Alana LB Andrela, Lígia V Bortoloti, Stepheny C de Campos,
Fábio HR Fagundes, Márcio Piovani, Camila A Cotrim, Wagner Vilegas and Marcos H Toyama 983
Propagation and Conservation of Native Forest Genetic Resources of Medicinal Use by Means of in vitro and
ex vitro Techniques
Sandra Sharry, Marina Adema, María A. Basiglio Cordal, Blanca Villarreal, Noelia Nikoloff, Valentina Briones and
Walter Abedini 985
Genotoxic Evaluation of a Methanolic Extract of Verbascum thapsus using Micronucleus Test in Mouse
Bone Marrow
Franco Matías Escobar, María Carola Sabini, Silvia Matilde Zanon, Laura Noelia Cariddi, Carlos Eugenio Tonn and
Liliana Inés Sabini 989

Continued Overleaf
Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

Study of Antiviral and Virucidal Activities of Aqueous Extract of Baccharis articulata against Herpes suis virus
Cristina Vanesa Torres, María Julia Domínguez, José Luis Carbonari, María Carola Sabini, Liliana Inés Sabini and
Silvia Matilde Zanon 993
Evaluation of Cytogenotoxic Effects of Cold Aqueous Extract from Achyrocline satureioides by Allium cepa L test
María C. Sabini, Laura N. Cariddi, Franco M. Escobar, Romina A. Bachetti, Sonia B. Sutil, Marta S. Contigiani,
Silvia M. Zanon and Liliana I. Sabini 995
Toxic Plants Used in Ethnoveterinary Medicine in Italy
Lucia Viegi and Roberta Vangelisti 999
Diagnosis of Public Programs focused on Herbal Medicines in Brazil
Ely Eduardo Saranz Camargo, Mary Anne Medeiros Bandeira and Anselmo Gomes de Oliveira 1001
Identification of Thiosildenafil in a Health Supplement
Marcello Nicoletti 1003
Hypolipidemic Effect of Seed Oil of Noni (Morinda citrifolia)
Diana C. Pazos, Fabiola E. Jiménez, Leticia Garduño, V. Eric López and M. Carmen Cruz 1005
Composition of Egyptian Nerolì Oil
Ivana Bonaccorsi, Danilo Sciarrone, Luisa Schipilliti, Alessandra Trozzi, Hussein A. Fakhry and Giovanni Dugo 1009
Essential oil of Nepeta x faassenii Bergmans ex Stearn (N. mussinii Spreng. x N. nepetella L.): A Comparison Study
Niko Radulović, Polina D. Blagojević, Kevin Rabbitt and Fabio de Sousa Menezes 1015
Chemical Composition and Biological Activity of Salvia verbenaca Essential Oil
Marisa Canzoneri, Maurizio Bruno, Sergio Rosselli, Alessandra Russo, Venera Cardile, Carmen Formisano,
Daniela Rigano and Felice Senatore 1023
Chemical Composition and Antimicrobial Activities of the Essential Oils from Ocimum selloi and
Hesperozygis myrtoides
Márcia G. Martini, Humberto R. Bizzo, Davyson de L. Moreira, Paulo M. Neufeld, Simone N. Miranda,
Celuta S. Alviano, Daniela S. Alviano and Suzana G. Leitão 1027
Chemical Composition and Antibacterial Activity of the Essential Oil of Lantana camara var. moritziana
Nurby Rios Tesch, Flor Mora, Luis Rojas, Tulia Díaz, Judith Velasco, Carlos Yánez, Nahile Rios, Juan Carmona and
Sara Pasquale 1031
Activity against Streptococcus pneumoniae of the Essential Oil and 5-(3-Buten-1-ynyl)-2, 2'-bithienyl Isolated
from Chrysactinia mexicana Roots
Bárbara Missiam Mezari Guevara Campos, Anabel Torres Cirio, Verónica Mayela Rivas Galindo, Ricardo Salazar Aranda,
Noemí Waksman de Torres and Luis Alejandro Pérez-López 1035
Antimycotic Effect of the Essential Oil of Aloysia triphylla against Candida Species Obtained from
Human Pathologies
María de las Mercedes Oliva, María Evangelina Carezzano, Mauro Nicolás Gallucci and Mirta Susana Demo 1039
Secretory Cavities and Volatiles of Myrrhinium atropurpureum Schott var. atropurpureum (Myrtaceae): An
Endemic Species Collected in the Restingas of Rio de Janeiro, Brazil
Cristiane Pimentel Victório, Claudio B. Moreira, Marcelo da Costa Souza, Alice Sato and
Rosani do Carmo de Oliveira Arruda 1045
Chemical Composition and in vitro Antibacterial Activity of the Essential Oil of Phthirusa adunca from
Venezuelan Andes
Flor D. Mora, Nurby Ríos, Luis B. Rojas, Tulia Díaz, Judith Velasco, Juan Carmona A and Bladimiro Silva 1051

Manuscripts in Press 1054


Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

LIST OF AUTHORS

Abedini, W ............................ 985 de Campos, SC .......................983 Mencherini, T .........................951 Salvador, MJ ................... 969,977
Adema, M .............................. 985 de Lourenço, CC ....................977 Mendiondo, ME .....................965 Sansone, F .............................. 951
Almeida, AP .......................... 961 de Oliveira, AG ....................1001 Menezes, FS .........................1015 Sato, A .................................. 1045
Alviano, CS ......................... 1027 Demo, MS ............................1039 Miranda, SN .........................1027 Schipilliti, L ......................... 1009
Alviano, DS ......................... 1027 Derita, M ................................931 Mora, F .................................1031 Sciarrone, D ......................... 1009
Ambrosio, L .......................... 943 Díaz, T ........................ 1031,1051 Mora, FD ..............................1051 Senatore, F ........................... 1023
Andreazza, NL ...................... 977 Domínguez, MJ ......................993 Moreira, CB .........................1045 Sepúlveda-Arias, JC............... 925
Andrela, ALB ........................ 983 Dugo, G ................................1009 Moreira, DL ..................939,1027 Sharry, S ................................. 985
Aparicio, R ............................ 935 Neufeld, PM .........................1027 Silva, b ................................. 1051
Aquino, RP ............................ 951 Eberlin, MN............................969 Soares, VCG .......................... 983
Aranda, RS .......................... 1035 Ehrenfried, CA .......................969 Nicoletti, M ..........................1003 Souza, MC ............................ 1045
Arevalo, C ............................. 957 Escobar, FM ................... 989,995 Nikoloff, N .............................985 Stefanello, MEA ............. 969,977
Arruda, ACO ....................... 1045 Sutil, SB ................................. 995
Assafim, M ............................ 961 Fagundes, FHR.......................983 Oliva, MM ............................1039
Fakhry, HA ...........................1009 Ordoñez, R .............................965 Tesch, NR............................. 1031
Bachetti, RA .......................... 995 Falco, SD ................................943 Orozco, LM ............................925 Tommasi, ND ......................... 943
Bandeira, MAM .................. 1001 Fernández, MC .......................973 Tonn, CE ................................ 989
Bizzo, HR ............................ 1027 Fidalgo, LM............................973 Parra, MG ...............................973 Torrenegra G., RD ................. 947
Blagojević, PD .................... 1015 Formisano, C ........................1023 Pascoal, ACRF ................969,977 Torres, CV.............................. 993
Bonaccorsi, I........................ 1009 Frattani, FS .............................961 Pasquale, S ...........................1031 Torres, NW .......................... 1035
Bonacorsi, C .......................... 983 Pazos, DC .............................1005 Toyama, MH .......................... 983
Bortoloti, LV ......................... 983 Galindo, VMR ......................1035 Peña, A ...................................935 Trozzi, A .............................. 1009
Briones, V.............................. 985 Gallucci, MN ........................1039 Pérez-López, LA ..................1035
Bruno, M ............................. 1023 Garduño, L ...........................1005 Piaz, FD ..................................943 Usubillaga, A ......................... 935
Gualtieri, M ............................943 Piccinelli, AL ..................957,973
Camargo, EES ..................... 1001 Guimarães, EF ........................939 Picerno, P ...............................951 Vangelisti, R .......................... 999
Campone, L ........................... 957 Piovani, M ..............................983 Velasco, J ....................1031,1051
Campos, BMMG ................. 1035 Hernández, IM........................973 Prota, L ...................................951 Veloza, LA ............................. 925
Canzoneri, M ....................... 1023 Velozo, LSM .......................... 939
Carbonari, JL ......................... 993 Isla, MI ...................................965 Rabbitt, K .............................1015 Victório, CP ......................... 1045
Cardile, V ............................ 1023 Radulović, N ........................1015 Viegi, L .................................. 999
Carezzano, ME .................... 1039 Jiménez, FE ..........................1005 Ramos, IS ...............................973 Vilegas, W.............................. 983
Cariddi, LN..................... 989,995 Juárez, BE ..............................965 Rastrelli, L ............... 951,957,973 Villarreal, B ............................ 985
Carmona A,J ........................ 1051 Rigano, D .............................1023 Villasmil, T ............................ 935
Carmona, J ........................... 1031 Kaplan, MAC .........................939 Rios, N.........................1031,1051
Chaves, DSA ......................... 961 Rodríguez A, OE ....................947 Yánez, C ............................... 1031
Cirio, AT ............................. 1035 Lauro, MR ..............................951 Rojas, J ..........................935,1031
Contigiani, MS ...................... 995 Leitão, SG ............................1027 Rojas, LB .............................1051 Zacchino, S ............................ 931
Cordal, MAB ......................... 985 Lepore, L ................................943 Rosselli, S.............................1023 Zampini, C ............................. 965
Costa, SS ............................... 961 López, VE ............................1005 Ruiz, I .....................................957 Zanon, SM................989,993,995
Cotrim, CA ............................ 983 Russo, A ...............................1023 Zingali, RB ............................. 961
Cotugno, R ............................ 943 Malafronte, N .........................943
Cruz, MC ............................. 1005 Marques, AM .........................939 Sabini, LI ................. 989,993,995
Cuesta-Rubio, O .................... 973 Martini, MG .........................1027 Sabini, MC .............. 989,993,995
2011
NPC Natural Product Communications Vol. 6
No. 7
Use of Dimethyldioxirane in the Epoxidation of the 925 - 930

Main Constituents of the Essential Oils Obtained from


Tagetes lucida, Cymbopogon citratus, Lippia alba and
Eucalyptus citriodora
Luz A. Velozaa*, Lina M. Orozcoa and Juan C. Sepúlveda-Ariasb
a
Laboratorio de Fitoquímica, Escuela de Química, Universidad Tecnológica de Pereira, A.A. 097,
Vereda La Julita Pereira, Risaralda – Colombia
b
Laboratorio de Fisiología Celular e Inmunología, Facultad de Ciencias de la Salud, Universidad
Tecnológica de Pereira, A.A. 097, Vereda La Julita, Pereira, Risaralda – Colombia

lveloza@utp.edu.co

Received: December 10th, 2010; Accepted: March 16th, 2011

Dimethyldioxirane (DMDO), a widely used oxidant in organic synthesis is considered an environmentally friendly oxygen transfer reagent
because acetone is the only byproduct formed in its oxidation reactions. This work describes the isolation of the main constituents (terpenes)
in the essential oils obtained from Tagetes lucida, Cymbopogon citratus, Lippia alba and Eucalyptus citriodora, their epoxidation with
DMDO in acetone solution and the characterization of the resulting epoxides by GC-MS (EI) and NMR. This is one of the first reports
involving the application of dioxirane chemistry to essential oils in order to generate modified compounds with potential uses in several areas
of medicine and industry.

Keywords: dimethyldioxirane, epoxidation, estragole, citral, carvone, citronellal.

The use of dimethyldioxirane (DMDO), a member of a Typically, enones are epoxidized with alkaline hydrogen
new class of three-membered ring peroxide containing peroxide (H2O2) [8], but in some cases the hydrolytically-
oxidants, has increased notably in recent years due to its sensitive epoxide products open via a side reaction to form
ability to do oxygen atom transfer reactions with a wide a diol. The three types of olefins mentioned above have
range of substrates, including C=C bonds [1], C-H been successfully oxidized with DMDO in high regio- and
insertions in hydrocarbons [2], as well as oxidations of stereoselectivity.
atoms containing lone pairs of electrons, such as
sulfides [3] and primary and secondary amines [4]. DMDO Several methods for the generation of epoxides from
is also considered to be an environmentally friendly alkenes have been developed, such as the Sharpless [9] and
oxygen transfer reagent, and is attractive from the “green Jacobsen [10] catalytic epoxidations, the use of alternative
chemistry” [5] point of view due to its lack of toxic or routes involving the formation of intermediate halohydrins
harmful metals. DMDO’s reactions occur under extremely by using trichloroisocyanuric acid in aqueous acetone [11]
mild and neutral conditions, and the only byproduct of its and the use of peracids to generate both mono- and
reactions is acetone, which can be easily removed. Thus, bisepoxides, as well as cleaved oxidation products. A
DMDO reactions do not require complicated purification system involving a hydrogen peroxide-dinuclear
procedures to obtain pure products. manganese (IV) complex and a carboxylic acid has been
successfully employed for the efficient epoxidation of
This electrophilic oxygen transfer reagent is the reagent of terpenes such as limonene, citral, carvone and linalool,
choice for the epoxidation of both conjugated and while other sterically hindered terpenes (i.e., citronellal, -
unconjugated double bonds containing other functional and-pinene) were epoxidized in low yields [12]. The
groups, such as hydroxyls or carbonyls. Isolated double selective oxidation of monoterpenes (i.e., limonene,
bonds are selectively and easily oxidized under mild terpinene, neryl acetate, geranyl acetate, citral and
conditions (0-25°C and neutral pH) [6]. On the other hand, geranyl nitrile) with H2O2 catalyzed by
allylic alcohols are oxidized frequently with tert-butyl peroxotungstophosphate under biphasic conditions
hydroperoxide (TBHP) [7] in the presence of transition produced mono- and bisepoxides in good yields [13].
metals, which are necessary to initiate the reaction. While alkene epoxidation has found widespread use in the
926 Natural Product Communications Vol. 6 (7) 2011 Veloza et al.

total synthesis of natural products, there are very few As an important application of dioxirane chemistry to
reports in the literature of regioselective epoxidations of essential oils, this work describes the epoxidation of the
natural products with DMDO. Therefore, in this work, we main constituents of the essential oil from Tagetes lucida,
have carried out the DMDO epoxidation of the main Cymbopogon citratus, Lippia alba and Eucalyptus
terpene constituents in the essential oils extracted from citriodora with acetone solutions of DMDO. In general,
Tagetes lucida, Cymbopogon citratus, Lippia alba and the importance of the derivatives generated is due to their
Eucalyptus citriodora, which makes this one of the first polyfunctionality and the high reactivity of the oxirane
reports where dioxirane chemistry has been applied to ring. Such terpene epoxides can be readily transformed
isolated components of essential oils. into alcohols, aldehydes, ketones and heterocycles, which
give these terpene epoxides a promising position in terpene
Tagetes lucida (Asteraceae) is an endemic plant that is chemistry with future applications in industry and
widely distributed in Central and South America, and is medicine.
known in Colombia as “Estragón de invierno” [14]. Its
extracts are reported to possess bactericidal [15], The chemical composition and identity of the main
antimycotic [16] and antioxidant [17] activities, but this constituents of the essential oils obtained from Tagetes
plant has not been well studied from the chemical point of lucida, Cymbopogon citratus, Lippia alba and Eucalyptus
view. In Colombia, the essential oil extracted from this citriodora were determined by GC-MS (EI). The main
plant contains a large amount (93.8%) of a constituent of the oil from T. lucida was estragole, which
phenylpropanoid, estragole (1-allyl-4-methoxybenzene), had a relative abundance of 93.8% and a retention time of
which is present in many vegetables as a flavoring [18]. 7.7 minutes. Citral was the main component of the oil from
C. citratus. It had a relative abundance of 74% and
Cymbopogon citratus (Poaceae) is known worldwide as retention times of 8.1 and 8.3 minutes for the isomers.
the herb lemongrass, and is widely used in folk medicine Carvone was the main constituent of the oil from L. alba,
due to its antimicrobial [19], anti-inflammatory, with a relative abundance of 42% and a retention time of
antispasmodic [20] and antifungal [21] activities. The 7.9 minutes. Citronellal was the main constituent of the oil
essential oil from lemongrass has been widely used in the from E. citriodora with a relative abundance of 74% and a
perfume and cosmetics industries. It has also been used in retention time of 6.7 minutes. All of the compounds were
chemical synthesis, due to its high citral (3,7-dimethyl-2,6- isolated in high purity (95%), which was confirmed by
octadienal) content. Citral exists as a natural mixture of GC-MS.
two isomeric aldehydes, geranial and neral. Recently,
some citral derivatives with potential bactericidal activity The epoxidation of estragole, citral, carvone and
were generated using “green” thiol conjugate additions citronellal with DMDO in an acetone solution
[22]. Citral is a polyfunctional molecule that mediates a afforded epoxyestragole 1, 6,7-epoxycitral 2a and 2b,
wide number of chemical transformations, mainly 8,9-epoxycarvone 3 and 6,7-epoxycitronellal 4 (Figure 1),
epoxidation reactions. with greater than 95% conversion (confirmed by GC-MS).
The resulting epoxides were stored at room temperature
Lippia alba (Verbenaceae) is a shrub widely distributed and evaluated at several points during the course of one
from Mexico to South America [23] and is known in year and found to be stable (data not shown).
Colombia as “Pronto alivio”. L. alba essential oil and some
organic and polar extracts have shown analgesic, anti- O
6
CHO

inflammatory [24], antifungal [25], and antibacterial [26] O O 7

properties. In Colombia the essential oil from this plant is 1 2a, 2b


9

distinguished by its high content of S-carvone [2-methyl-5-


O
(1-methylethenyl)-2-cyclohexene-1-one] (40-57 %), a 10 O

substance with a high-value for the perfume and cosmetics 7 8


7
6
CHO

industries and as a starting material for fine organic 3


O 9
9
synthesis [27]. 3 4

Figure 1: Epoxides obtained from the DMDO reaction.


Eucalyptus citriodora (Myrtaceae) is an aromatic specie
known as “lemon scented gum”. Water extracts from dried Estragole was chosen for DMDO epoxidation because it
leaves of E. citriodora are traditionally used as possesses allyl and methoxy substituents. The isolated
antipyretics, anti-inflammatory and analgesic [28]. E. double bond does not have electron-donating groups that
citriodora essential oil has been shown to contain high would promote epoxidation. In addition, the electron-
concentration (70-80%) of citronellal (3,7-dimethyl-6- donating methoxy group (–OMe) bound to the aromatic
octenal) [29], which is effective against bacterial and ring does not have a direct influence on the olefin’s
fungal infections [30]. Besides, this compound is attractive reactivity. Under the conditions employed for the reaction,
from the stereochemical point of view since it can be used 1 was obtained in a short period of time (0.5-1 h) in
in an efficient way to introduce a new stereogenic center in 85% yield (101 mg). When this substrate and a series of
more complex structures [31]. other substrates, including unsubstituted styrenes and
Dimethyldioxirane epoxidation of essential oils Natural Product Communications Vol. 6 (7) 2011 927

cycloalkenes, were epoxidized with aqueous H2O2 and a citral’s terminal double bond due to the presence of its
polystyrene-supported triphenylarsine reagent, the electron-donating substituents. The exclusive formation of
moderately unstable epoxide products were isolated in the monoepoxide shows that the oxidation of the aldehyde
poor yield [32]. function to a carboxylic acid did not occur under the
reaction conditions employed, which indicates that DMDO
Kim, et al. [33] synthesized trans-anethole oxide from promotes the epoxidation of the double bonds in
trans-anethole (an isomer of estragole) using an acetone preference to oxygen insertion.
solution of DMDO as the oxidizing reagent. This oxide
was stable for one year, and reaction’s yield was >95%. Other studies have shown that under the same conditions
This yield was much better than that obtained by Mohan used in our epoxidation of citral, the oxidation of geraniol
and Whalen [34] and Greca et al [35], who used m-CPBA (the allylic alcohol corresponding to the reduction of citral)
to oxidize the same substrate, but obtained low yields with DMDO produces the 2,3 and 6,7 epoxides, as well as
(38%) that were complicated by the presence of m- the bis-epoxide [36]. Clearly, the nature of the substrate is
CPBA’s acid byproduct. These results and estragole’s an important factor in determining the regioselectivity of
structural characteristics provide evidence supporting the the epoxidation reaction. In the case of geraniol, the
high reactivity of DMDO. Therefore, it is clear that this formation of the 2,3 epoxide has been explained in terms
oxygen transfer reagent is the reagent of choice for the of the stability gained from an intermolecular hydrogen
epoxidation of double bonds, including electron deficient bond formed by the hydroxyl and the DMDO molecule in
olefins. The use of this oxidant offers a variety of the epoxidation transition state. The lack of 2,3 epoxycitral
advantages; it is generated from readily available reagents, formation can be explained by the inductive electron-
it reacts to generate epoxides under mild conditions and its withdrawing effect of the carbonyl group, which lowers
only byproduct is acetone and prevents the need for work- the nucleophilicity of the adjacent double bond. The
up conditions that can cause the opening of the oxirane resonance effect of the -unsaturated aldehyde also
ring. contributes to the decreased reactivity of the double bond.
Finally, in the case of citral, the formation of a hydrogen
Another substrate chosen for DMDO epoxidation was bond between the substrate and the DMDO molecule is not
citral, which is widely distributed in nature as a mixture of possible.
E/Z isomers. This substrate can undergo a wide range of
reactions due to its multiple functional groups, including The treatment of citral with H2O2 in an alkaline medium in
two trisubstituted double bonds, one of which is adjacent the presence of a phase-transfer catalyst gives rise to the
to a carbonyl group. One benefit of using this substrate is formation of the 2,3 epoxide and 6-methyl-5-hepten-2-one,
that the regioselectivity in the epoxidation of the 6,7 which forms as a result of the epoxide’s decomposition
double bond versus the 2,3 double bond provides a [37]. Alternatively, treating citral with peracetic acid gives
measure of the relative rate of reaction of the two olefins the 6,7-epoxy derivative and the (E/Z)-2,6-dimethyl-5,6-
that can be read from the ratio of the two possible epoxy-1-heptenyl isomers resulting from a Baeyer-Villiger
monoepoxides (i.e., the 6,7 and 2,3 epoxides) because this side reaction [38]. The formation of the 6,7 derivative has
is a simple intramolecular competition experiment. also been reported in moderate yield (43%) by Woitiski
Although the two double bonds are trisubstituted, the [12] by reacting citral with a H2O2-dinuclear manganese
inductive electron- withdrawing effect of the carbonyl (IV) complex of oxalic acid.
group lowers the nucleophilicity of the 2,3 double bond,
and if electronic properties are the decisive factor in the Carvone is another substrate chosen for DMDO
reactivity, then preferential epoxidation of the 6,7 double epoxidation, which contains two electron-poor double
bond would be expected. bonds, the exocyclic double bond due to the low number of
electron-donating alkyl substituents and the endocyclic
The 6,7-epoxycitral 2a, 2b was obtained as a mixture of bond due to the bearing electron-withdrawing effect of the
diastereoisomers with an E/Z ratio of 50:50 which carbonyl group. These carvone’s structural characteristics
corresponds to the content in essential oil and in 87% yield allow to compare the reactivity of the two double bonds in
(92 mg). The preferential epoxidation of the 6,7 double an intramolecular competition experiment with this cyclic
bond is probably due to its greater nucleophilicity, which substrate. The results show the regioselectivity in the
is a consequence of its electron-donating alkyl reaction of carvone with DMDO and the formation of the
substituents. Conversely, the reactivity of the 2,3 double exocyclic monoepoxide 3 as the only product of the
bond is drastically reduced due to the electron- reaction in 80% yield (102 mg) and 50:50
withdrawing effect of the conjugated carbonyl group, diastereoselectivity. The selective epoxidation of the
which results in much lower nucleophilicity. This result carvone exocyclic double bond corroborates that DMDO is
shows the electrophilic character of DMDO and its a powerful reagent for the epoxidation of electron-poor
preference for electron-rich double bonds. In addition, the double bonds. The lack of endocyclic oxirane formation
high conversion to the 6,7 monoepoxide indicates that the demonstrates the low nucleophilicity of the enone double
reaction’s rate increases with the nucleophilic character of bond due to the carbonyl group electron-withdrawing
effect.
928 Natural Product Communications Vol. 6 (7) 2011 Veloza et al.

In the epoxidation of carvone under oxygen using Experimental


Nickel(II) acetylacetonate as catalyst, (R)-(-)-carvone
General: Gas Chromatography-Mass Spectrometry (GC-
exhibited low reactivity due to long times reaction to
MS) analyses were performed using a Shimadzu GC-2010
obtain the exocyclic monoepoxide [39]. When the
gas-chromatograph coupled to a selective mass detector
anhydrous hydrogen peroxide/alumina system is used, a
(Shimadzu QP-2010) and equipped with an Rtx-5Sil-MS
high regioselectivity of the exocyclic monoepoxide
(93.8%) and a low substrate conversion (8.7%) were column (30 m x 0.25 mm i.d., 0.25 m film thickness)
observed, however, yields decreased to 0.8% in the operating in electronic ionization mode at 70 eV. Helium
absence of alumina catalyst [40]. was used as the carrier gas. All data processing was done
on the Shimadzu Labsolutions software (GCMS Solution
Citronellal is an aliphatic olefin which contains an version 2.5), which includes the Wiley Registry of Mass
aldehyde group and a terminal trisubstituted double bond. Spectral Data, 7th Edition (Wiley Interscience, New York).
1
This substrate can be used as a chemoselectivity probe for H NMR spectra were acquired on a Bruker Avance
DMDO epoxidation in terms of the aldehyde oxidation to DRX400 (400.13 MHz) spectrometer at 25 °C using
carboxylic acid versus the double bond epoxidation. The CDCl3 as the solvent and TMS as an internal standard.
results show the double bond oxidation leading to the
formation of the diastereomeric epoxides in 84% yield Essential oils extraction: Essential oils were obtained
(272 mg) and 50:50 ratio, confirming the high reactivity from plant leaves by microwave-assisted hydrodistillation
and chemoselectivity of DMDO to nucleophilic olefins. (MWHD) at CENIVAM (Centro Nacional de
Similar results were obtained for the epoxidation of Investigaciones para la Agroindustrialización de Especies
Citronellal with tert-butyl hydroperoxide (TBHP) and Vegetales Aromáticas y Medicinales Tropicales),
molybdenum catalyst in nonpolar and aprotic solvents, Universidad Industrial de Santander, Bucaramanga
giving a mixture of diastereomeric epoxides with a 50:50 (Colombia).
ratio and 71% yield [41].
Terpenes isolation: The essential oils from T. lucida (800
Essential oils are an important source of terpenes that can mg) and L. alba (812 mg) were fractionated over a silica
be modified chemically to generate a wide variety of gel column eluting with n-hexane-chloroform (90:10) and
polyfunctional compounds. From the chemical point of increasing the polarity of the solvent to 50:50. Estragole
view, epoxidation of the main constituents of Tagetes (119 mg) and carvone (128 mg) were obtained with a
lucida, Cymbopogon citratus, Lippia alba and Eucalyptus purity of 95% as determined by GC-MS. The structure of
citriodora with DMDO generates oxirane derivatives and each compound was identified by comparison of their
provides evidence for the high reactivity of this new mass spectrum (Wiley Registry of Mass Spectral Data) and
oxygen transfer reagent. Although a large number of was further confirmed by comparison to 1H NMR data
compounds have been epoxidized with DMDO, to our reported in the literature.
knowledge, this is the first report of the synthesis of
estragole, citronellal and citral epoxides using DMDO. The essential oils from C. citratus (3 mL) and E.
Epoxide stability is an important characteristic when citriodora (3 mL) were distilled under reduced pressure
carrying out tests of biological activity. (60ºC/550 mm Hg and 90ºC/550 mm Hg, respectively).
Citral (106 mg) and citronellal (324 mg) were obtained
The high conversion of estragole and the good yield of with a purity of 95% as determined by GC-MS. The
epoxyestragole indicate that unactivated olefins can be structure of citral and citronellal were identified by
efficiently oxidized with DMDO. The epoxidation of citral comparison of their mass spectrum (Wiley Registry of
proved to be regioselective, providing only 6,7 epoxide as Mass Spectral Data) and was further confirmed by
a result of the higher nucleophilicity of the 6,7 double comparison to 1H NMR data reported in the literature.
bond relative to the 2,3 double bond, which is electron
deficient due to the electron-withdrawing effect of the Preparation of DMDO in acetone solution: A
conjugated carbonyl group. In addition, the use of citral concentrated solution of DMDO in acetone (0.11 M) was
shows that DMDO oxidizes double bonds in preference to prepared using Oxone (Caroate, 2KHSO5.KHSO4.K2SO4),
oxidizing aldehydes to carboxylic acids. For carvone, the acetone and NaHCO3 according to a previously reported
nucleophilicity of the double bond adjacent to the carbonyl literature procedure [42]. The DMDO concentration was
group is quite low due to the electronic-withdrawing effect determined by UV/Vis spectrophotometry.
of this group, which is an important reactivity factor. The
DMDO epoxidation of citronellal confirms the General procedure for the epoxidation of terpenes with
chemoselectivity of this oxidant agent in terms of the DMDO: Estragole, citral, carvone and citronellal were
aldehyde oxidation to carboxylic acid versus the double dissolved in acetone (1 mL) and 1.0-1.2 equiv. of DMDO
bond epoxidation. Our results indicate that it is important (0.11 M solution in acetone) was rapidly added at 25 °C.
to consider both the regio and chemoselectivity of the The solution was stirred at this temperature and monitored
DMDO in the epoxidation of natural compounds. by TLC and GC-MS until the peroxide test (KI/HOAc)
was negative (total reaction time 0.5-1 h). The solvent was
Dimethyldioxirane epoxidation of essential oils Natural Product Communications Vol. 6 (7) 2011 929

removed under reduced pressure to give the respective 8,9-Epoxycarvone (3)


epoxides in high purity. The structure of the epoxides was 1
H-NMR (400 MHz, CDCl3) Isomer A:  6.72-6.76 (1H,
confirmed with spectral data and comparison with spectral m, H-3), 2.71 (1H, d, J=4.4 Hz, H-9), 2.03-2.57 (6H, m, H-
data reported in the literature of 1 [43], 2a,2b [44], 3[45] 4, H-5, H-6, H-9), 1.77-1.80 (3H, m, H-7), 1.33 (3H, s, H-
and 4 [44]. 10).
Isomer B:  6.72-6.76 (1H, m, H-3), 2.68 (1H, d, J=4.8 Hz,
Epoxyestragole (1) H-9), 2.03-2.57 (6H, m, H-4, H-5, H-6, H-9), 1.77-1.80
H-NMR (400 MHz, CDCl3): 7.16 (2H, d, J=8.0 Hz,
1
(3H, m, H-7), 1.32 (3H, s, H-10).
H-5, H-9), 6.84-6.87 (2H, m, H-6, H-8), 3.79 (3H, s,
H-10), 3.09-3.11 (1H, m, H-2), 2.74-2.89 (3H, m, H-1, 6,7-Epoxycitronellal (4)
H-3), 2.52 (1H, dd, J=2.8 Hz, J=5.0 Hz, H-3). 1
H-NMR (400 MHz, CDCl3):  9.77 (1H x 2, m, H-1),
2.68-2.71 (1H x 2, m, H-6), 2.39-2.46 (2H, m, H-2’), 2.24-
6,7-Epoxycitral 2.30 (2H, m, H-2), 2.08-2-18 (1H x 2, m, H-3), 1.52-1.57
1
H-NMR (400 MHz, CDCl3) 2a epoxygeranial:  10.0 (4H x 2, m, H-4, H-5), 1.27, 1.31 (6H x 2, s, H-8, H-10),
(1H, d, J=8.0 Hz, H-1), 5.91-5.93 (1H, m, H-2), 2.71-2.77 1.00 (3H x 2, d, J=6.8 Hz, H-9).
(3H, m, H-4, H-6), 2.01 (3H, s, H-9), 1.64-1.83 (2H, m,
H-5), 1.28 and 1.31 (6H, s, H-8, H-10). Supplementary data: 1H-NMR spectra for compounds 1,
2b epoxyneral:  9.97 (1H, d, J=8.0 Hz, H-1), 5.91-5.93 2a, 2b, 3 and 4 are contained in the supplementary data.
(1H, m, H-2), 2.71-2.77 (1H, m, H-6), 2.29-2.48 (2H, m,
H-4), 2.20 (3H, s, H-9), 1.64-1.83 (2H, m, H-5), 1.28 and Acknowledgments - The authors gratefully acknowledge
1.31 (6H, s, H-8, H-10). financial support from COLCIENCIAS-CENIVAM
(contrato RS-432-2004), Universidad Tecnológica de
Pereira and Red ALMA MATER.

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2011
NPC Natural Product Communications Vol. 6
No. 7
Validation of the Ethnopharmacological Use of Polygonum 931 - 933

persicaria for its Antifungal Properties


Marcos Derita*and Susana Zacchino

Pharmacognosy Area, Faculty of Biochemical and Pharmaceutical Sciences, National University of


Rosario, Suipacha 531, Rosario, 2000, Argentina

mgderita@hotmail.com

Received: November 13th, 2010; Accepted: March 16th, 2011

Polygonum L. genus (Polygonaceae) is represented in Argentina by 21 species and some of them have been used in the traditional medicine
of our country to treat affections related with fungal infections, such as skin ailments and vaginal diseases. With the aim of contributing to the
correct ethnopharmacological use of this genus, in the present work we describe the antifungal properties of P. persicaria (species not studied
up to now) and the bio-guided isolation of the main active compounds. Results showed that dichloromethane extracts was the most active
with MICs (Minimun Inhibitory Concentrations) between 31.2 – 1000 µg/mL, validating the ethnopharmacological use of P. persicaria to
treat affections related with fungal infections in the Argentinean traditional medicine.

Keywords: Validation, Ethnopharmacological use, Polygonum, Antifungal activity.

Since the early 1980s, fungal infections have emerged as A


major causes of morbi-mortality, mainly among 11

immunocompromised patients. The majority of deaths


11
15 CHO
15 CHO 12
12 1
1 CHO
were associated with species of Candida, Aspergillus and CHO 9
9 2 10
2 8
10 8
5

Cryptococcus [1]. Instead, dermatophytes such as


5 3
3 4 7
4 7
6 6
H H

Trichophyton and Microsporum spp. produce superficial 13 14 (1) 13 14 (2)


B
infections (tineas) which are usually not threatening but 5

dramatically diminish the quality of life of human beings


6 4

B
H 5´ 1

[2]. Although it appears to be an array of antifungal agents


1 2´
8 3´ H3CO OCH3 3
H3CO O 4´ 
7
6´ 2
8a 2 1´ A
(polyenes, azoles, allylamines and the recent 6
4a
4
3




´

echinocandins) there are, in fact, few therapeutic options.


5 5´ 2´
H

OH O
H
(3) OH O (4)
Decreased susceptibilities of yeasts to the currently 6
5

available antifungal agents [3] added to the increase in the HO



OCH3
1
3

number of reported cases of resistance [4], have led to a


4´ 
6´ 2

general consensus that new efforts for detecting novel


3´ 
´

antifungal entities remain a priority. In this context, the OH O (5)


study of plants with history of ethnopharmacological use Figure 1: A) Sesquiterpene [polygodial (1) and isopolygodial (2)] and B)
for ailments related to fungal infections, can serve two flavonoids [pinostrobin (3), flavokawin B (4) and cardamonin (5)] isolated
from P. persicaria DCM extract.
goals: validation of the use of traditional medicines and
finding new leads [5]. of two sesquiterpene dialdehydes: polygodial (1),
isopolygodial (2), and three flavonoids: pinostrobin (3),
Polygonum L. genus (Polygonaceae) is represented in
flavokawin B (4) and cardamonin (5) (Figure 1).
Argentina by 21 species and some of them have been used
Compounds 1 and 2 were previously isolated from Drymis
to treat affections related with fungal infections, such as
spp. [9,10], P. punctatum [7] and P. acuminatum [8], while
skin ailments and vaginal diseases [6]. Previous studies of
compounds 3-5 were previously isolated from
this genus reported that P. punctatum possessed antifungal
Boesenbergia pandurata, Myrica pensilvanica, P.
properties against yeasts and dermatophytes [7]. With the
ferrugineum and Piper spp [11-13].
aim of contributing to the correct ethnopharmacological
use of this genus, in a previous work we described
Compounds 1-5 were evaluated for their antifungal
the antifungal properties of P. acuminatum [8] and
activities with the microbroth dilution assay recommended
in this work we describe those of P. persicaria (species
by the Clinical and Laboratory Standards Institutes (CLSI)
not studied up to now) and the bio-guided isolation
932 Natural Product Communications Vol. 6 (7) 2011 Derita M. et al.

Table 1: Antifungal activity (MICs in µg/mL) of P. persicaria extracts.


Species Extract Antifungal activity (MICs in µg/mL)
Ca Sc Cn Afu Afl An Mg Tr Tm

P. persicaria Hex I I 1000 I I I 1000 500 1000


DCM 1000 500 500 1000 1000 1000 125 62.5 31.2
EtOAc 1000 1000 1000 I I I 1000 500 1000
MeOH I I I I I I I I I
Standards drugs Ketoconazole 0.50 0.50 0.25 0.12 0.50 0.25 0.04 0.02 0.02
Amphotericin 1.00 0.50 0.25 0.50 0.50 0.50 0.12 0.07 0.07
Terbinafine - - - - - - 0.04 0.01 0.04
Ca: Candida albicans ATCC 10231; Sc: Saccharomyces cerevisiae ATCC 9763; Cn: Cryptococcus neoformans ATCC 32264; Afu: Aspergillus fumigatus ATCC 26934;
Afl: Aspergillus flavus ATCC 9170; An: Aspergillus niger ATCC 9029; Mg: Microsporum gypseum C 115; Tr: Trichophyton rubrum C 113; Tm: Trichophyton
mentagrophytes ATCC 9972. I: Inactive = MIC > 1000 µg/mL.

Table 2: Antifungal activity (MICs in µg/mL) of compounds isolated from P. persicaria.

Compounds Antifungal activity (MICs in µg/mL)


Ca Sc Cn Afu Afl An Mg Tr Tm
1 3.90 15.6 7.8 250 250 250 62.5 7.8 7.8
2 250 125 125 250 250 250 62.5 62.5 31.2
3 250 250 250 125 125 250 62.5 62.5 62.5
4 I I 250 250 125 250 125 125 125
5 250 250 250 250 250 250 62.5 15.6 15.6
Standards drugs Ketoconazole 0.50 0.50 0.25 0.12 0.50 0.25 0.04 0.02 0.02
Amphotericin 1.00 0.50 0.25 0.50 0.50 0.50 0.12 0.07 0.07
Terbinafine - - - - - - 0.04 0.01 0.04
Ca: Candida albicans ATCC 10231; Sc: Saccharomyces cerevisiae ATCC 9763; Cn: Cryptococcus neoformans ATCC 32264; Afu: Aspergillus fumigatus ATCC 26934;
Afl: Aspergillus flavus ATCC 9170; An: Aspergillus niger ATCC 9029; Mg: Microsporum gypseum C 115; Tr: Trichophyton rubrum C 113; Tm: Trichophyton
mentagrophytes ATCC 9972. I: inactive = MIC > 250 µg/mL).

[14] and results are shown in Table 2. They were active useful in guiding the discovery of antifungal compounds
against yeasts, Aspergillus spp. and dermatophytes with against dermatophytes, as it was demonstrated in a recent
MICs between 3.90 - 250 µg/mL survey among seven Latinaoamerican countries [15].

As it can be observed in Table 2, the five compounds Experimental


isolated from P. persicaria, drimanes as well as Extracts preparation and compounds isolation: Air-dried
flavonoids, all showed antifungal activity. Among them, aerial parts of each species (100 g) were powdered and
polygodial (1) showed the best activity against yeasts and successively macerated (3×24 h each) with Hexane (Hex),
dermatophytes with MICs between 3.9 to 62.5 µg/mL and dichloromethane (DCM), ethyl acetate (EtOAc) and
it was almost inactive against species of Aspergillus genus. methanol (MeOH) with mechanical stirring to obtain the
Its epimer, isopolygodial (2), showed a lower antifungal corresponding extracts, after filtration and evaporation.
activity (MICs between 31.2 to 250 µg/mL), suggesting Bioassay-guided fractionation of DCM extract allowed us
that the C-9 configuration plays an important role in the to isolate the compounds responsible for the antifungal
antifungal activity, as we have been found in a previous activity. 1.1 g of P. persicaria DCM extract were
paper [8]. submitted to column chromatography using mixtures of
Hex: AcOEt in increasing polarity as elution solvents. We
Regarding flavonoids 3-5, there is not a clear difference obtained 10 fractions; three of them were actives (fractions
among the antifungal activities of them against yeasts 6-8). From 150 mg of fraction 6, by repeated column
and Aspergillus spp. Nevertheless, chalcone 5 showed chromatography, we obtained 55 and 30 mg of compounds
a high antifungal activity against T. rubrum and T. 1 and 2 respectively. From 170 mg of fraction 7, by
mentagrophytes with MICs = 15.6 µg/mL, eight times repeated column chromatography, we obtained 50, 46 and
higher than the activity showed by chalcone 4 against the 25 mg of compounds 3, 4 and 5 respectively. Additionally,
same strains. This striking difference in activity against from 70 mg of fraction 8, we obtained 10 mg of compound
Trichophyton spp. could be attributed to the phenolic OH 5. All the compounds were characterized by UV-visible,
present in compound 5 which is absent in 4. IR, 1H NMR and 13C NMR spectroscopy.
These results show that the antifungal activity of P.
Antifungal assay: For the antifungal evaluation, strains
persicaria could be attributed to polygodial but it is clear
from the American Type Culture Collection (ATCC,
that the rest of the isolated compounds could contribute to
Rockville, MD, USA) and Centro de Referencia en
the antifungal behavior of this traditional used species. In
Micología, CEREMIC [C, Faculty of Biochemical and
addition, these results validate the ethnopharmacological
Pharmaceutical Sciences, Suipacha 531 (2000)-Rosario,
use of P. persicaria to treat affections related to fungal
Argentina] were used: Candida albicans (Ca) ATCC
infections in the Argentinean traditional medicine and add
10231, Saccharomyces cerevisiae (Sc) ATCC 9763,
a new evidence that the ethnopharmacological approach is
Cryptococcus neoformans (Cn) ATCC 32264, Aspergillus
Ethnopharmacological use of Polygonum Natural Product Communications Vol. 6 (7) 2011 933

flavus (Afl) ATCC 9170, Aspergillus fumigatus (Afu) techniques according to the guidelines of CLSI for yeasts:
ATTC 26934, Aspergillus niger (An) ATCC 9029, document M27-A2 and for filamentous fungi, M38A. For
Trichophyton rubrum (Tr) C 110, Trichophyton the assay, stock solutions of extracts or pure compounds
mentagrophytes (Tm) ATCC 9972 and Microsporum (100 µL) were two-fold diluted with the culture medium.
gypseum (Mg) C 115. Strains were grown on Sabouraud- A volumen of 100 µL of inoculum suspension [adjusted to
chloramphenicol agar slants for 48 h at 30 ºC, maintained 1–5 × 104 cells/spores as Colony Forming Units
on slopes of Sabouraud-dextrose agar (SDA, Oxoid) and (CFU/mL)] was added to each well with the exception of
subcultured every 15 days to prevent pleomorphic the sterility control where sterile water was added to the
transformations. Inocula of cell or spore suspensions were well instead. Ketoconazole (Sigma Chem. Co., St. Louis,
obtained and quantified following reported procedures MO), Terbinafine (Novartis) and Amphotericin B (Sigma)
(CLSI).[14] were used as positive controls.

Minimum Inhibitory Concentration (MIC) of each extract Acknowledgments - CONICET, ANPCyT, UNR,
or compound was determined by using broth microdilution ERASMUS MUNDUS, UNIBO.

References

[1] Pfaller M, Diekema D. (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clinical Microbiology
Reviews, 20, 133-163
[2] Weitzman I, Summerbell R. (1995) The dermatophytes. Clinical Microbiology Reviews, 8, 240-259
[3] Hsueh P, Lau Y, Chuang Y, Wan J, Huang W, Shyr J, Yan J, Yu K, Wu J, Ko W, Yang Y, Liu Y, Teng L, Liu Ch, Luh K. (2005)
Antifungal susceptibilities of clinical isolates of Candida species, Cryptococcus neoformans, and Aspergillus species from Taiwan:
Surveillance of multicenter antimicrobial resistance on Taiwan program data from 2003. Antimicrobial Agents and Chemotherapy,
49, 512-517
[4] White T, Holleman S, Dy F, Mirels L, Stevens D. (2002) Resistance mechanisms in clinical isolates of Candida albicans.
Antimicrobial Agents and Chemotherapy, 46, 1704-1713
[5] Verpoorte R. (2000) Pharmacognosy in the new millennium: leadfinding and biotechnology. Journal of Pharmacy and
Pharmacology, 52, 253-262
[6] Del Vitto L, Petenatti E, Petenatti M. (2003) Materia Medica Vegetal. Plantas medicinales nativas y exóticas empleadas en
fitomedicinas, homeopáticos, galénicos. Servicio Técnico Herbario UNSL, San Luis, pp. 1-63
[7] De Almeida Alves T, Lacerda Ribeiro F, Kloos H, Zani C. (2001) Polygodial, the fungitoxic component from the Brazilian
medicinal plant Polygonum punctatum. Memórias do Instituto Oswaldo Cruz, 96, 831-833
[8] Derita M, Leiva M, Zacchino S. (2009) Influence of plant part, season of collection and content of the main active constituent, on
the antifungal properties of Polygonum acuminatum Kunth.. Journal of Ethnopharmacology, 124, 377-383
[9] Cechinel Filho V, Schlemper V, Santos A, Pinheiro T, Yunes R, Mendes G, Calixto J, Delle Monache F. (1998) Isolation and
identification of active compounds from Drymis winteri barks. Journal of Ethnopharmacology, 62, 223-227
[10] Muñoz-Concha D, Vogel H, Yunes R, Razmilic I, Bresciani L, Malheiros A. (2007) Presence of polygodial and drimenol in Drymis
populations from Chile. Biochemical Systematics and Ecology, 35, 434-438
[11] Hodgetts K. (2001) Approaches to 2-substituted chroman-4-ones: synthesis of (-)-pinostrobin. Tetrahedron Letters, 42, 3763-3766.
[12] Burke B, Nair M. (1986) Phenylpropene, benzoic acid and flavonoids derivatives from fruits of Jamaican Piper species.
Phytochemistry, 25, 1427-1430
[13] López S, González Sierra M, Gattuso S, Furlán R, Zacchino S. (2006) An unusual homoisoflavanone and a structurally-related
dihydrochalcone from Polygonum ferrugineum. Phytochemistry, 67, 2152-2157
[14] CLSI (Clinical and Laboratory Standards Institute). (2002) Methods M 27-A2, Vol. 22 (15): 1-29 and M 38-A, Vol. 22 (16): 1-27.
Wayne Ed.
[15] Svetaz L, Zuljan F, Derita M, Petenatti E, Tamayo G, Cáceres A, Cechinel Filho V, Giménez A, Pinzón R, Zacchino S, Gupta M.
(2010) Value of the ethnomedical information for the discovery of plants with antifungal properties. A survey among seven Latin
American countries. Journal of Ethnopharmacology, 127, 137-158
2011
NPC Natural Product Communications Vol. 6
No. 7
On the Isomerization of ent-Kaurenic Acid 935 - 938

Julio Rojasa, Rosa Apariciob, Thayded Villasmilc, Alexis Peñac and Alfredo Usubillagab*
a
Postgrado de Quimica de Medicamento, Facultad de Farmacia y Bioanálisis,
Universidad de Los Andes, Mérida, Venezuela
b
Instituto de Investigaciones, Facultad de Farmacia y Bioanálisis, Universidad de Los Andes, Mérida,
Venezuela
c
Postgrado Interdisciplinario de Química, Facultad de Ciencias, Universidad de Los Andes, Mérida,
Venezuela

usubillaga@intercable.net.ve

Received: December 12th, 2010; Accepted: March 15th, 2011

Kaurenic acid (1a) is a tetracyclic diterpene that has an exocyclic double bond at  Isokaurenicacid (2a) has an endocyclic 15double
bondThis compound has been isolated from Espeletia tenore (Espeletinae), a resinous plant from the Venezuelan Andes, but its occurrence
is rare. In order to obtain a larger amount of 2a, the isomerization of 1a, which is easily obtained from other Espeletinae, was tried. Kaurenic
acid methyl ester (1b) was treated with dil. HCl in CH3Cl/EtOH, after 6 h under reflux a yield of 41.5% isokaurenic acid methyl ester (2b)
was obtained but 35.7% 16-ethoxy-kauran-19-oic acid methyl ester (3b) had formed as a byproduct. Treating 1b with CF3COOH in
refluxing CH2Cl2 permitted to obtain a yield of 66.6 % of 2b in 4 h and only traces of 16-hydroxy-kauran-19-oic acid methyl ester (3a) as
a byproduct. Both isomers were separated on a silica gel column impregnated with 20% AgNO3. Treating 2b with KOH in refluxing DMSO
yielded pure isokaurenic acid, no back isomerization was observed.

Keywords: Ent-kaur-16-en-19-oic acid, ent-kaur-15-en-19-oic acid, CF3COOH, methyl esters, silver nitrate chromatography.

Ent-kaurenic acid (1a, Figure 1) is a tetracyclic diterpene 20 11 13


17

that has been reported to have antimicrobial [1] and 1


15
16

8 R
antiparasitic activity [2]. It also shows cytotoxicity against 4

several cancer cell lines [3]. The occurrence of kaurenic 19


COOR
acid, which has an exocyclic double bond at Δ16, is COOR COOCH3

1a R= H 2a R= H 3a R= OH
widespread in the plant kingdom, while the occurrence of 1b R= CH3 2b R= CH3 3b R= OC2H
5
its isomer ent-kaur-15-en-19-oic acid (2a), also called Figure 1
isokaurenic acid, is rare. This substance (2a) was first
obtained by Ekong and Ogan by lithium reduction of Since it had been observed that, during isolation of the
Xylopic acid [4a], but 2a has been isolated as a natural acidic fraction of some Espelettinae, treatment with HCl
product from Espeletia tenore, a midget Espeletiinae from produced traces of isokaurenic acid, isomerization of 1b
the Venezuelan Andes [4b]. in CHCl3/EtOH solution was tried adding 5 drops of
HCl:H2O (10:1) at room temperature. The course of the
Since the quantity of isokaurenic acid that could be reaction was followed by gas chromatography. As it is
obtained from E. tenore is small, isomerization of kaurenic shown on Table 1, after one hr of reaction 35.4% of the
acid was studied in order to obtain it in sufficient quantity original amount of 1b had isomerized into 2b, while the
to explore its biological properties. Double bond migration relative concentration of 1b had diminished to 55.6%, but
in olefins could be base-catalyzed or acid-catalyzed at the same time 9.0% of ent-16α-ethoxy-kauran-19-oic
[5a,5b]. Base-catalyzed isomerization can be effected in acid (3b) had formed as a by-product. After 4 hr of
homogeneous solution or in the presence of basic reaction a maximal yield of 2b was achieved (46.4%), but
heterogeneous catalysts. Isomerization of 1b was tried after 6 hr the relative concentration of 2b had diminished
with sodium on alumina according to Shabtai and Gil-Av and settled at 41.4%. At the same time the by-product (3b)
[6], but no reaction was observed after 24 hours. represented 35.7% of the reaction mixture.
Isomerization was also attempted treating 1b with iodine
in benzene solution under reflux, according to Barnes and Since formation of 3b was undesirable, isomerization
MacMillan [7] but only 10.6% of 2b was obtained after 6 was tried using CF3COOH in dry CH2Cl2.The course of
hr. the reaction is shown on Table 2. After 1.0 hr of reaction
936 Natural Product Communications Vol. 6 (7) 2011 Rojas et al.

Table 1: Acid-isomerization of ent-kaurenic acid using HCl in


CHCl3/EtOH.
Time (hr) % 2b % 1b % 3b
0 -------- 100 ------
1 35.4 55.6 9.0
2 46.4 35.3 18.2
4 41.8 24.7 33.5
6 41.5 22.8 35.7

54.4% of isomerization had been achieved. After 2.0 hr the


yield was 63.5%. After 4.0 hr the reaction had arrived to
equilibrium where relative concentration of 2b was 66.6%
and concentration of 1b was down to 33.3%. But after 4.0
hr it was observed that 0.1% of 3a had been formed,
probably caused by the presence of traces of water. Figure 2

Table 2: Acid-isomerization of ent-kaurenic acid using CF3COOH. Experimental


Time (hr) % 2b % 1b % 3b General procedures: Melting points were determined on a
0 -------- 100 ------ Fisatom 430 D apparatus and are uncorrected. IR spectra
1 54.4 45.6 0 were measured on a Shimadzu Affinity instrument as KBr
2 63.5 36.5 0 discs. NMR spectra were recorded with a Bruker Avance
3 66.2 33.8 t 400 MHz instrument for solutions in CDCl3, 1H, 13C,
4 66.6 33.3 0.1 DEPT, H-H COSY, HMQC, and HMBC experiments
t : traces were performed. GC was made on a Perkin Elmer
Autosystem gas chromatograph equipped with FID
Figure 2 shows the increase of 2b as a function of time and detector. A 5% phenylmethyl polysiloxane capillary
the abatement of kaurenic acid methyl ester concentration column was used (30 m, 0.25 mm i.d., film thickness 0.25
(1b) to end up with a constant concentration mixture made m). The oven temperature was programmed from 250°C
up of 66.6% 2b and 33.3% 1b. Absolute exclusion of to 300°C at 10°C/min., and kept isothermal at the higher
water would hinder the formation of 16-hydroxy-kauran- temperature for 10 min. The injector and detector
19-oic acid methyl ester (3a). But from a practical temperatures were 200°C and 300°C respectively. The
point of view two hours of reaction is enough to obtain carrier gas was helium at 0.9 mL/ min. The samples (1.0
a good yield (63.5%) of the desired product. Separation L) were injected using a split ratio of 1:10. For mass
of both isomers is accomplished over a silica gel spectrometry an Agilent MSD 5973 instrument equipped
column impregnated with 20% silver nitrate. Taking into with a DB-5MS capillary column (30 m, 0.25 mm, 0.25
consideration that silver nitrate chromatography is required m film).The oven temperature program was the same
to separate both isomers, the methyl ester of kaurenic acid used for GC analysis. Injector temperature, carrier gas, and
(1b) was used instead of the free acid (1a). Isokaurenic sample injection conditions were also the same but a split
acid (2a) was recovered refluxing 2b with KOH in DMSO ratio of 1:50 was used. Analytical TLC was performed on
solution. Merck aluminum-backed silica gel foils (F254). Flash
chromatography was performed on Merck silica gel Grade
According to Hubert and Reimlinger [5b] the acid 9385 (230-400 mesh) by gradient elution with hexane-
catalyzed isomerization takes place via carbonium ions. In EtOAc or hexane-diethyl ether mixtures. Kaurenic isomers
the case of kaurenic acid, presence of an acid causes the were separated on a silica gel column impregnated with
formation of a carbonium ion at C-16. The catalyst acts as 20% AgNO3.
a proton donor and acceptor. Migration of the double
bonds occurs because a proton is transferred to the C-17 Isolation of kaurenic acid (1a): Espeletia semiglobuta
exocyclic methylene moiety, which becomes a methyl was collected at Paramo of Piedras Blancas in February
group, and at the same time, a proton is lost from C-15 2009. A voucher specimen (AU-30) was deposited at the
generating a Δ15 double bond. But this process is MERF Herbarium. The leaves (10 Kg) were air dried,
reversible and the reaction proceeds until it reaches a ground and extracted with a hexane-diethyl ether mixture
thermodynamic equilibrium. In this case a Δ15 double (3:1) at room temperature. The extract was shaken with
bond is thermodynamically more stable and therefore its 0.5 N NaOH solution. The aqueous layer was made acidic
formation is enhanced leading to the migration of 2/3 of by addition of dil. HCl and shaken with hexane to recover
the original exocyclic double bond to a Δ15 position. the acid fraction. Kaurenic acid was purified by flash
chromatography over silica gel using hexane and hexane-
The rate of formation of 3a increases with reaction time. diethyl ether (9:1) as solvent. Chromatographic fractions
After 72 h under reflux the yield of 3a was 13%. were inspected by TLC, fractions containing pure kaurenic
acid were combined and crystallized Pure kaurenic acid
Isomerization of Kaurenic acid Natural Product Communications Vol. 6 (7) 2011 937

(13.5 g) was obtained, mp 175-178°C (lit [8] 179-181). When both isomers had eluted the column was treated with
It was compared with an authentic sample (TLC, hexane-EtOAc (10:1) to elute 3b.
1
H-NMR) [4b]. MP: 140°C.
IR (cm-1): 2981, 2932, 2854, 1724, 1466, 1443, 1234,
Kaurenic acid methyl ester (1b): A solution of kaurenic 1157, 1068, 987.
acid (1a, 20 mmol) was dissolved in Et2O and mixed with 1
H NMR (400 MHz, CDCl3): 0.77 (1H, dt, J = 4; 14 Hz,
freshly distilled diazomethane (about 30 mmol) obtained H-1a), 0.81 (3H, s, H-20), 0.96 (1H, m, H-9), 1.02 (1H,
from nitrosomethylurea [9a,9b]. After 24 h at room dd, J= 2; 14 Hz, H-5), 1.10 (3H, t, J = 7 Hz, Me), 1.17
temperature the solvent and excess diazomethane were (3H, s, H-18), 1.26 (3H, s, H-17), 1.32 (1H, m, H-15a),
distilled off at low pressure. Kaurenic acid methyl ester 1.38 (1H, m, H-14a), 1.5 (2H, m, H-12), 1.51 (2H, m, H-
(1b) crystallized from hexane, MP 75°C. GC retention 11), 1.52 (1H, m H-3a), 1.53 (2H, m, H-2), 1.75 (1H, m,
time 3.80 min. MS (EI, 70 eV): m/z (%) = 316.2 (51), 302 H-3b), 1.78 (2H, m, H-6), 1.84 (1H, m, H-1b), 2.02 (1H,
(35), 284 (62), 257 (199), 241 (79), 213 (33), 187 (2), 121 bs, H-13), 2.15 (2H, m, H-7), 3.31 (2H, q, J = 7 Hz,
(45), 91 (47). OCH2), 3.63 (3H, s, OMe).
13
C NMR (100 MHz, CDCl3): 178.2 (COO), 83.9 (C-17),
Attempted isomerization of 1b with sodium on alumina: 57.1 (C-5), 56.8 (OCH2), 56.2 (C-9), 55.2 (C-15), 51.2
The catalyst was prepared according to Shabtai and Gil-Av (OCH3), 44.9 (C-4), 43.9 (C-8), 42.3 (C-14), 44.0 (C-13),
[6]. Alumina was heated at 300oC during 24 h . Pretreated 40.9 (C-1), 39,6 (C-10), 38.3 (C-7), 37.2 (C-3), 28.9
alumina (5.0 g) was mixed with 1.0 g of sodium at 140°C (C-18), 26.8 (C-12), 22.3 (C-6), 19.3 (C-2), 19.2 (C-17),
with stirring under argon atm. The catalyst was cooled at 18.2 (C-11), 16.7 (CH3), 15.5 (C-20).
2-3°C and a solution of 1b (500 mg) in dry hexane was MS (EI, 70 eV): m/z (%) = 362 (4), 347 (34), 316 (85), 274
added. Samples were taken after continuous stirring at 1.0 (100), 257 (55), 217 (54), 121 (70).
h, 6.0 h, and 24 h, and analyzed by GC-MS after 1.0 h, 6.0
h, and 24 h, but no isomerization occurred and only the Isomerization of 1b with CF3COOH in CH2Cl2: To a
peak of 1b, with a retention time of 3.79 min., was solution of kaurenic acid methyl ester (5.0 mmol) in dry
observed on the gas chromatogram. CH2Cl2 (100 mL) 10 drops of trifluoroacetic acid were
added and the mixture was heated under reflux in an argon
Isomerization of 1b with iodine in benzene solution: atmosphere. Aliquot samples (5 mL) were taken at 1h, 2h,
Kaurenic acid methyl ester (1b, 300 mg) was dissolved in and 4h. They were washed with H2O; the organic layer
dry benzene containing 20 mg of iodine and it was heated was dried over Na2SO4 and evaporated to dryness.
under reflux for 6 hr. The solution was cooled and shaken Samples (5mg) were dissolved in Et2O and analyzed by
twice with aqueous sodium thiosulphate. The organic layer gas chromatography. Table 2 shows the results of this
was taken to dryness. A 5 mg sample of the solid was reaction as a function of time. To recover the isokaurenic
dissolved in diethyl ether and analyzed by GC. It was acid methyl ester the rest of the reaction mixture was
observed that after six hours of reaction only 10.6% of 1b washed with H2O, dried over Na2SO4, filtered, and the
had isomerized into 2b which had a retention time of 3.55 solvent was evaporated to dryness. The reaction product
min. was dissolved in hexane and chromatographed on a
column of silica gel impregnated with 20% of AgNO3. The
Isomerization of 1b with diluted HCl in CHCl3/EtOH: column was eluted with hexane; 100 mL fractions were
Kaurenic acid methyl ester (2.0 mmol) was dissolved in a taken and inspected by GC. Fractions 6-12 eluted pure 2b
mixture 50 mL of CHCl3 and 10 mL of EtOH. After (722 mg). MP 74-75°C. GC retention time 3.55 min. It was
addition of 5 drops of 10% HCl, the mixture was heated identical to an authentic sample obtained by methylation of
under reflux. Aliquot samples (5 mL) were taken at 1h, 2h, 2a isolated from Espeletia tenore [4b] (IR, 1H NMR, MS).
4h, and 6 h, washed with dil. NaHCO3, and with H2O. The Fractions 13-18 eluted a mixture of 1b and 2b (330 mg),
organic layer was dried over Na2SO4 and evaporated to and fractions 19-27 pure 1b (275 mg).To a solution of 500
dryness. Samples (5 mg) were dissolved in Et2O and mg of 2b in 50 mL of DMSO 300 mg of KOH were added
analyzed by GC. Results are shown on table 1. and the mixture heated under reflux for 2 h. The solution
was cooled; 50 mL of H2O was added, and taken to pH 3.0
Isolation of ent-16-ethoxy-kauran-19-oic acid methyl by addition of dil. HCl. The solution was then shaken with
ester (3b): The rest of the solution (40 mL) of the previous 100 mL of hexane. The hexane layer was shaken twice
isomerization reaction was washed with dil. NaHCO3 and with 20 mL of H2O, dried over Na2SO4, and the solvent
H2O. The chloroform layer was treated with dry Na2SO4, distilled under vacuum. The isokaurenic acid (455 mg) was
filtered, and mixed with 1 g of silica gel. CHCl3 was crystallized from hexane, MP 169-171°C. Identical to 2a
evaporated under vacuum and the silica gel containing the isolated from Espeletia tenore (IR, 1H NMR )[4b]. A
product of isomerization was added to the top of a flash sample (5 mg) was methylated and examined by GC. Only
column charged with 12 g of silica gel. The column was one peak was observed at 3.55 min (100%).
eluted with hexane which yielded a mixture of 1b and 2b.
938 Natural Product Communications Vol. 6 (7) 2011 Rojas et al.

Isolation of ent-16-hydroxy-kauran-19-oic acid methyl (1H, m, H-14b), 1.75 (2H, m, H-6), 1.80 (1H, bs, H-13),
ester (3a): To a solution of kaurenic acid methyl ester (2.0 1.82 (1H, m, H-2b), 1.84 (1H, m, H-1b), 1.89 (1H, m,
mmol) in CH2Cl2 five drops of trifluoroacetic acid were H-7b), 3.63 (3H, s, OCH3).
13
added and the mixture was heated under reflux. After 72 h. C NMR (100 MHz, CDCl3): 174.3 (COO), 79.7 (C-16),
the reaction mixture was cooled and shaken with H2O. The 58.1 (C-15), 57.3 (C-5), 56.3 (C-9), 51.4 (OCH3), 49.2
organic layer was dried over Na2SO4, filtered, and (C-13), 45.6 (C-8), 44.1 (C-4), 42.4 (C-7), 41.0 (C-1), 39.8
evaporated to dryness. A 5 mg sample was dissolved in (C-10), 38.4 (C-3), 37.9 (C-14), 29.0 (C-18), 27.1 (C-12),
Et2O and inspected by GC. The gas chromatogram showed 24.8 (C-17), 22.4 (C-6), 19.4 (C-2), 18.6 (C-11), 15.7
peaks at 3.54 (2b, 60%), 3.79 (1b, 27%), and 4.94 min (3a, (C-20).
13%). Flash chromatography over a silica gel column MS (EI, 70 eV): m/z (%) = 334 (11), 316 (100), 302 (26),
afforded 42 mg of 3a. 276 (67), 257 (73), 217 (30), 180 (32), 121 (85).
MP: 159-160°C.
IR (KBr, cm-1): 3412, 2982, 2953, 2868, 1728, 1424, 1157, Acknowledgments – This work was possible thanks to the
925. financial support of Mision Ciencia (Grant 2007000881).
1
H NMR (400 MHz, CDCl3): 0.77 (1H, dt, J = 4; 14 Hz, The authors are indebted to Dr. Andres Leon for IR spectra
H-1a), 0.80 (3H, s, H-20), 0.96 (1H, m, H-9), 1.01 (1H, m, and to Dr. Ali Bahsas and Dr. Sonia Koteich for NMR
H-5), 0.97 (1H, m, H-3a), 1.15 (3H, s, H-18), 1.35 (3H, s, analyses.
H-17), 1.40 (1H, m, H-2a), 1.42 (1H, m, H-14a), 1.5 (2H,
m, H-11), 1.53 (2H, m, H-15), 1.57 (1H, m, H-7a), 1.58

References
[1] Mitscher LA, Rao GS, Veysoglu T, Drake S, Haas T. (1983) Isolation and identification of trachyloban-19-oic and kaur-16-en-19-
oic acids as antimicrobial agents from the prairie sunflower, Heliantus annuus . Journal of Natural Products, 46, 745-746.
[2] Alves TMA, Chaves PPG, Santos LMST, Nagem TJ, Murta SMF, Cevarolo IP, Romanha AJ, Zani CI. (1995) A diterpene from
Mikania obtusata, active on Trypanosoma cruzi. Planta Medica, 61, 85-86.
[3] Fatope MO, Audo OT, Takeda O, Zeng L, Shi G, Shimada H, McLaughlin JL. (1996) Bioactive ent-kaurene diterpenoids from
Annona senegalensis. Journal of Natural Products, 59, 301-303.
[4] (a) Ekong EU, Ogan AU (1968) Chemistry of the constituents of Xylopia aethiopica. The .structure of xylopic acid, a new
diterpene acid. Journal of the Chemical Society (C), 311-312; (b) Usubillaga A, Morales Mendez A. (1970) Derivados del Kaureno
en la Espeletia tenore. Revista Latinoamericana de Química, 1, 128-131.
[5] (a) Hubert AJ, Reimlinger H. (1969) The isomerization of olefins. Part I. Base catalysed isomerization of olefins. Synthesis, 97-112.
(b) Hubert AJ, Reimlinger H. (1969) The isomerization of olefins. Part II. Thermal and catalytic isomerization of olefins using
acids, metals, metal complexes, or boron compounds as catalysts. Synthesis, 405-430.
[6] Shabtai J, Gil-Av E. (1963) A convenient method for the preparation of 1-methylcyclobutene. Journal of Organic Chemistry, 28,
2893-2894.
[7] Barnes MF, MacMillan J. (1967) The Garryfoline-Cuauchichicine rearrangement: A study of the mechanism in the (-)-kaurenols.
Journal of the Chemical Society C, 361-366.
[8] Henrick CA, Jefferies PR. (1964) The chemistry of the Euphorbiaceae. VII. The diterpenes of Ricinocarpus stylosus Diels.
Australian Journal of Chemistry, 17, 915-933.
[9] (a) Amstutz ED, Myers MM. (1957) Nitrosomethylurea from acetamide. In Organic Syntheses. Blatt AH (Ed). John Wiley and
Sons, Inc. Collective Vol. II, 462-463. New York; (b) Arndt F (1957) Diazomethane. In Organic Syntheses. Blatt AH (Ed). John
Wiley and Sons, Inc. Collective Vol. II, 165-166. New York
2011
NPC Natural Product Communications Vol. 6
No. 7
Aristolactams from roots of Ottonia anisum (Piperaceae) 939 - 942

André M. Marquesa, Leosvaldo S. M. Velozoa, Davyson de L. Moreirab, Elsie F. Guimarãesc and


Maria Auxiliadora C. Kaplana
a
Núcleo de Pesquisas de Produtos Naturais (NPPN), Centro de Ciências da Saúde, Bloco H,
Universidade Federal do Rio de Janeiro (UFRJ). CEP: 21941-590 - Rio de Janeiro, RJ, Brazil
b
Departamento de Produtos Naturais, Far-Manguinhos, FIOCRUZ. CEP: 21041-250 - Rio de Janeiro,
RJ, Brazil
c
Instituto de Pesquisa Jardim Botânico do Rio de Janeiro. CEP: 22.460-030 - Rio de Janeiro, RJ,
Brazil

dmoreira@far.fiocruz.br

Received: December 15th, 2010; Accepted: March 14th, 2011

The Piperaceae species are known worldwide for its medicinal properties and its chemical compounds. In Brazil, many species of this family
are distributed mainly in Amazon Region and in the Atlantic Forest. The genus Ottonia is known as source of amides, flavonoids,
arylpropanoids and terpenes with record biological activities. Six aristolactams, including, aristolactam BII, piperolactam C, goniothalactam,
stigmalactam, aristolactam AII and aristolactam BIII were isolated from roots of this species. GC-MS, 1H NMR and NOESY techniques were
used to characterize these compounds. This is the first report about the occurrence of aristolactams in the Ottonia anisum Sprengel.

Keywords: Piperaceae, Ottonia anisum, aristolactams, alkaloids.

The family Piperaceae is composed approximately by 2000 of aristolactams in Ottonia species. Aristolactams belong
species with wide tropical and subtropical distribution and to a large and important group of naturally occurring
great representation in Central and South Americas, alkaloids that possess the phenanthrene lactam skeleton
occurring in Mexico, Panama, Peru, Costa Rica, Argentina [5]. The phenanthrene chromophore group is found in the
and from North to Southern Brazil. The Piper species are Aristolochiaceae together with the aristolochic acids and
mostly tropical plants of worldwide occurrence and are 4,5-dioxoaporphines alkaloids. Aristolactams have been
represented by ca. 700 species. In Brazil, 260 species are reported from plants of the Annonaceae, Monimiaceae,
distributed mainly in Amazon Region and in the Atlantic Menispermaceae, Piperaceae and Saururaceae families
Forest of the country [1a-c]. The phytochemical [6a,b]. The phenanthrene lactam core is frequently found
investigation of Piper has led to the isolation of a large in biologically active natural products [7a-d]. Among
number of bioactive compounds such as alkaloids, amides, them, aristolactams and aporphines constitute an
propenylphenols, piperolides, flavonoids, chromenes, important alkaloid group due to their unique structural
prenylated benzoic acid derivatives and lignoids. features and potent biological activities, such as anti-
Furthermore, literature records registered the occurrence of inflammatory, to treat arthritis, gout, rheumatism, antiPAF,
interesting secondary metabolites in species of the genus antimycobacterial, and neuro-protective [8a-e].
Peperomia (benzoic acid derivatives and seconeolignans),
Ottonia (amides, flavonoids and arylpropanoids) and Major secondary compounds from non polar root extracts
Pothomorphe (catechol derivatives) [2a-e]. The chemistry of O. anisum were isolated by chromatographic techniques
of Piperaceae from Rio de Janeiro State, Brazil, has been and identified as 3,4-dimethoxy-aristolactam (aristolactam
addressed with great success. Previous phytochemical BII, 1), 2,3,4-trimethoxy-aristolactam (piperolactam C, 2),
investigations of Piperaceae species from Brazilian 6-hydroxy-3,4-dimethoxy-aristolactam (stigmalactam, 3,
Atlantic Forest performed by our research group led to the 6-hydroxy-2,3,4-trimethoxy-aristolactam (goniothalactam,
isolation of several compounds, such as kaplanin, 4), 3-hydroxy-4-methoxy-aristolactam (aristolactam AII,
lhotzchromene and blandachromenes I and II [3a-c]. In 5), 3,4,6-trimetoxy-aristolactam (aristolactam BIII, 6)
order to continue with the phytochemical studies of native (Figure 1). These compounds 1-6 were identified by
Piperaceae species from Southeast Brazil, Ottonia anisum comparison of physical and spectroscopic data (MS and
have been successfully studied [4]. The O. anisum root NMR) with literature records [9a-c]. Compound (1) was
extracts were chemically investigated to afford six obtained as a yellow needle crystalline solid. The
aristolactams. This is the first work regarding the presence molecular formula was confirmed by GC-MS measurement
940 Natural Product Communications Vol. 6 (7) 2011 Moreira et al.

R1 A singlet was at  7.86 (1H, s) corresponds to H-2. The


O
R2
2
1 NOESY spectrum of compound (3) showed the methoxyl
3
NH
groups attached to C-3 and C-4 due to the spatial
proximity of H-2/CH3O-3, CH3O-3/CH3O-4, CH3O-4/H-5.
R3 4 (1) R1=R4=H; R2=R3=OCH3; NOESY correlations between H-7 and H-8 were also
(2) R1=R2=R3=OCH3; R4=H;
5 9 (3) R1=H; R2=R3=OCH3; R4=OH; observed, as well as between H-8/H-9 (Figure 1)
(4) R1=R2=R3= OCH3; R4=OH; confirming (3) as goniothalactam. Compound (4) was
6
8
(5) R1=R4=H; R2=OH; R3=OCH3; isolated as brownish yellow needles. The mass spectrum
R4
7
(6) R1=H; R2=R3=R4=OCH3; m/z [M+] 325 established the molecular formula
C18H15NO5. The 1H NMR spectrum of compound (4)
Figure 1: Aristolactams isolated from O. anisum. Arrows show the
NOESY correlations.
showed signals related to four aromatic protons, a broad
D2O-exchangeable proton at  10.79 and three methoxy
m/z [M+] 279, suggesting the Cl7Hl3NO3. The 1H NMR groups attached to aromatic ring at  3.93 (3H, s), 4.04
spectrum of compound (1) showed signals related to six (3H, s) and  4.38 (3H, s). The signal at  8.58 (1H, d, J =
aromatic protons, a broad D2O-exchangeable proton at  3 Hz) assigned to H-5, and the signals at  7.08 (1H, dd,
10.67 and protons of two methoxy groups attached to J = 6.0, 3.0 Hz) and 7.79 (1H, d, J = 6.0, Hz)
aromatic ring at  4.06 (3H, s) and 4.04 (3H, s). The corresponding to H-7 and H-8 respectively. The NOESY
signal registered at  9.17 (1H, dd, J = 6.0, 3.0 Hz) was spectrum was quite similar to the compound (2), showing
assigned to H-5, and the signals at  7.52 (2H, m) and the spatial correlation between the methoxyl groups at C-2,
7.94 (1H, dd, J = 6.0, 3.0 Hz) corresponding to H-6, H-7 C-3 and C-4. These data are in accordance with
and H-8 respectively (Table 1). A singlet was identified at stigmalactam, 4. The compound (5) was isolated as a pale
yellow powder. The same aristolactam skeleton was
 7.86 (1H, s) corresponding to H-2. The NOESY
observed by the MS and NMR analysis. The mass
spectrum of compound (1) showed correlations between
spectrum m/z [M+] 265 established the molecular formula
H-2/CH3O-3, CH3O-3/CH3O-4, CH3O-4/H-5, as well as
C16H11NO3. The 1H NMR spectrum of compound (5)
between H-8/H-9 (Figure 1). Significant correlations
showed signals related to six aromatic protons, a broad
between H-5 ( 9.17) and H-6 ( 7.58), H-7 ( 7.58) and
D2O-exchangeable proton at  10.69 and a single methoxy
H-8 ( 7.94) were also observed. Considering these data as
group attached to aromatic ring at  4.03 (3H, s). The
well as literature records, the structure of compound (1)
was identified as aristolactam BII. The NMR analysis of signal at  9.25 (1H, dd, J = 6.0, 3.0 Hz) assigned to H-5,
the compounds (2-6) led us to conclude that these the signals at 7.96 (1H, dd, J = 6.0, 3.0 Hz)
constituents have the same aristolactam skeleton of corresponding to H-8 and the signals at  7.54 (2H, m)
compound (1). Compound (2) was isolated as a yellow corresponding to H-6 and H-7. Spatial correlations were
powder. The GC-MS established the molecular formula found between the hydroxyl group at C-3 and the H-2 and
that was confirmed by m/z [M+] 309. The 1H NMR CH3O-4. NOESY correlations between H-5, H-6, H-7 and
spectrum of compound (2) showed signals related to five H-8 were also confirmed, as well as between H-8/H-9.
aromatic protons, a broad D2O-exchangeable proton at  Analysis of GC-MS, 1H NMR and NOESY spectral data
10.96 and protons of three methoxy groups attached to showed chemical shifts very similar to the analogous
aromatic ring at  3.92 (3H, s), 4.17 (3H, s) and  4.42 previous analized compounds and allowed to confirmed
(5) as aristolactam AII. Compound (6) was isolated as a
(3H, s). The signals at  9.12 (1H, dd, J = 6.0, 3.0 Hz) was
yellow green powder with m/z [M+] 265 obtained from
assigned to H-5, and the signals at  7.52 (2H, m)
GC-MS. The 1H NMR spectrum of compound (6) showed
corresponding to H-6, H-7 besides  7.94 (1H, dd, J = 6.0, signals related to five aromatic protons, a broad D2O-
3.0 Hz) assigned to H-8. The NOESY spectrum of (2)
exchangeable proton at  7.67 and three methoxy groups
confirms the spatial correlation of the protons between
attached to aromatic ring at  3.96 (3H, s), 4.18 (3H, s)
CH3O-2/CH3O-3, CH3O-3/CH3O-4, CH3O-4/H-5, as well
as between H-8/H-9. NOESY correlations between and 4.45 (3H, s). The signals at  8.65 (1H, d, J = 3 Hz)
H-5, H-6, H-7 and H-8 were also observed (Figure 1). was assigned to H-5, and the signals at  7.15 (1H, dd,
These data confirmed compound (2) as piperolactam C. J = 6.0, 3.0 Hz) and 7.77 (1H, d, J = 6.0 Hz)
corresponding to H-7and H-8, respectively. A singlet
Compound (3) was isolated as yellow powder. GC-MS observed at  7.80 was assigned to H-2. NOESY
analysis for compound (3) showed a molecular ion at m/z correlations between the methoxyl group at  3.96 and H-5
[M+] 295. The 1H NMR spectrum of compound (3) and H-7 was confirmed. This is in agreement with the
showed five aromatic protons, a broad D2O-exchangeable aristolactam skeleton and suggested that the signal at
proton at  10.64 and two methoxy groups attached to 3.96 refers to the methoxyl group located at position C-6,
aromatic ring at  4.02 (3H, s) and 4.04 (3H, s). Signal at confirming compound (6) as aristolactam BIII.
 8.57 (1H, d, J = 3 Hz) was assigned to H-5, and the
signals at  7.08 (1H, dd, J = 6.0, 3.0 Hz) and  7.76 (1H, Nowadays, many communities in the North of Brazil still
d, J = 6.0 Hz) were assigned to H-7 and H-8, respectively. remain using O. anisum popularly in the treat toothache
Aristolactams from Ottonia anisum Natural Product Communications Vol. 6 (7) 2011 941

Table 1: 1H NMR spectroscopic data of compounds 1-5 in DMSO-d6 and compound 6 in CDCl3, H J (Hz).
Position 1 2 3 4 5 6
5 9.17 (1H) dd (6.0, 3.0) 9.12 (1H) dd (6.0, 3.0) 8.57 (1H) d (3.0) 8.58 (1H) d (3.0) 9.25 (1H) dd (6.0, 3.0) 8.65 (1H) d (3.0)
7 7.58 (1H) m 7.52 (1H) m 7.08 (1H) dd (6.0, 3.0) 7.08 (1H) dd (6.0, 3.0) 7.54 (1H) m 7.15 (1H) dd (6.0, 3.0)
8 7.94 (1H) dd, (6.0, 3.0) 7.94 (1H) dd, (6.0, 3.0) 7.76 (1H) d (6.0) 7.79 (1H) d (6.0) 7.96 (1H) dd (6.0, 3.0) 7.77 (1H) d (6.0)
9 7.13 (1H) s 7.12 (1H) s 7.07 (1H) s 7.15 (1H) s 7.09 (1H) s 7.07 (1H) s
R1 7.86 (1H) s 3.92 (3H) s 7.86 (1H) s 3.93 (3H) s 7.82 (1H) s 7.80 (1H) s
R2 4.06 (3H) s 4.42 (3H) s 4.04 (3H) s 4.04 (3H) s - 4.45 (3H) s
R3 4.04 (3H) s 4.17 (3H) s 4.02 (3H) s 4.38 (3H) s 4.03 (3H) s 4.18 (3H) s
R4 7.58 (1H) m 7.52 (1H) m - - 7.54 (1H) m 3.96 (3H) s
N-H 10.67 (1H) s 10.96 (1H) s 10.64 (1H) s 10.79 (1H) s 10.69 (1H) s 7.67( 1H) s

and as local anesthetic, even if a survey of the literature Extraction and Isolation: Air-dried and powdered roots
data shows that some of natural compounds have (650 g) were extracted with MeOH. The solvent was
carcinogenic and nephrotoxic properties. Aristolactams are evaporated to dryness under reduced pressure to give the
known to be nephrotoxic, carcinogenic and mutagenic MeOH extract (15g) which was partitioned successively
[8b,d] [10a-f]. However, naturally occurring aristolactams between n-hexane, followed by dichloromethane, ethyl
such as cepharanone B (aristolactam BII), aristolactam acetate and n-butanol. Hexane and dichloromethane
BIII, piperolactam A and goniothalactam have shown fractions were combined providing about 2g that were
potent inhibitory activity against human cancer cells. For subjected to a column chromatography on Sephadex LH
example, aristolactam BII inhibits T and B lymphocyte 20, eluted with a MeOH/CHCl3 (7:3) system furnishing 30
proliferation as well as shows cytotoxic activity, while fractions. The last 10 fractions were combined furnishing
aristolactam FI (piperolactam A,) displays inhibitory 200 mg of a very strong UV fluorescence fraction. This
effects on NO generation by RAW264 [5]. Several sample was subjected to preparative normal phase TLC
synthetic aristolactam derivatives exhibited potent eluted with hexane/ethyl acetate (3:2) yielding six fractions
antitumor activities against a broad array of cancer cell with different UV fluorescence color. Aristolactam 1 (17.0
lines with submicromolar range and some are equally mg), 2 (7.0 mg), 3 (4.0 mg), 4 (2.0 mg), 5 (4.5 mg) and 6
potent toward multidrug resistant cell lines compared to (3.0 mg) were isolated and analyzed by GC-FID, GC-MS
the commercially available drug [8a,d]. It is noteworthy and 1H and NOESY NMR techniques.
that the methoxy-substituted compounds are more potent
than the hydroxyl-substituted ones. The tetra-methoxy GC-FID analysis: Qualitative analyses were carried out on
substituted phenanthrene lactam has highly potent a GC 2010 Shimadzu apparatus with a DB-1MS fused
cytotoxic activity [11]. Although the cytotoxicity of silica capillary column (30 m x 0.25 mm x 0.25 m film
aristolactams is well known, structure-activity thickness). The operating temperatures used were: injector
relationships have not been explored mainly as a 260oC, detector 290oC and column oven 60°C up to 290oC
consequence of the synthetic difficulties associated with (10oC min-1). Hydrogen at 1.0 mL min-1 was used as
preparing a diverse array of aristolactam analogues. carrier gas.
Therefore, it is important the continuous knowledge about
the native Piperaceae species of Brazil since many of them GC-MS analysis: Qualitative analysis was carried out on a
have been used in folk medicine to treat many conditions. GC-MS QP 5000 Shimadzu machine with a ZB-5MS
To the best of our knowledge this is the first report of fused silica capillary column (30 m x 0.25 mm x 0.25 m
aristolactams in the genus Ottonia. film thickness) under the same experimental conditions
reported for GC-FID analysis. The aristolactams mass
Experimental spectra were matching with WILEY 275 and National
Plant material: Branches and roots of Ottonia anisum Institute of Standards and Technology (NIST 3.0) libraries
Sprengel were collected in Duque de Caxias, Rio de provided with the computer controlling the GC–MS
Janeiro, RJ, Brazil in april of 2007. A Voucher specimen system. The results were also confirmed by comparison of
was identified by Dr. Elsie Franklin Guimarães and is data of the isolated compounds with mass and
deposited at the RB Herbarium of Jardim Botânico do Rio fragmentation reported in the literature [9a-c].
de Janeiro, under the number RB393494.
Nuclear Magnetic Resonance Spectroscopy: The pure six
Chromatographic materials: Sephadex LH-20 constituents obtained were analyzed by 1H NMR and
(Pharmacia) was used for column chromatographic NOESY recorded on a Varian VNMRS 500 spectrometer.
separation. Silica gel PF254 (Merck) was used for TLC Chemical shifts were determined in DMSO-d6 and CDCl3,
preparative/purification. All compounds were visualized using TMS as internal standard. The signals of NMR
on analytical TLC under UV light (254 and 365 nm) and analysis were compared with literature data [9a-c].
by spraying with ceric sulfate solution followed by
heating. Acknowledgments – This work was supported by CNPq.
942 Natural Product Communications Vol. 6 (7) 2011 Moreira et al.

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2011
NPC Natural Product Communications Vol. 6
No. 7
Anti-angiogenic Activity Evaluation of Secondary 943 - 946
Metabolites from Calycolpus moritzianus Leaves
Laura Leporea, Maria J. Gualtieria, Nicola Malafrontea, Roberta Cotugnoa, Fabrizio Dal Piaza,
Letizia Ambrosioa, Sandro De Falcob and Nunziatina De Tommasia*
a
Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo,
84084 Fisciano, Salerno, Italy
b
Angiogenesis Lab, Institute of Genetics and Biophysics ‘Adriano Buzzati-Traverso’, CNR,
Via Pietro Castellino 111, 80131 Napoli, Italy

detommasi@unisa.it

Received: November 13th, 2010; Accepted: March 25th, 2011

Angiogenesis is a crucial step in many pathological conditions like cancer, inflammation and metastasis formation; on these basis the search
for antiangiogenic agents has widened. In order to identify new compounds able to interfere in the Vascular Endothelial Growth Factor
Receptor-1 (VEGFR-1, also known as Flt-1) recognition by VEGFs family members, we screened Calycolpus moritzianus (O. Berg) Burret
leaves extracts by a competitive ELISA-based assay. MeOH and CHCl3 extracts and several their fractions demonstrated to be able to prevent
VEGF or PlGF interaction with Flt-1, with an inhibition about 50% at concentration of 100 g/mL. Phytochemical and pharmacological
investigation of the active fractions led to the isolation of flavonoids, and terpenes.

Keywords: Calycolpus moritzianus, Angiogenesis, VEGFR1/Flt-1, VEGF and PlGF, bioassay-oriented study.

In the last decade the inhibition of angiogenesis and


vascular targeting has been the focus of new treatment
strategies against the cancer. Among the long list of
growth factors involved in the angiogenic process, VEGF-
A has been considered for years the most important
mediator of tumor angiogenesis [1]. Consequently, several
strategies have been developed to inhibit the release of this
growth factor, or to interfere in its interaction with
receptors, VEGF receptor 1 (Flt-1) and VEGF receptor 2
(Flk-1 in mouse, KDR in human) [2]. Recent data support
the concept that tumor infiltration by bone marrow-derived
myeloid cells confers resistance to current antiangiogenic
drugs targeting primary VEGF-A and its receptors
(VEGF(R)s) [3]. For this reason, novel targets out of
VEGF-A have been studied to diversify antiangiogenic
treatments and to overcome resistance [4]. Genetic and
pharmacological studies have identified Flt-1 and Placental
Growth Factor (PlGF) as possible therapeutic targets for Figure 1: Inhibitory effect of C. moritzianus leaves extracts were assayed on
PlGF/Flt-1 (A) and VEGF/Flt-1 interaction (B). The extracts were used at 500-
anticancer therapy [5]. Furthermore, has been proven that a 100-20 µg/mL. As control a specific inhibiting peptide was used (CP). The
combination of lower amount of VEGF(R)s inhibitors and white bar refers to ELISA experiment carried out without inhibitors. Each
compounds able to block PlGF showed equal antitumor experiment was performed three times and average values ± SD were reported.
efficacy compared to the standard dose of VEGF(R)s [5].
Accordingly to these data, the research of new natural
These findings suggested that molecules able to inhibit the compounds which may inhibit both PlGF and VEGF-A
activity of both PlGF and VEGF-A driven angiogenesis activity, has been the target of the present study. We
may be an opportunity for patients with cancer who may carried out a screening of Calycolpus moritzianus
suffer excessive or prohibitive adverse effects from (O. Berg) Burret leaves extracts by a competitive ELISA-
VEGF(R)s inhibitors. based assay [6]. We aimed to identify natural molecules as
inhibitors of PlGF and VEGF-A recognition by Flt-1.
944 Natural Product Communications Vol. 6 (7) 2011 Lepore et al.

Figure 2: Inhibitory properties of plant fractions from MeOH extract were assayed on PlGF/Flt-1 interaction (A) and VEGF/Flt-1 interaction (B). The fractions were
tested at 500-100-20 µg/mL. As control a specific inhibiting peptide was used (CP). The white bar refers to ELISA experiment carried out without inhibitors. Each
experiment was performed three times and average values ± SD were reported.

Figure 3: Inhibitory activity of compounds isolated from active methanolic fractions assayed at concentration of 100 µg/ml on PlGF/Flt-1 (A) and VEGF/Flt-1 (B). As
control a specific inhibiting peptide was used (CP). The white bar refers to ELISA experiment carried out without inhibitors. Each experiment was performed three times
and average values ± SD were reported.

Several extracts from C. moritzianus leaves have been as main components, which were identified as quercetin
tested at the doses of 0.5, 0.1, and 0.02 mg/mL. 3-O-β-D-glucopyranoside 1 [7], kampferol-3-O-β-D-
glucopyranoside 2 [7], quercetin-7-O-β-D-glucopyranoside
Methanol and chloroform residues exhibited a good 3 [8], kaempferol-3-O-β-D-rhamnopyranoside 4 [9],
activity in the inhibition on both PlGF/Flt-1 and VEGF- quercetin-3-O-β-D-rhamopyranoside 5 [9], and quercetin 6
A/Flt-1 interaction, with a binding reduction higher [10].
than 60% at 20 µg/mL (Figure 1).Therefore these extracts
were submitted to a bioassay-oriented fractionation. C. The main compound identified in Fr. H was quercetin
moritzianus MeOH extract was fractioned by sephadex (85% w/w abundance). All isolated compounds were
column chromatography giving 9 fractions (A-I), while identified by means of 1D- and 2D-NMR spectroscopy,
CHCl3 extract was separated using silica gel column ESI-MS analysis, and by comparison of their data with
chromatography giving 7 fractions (AA-GG). The effect of those reported in the literature.
the obtained fractions was tested on both PlGF/Flt-1 and
VEGF-A/Flt-1, leading to the results reported in Figure Among the pure compounds assayed, only quercetin
2-4. showed a moderate activity (60% inhibition of PlGF/Flt-1
interaction at 100 µg/mL), compounds 1, 3, 5 its
C. moritzianus MeOH extract fractions were assayed in glycosides were inactive (Figure 3). These data suggest
dose-dependent experiments at concentration ranging that the glycosylation at C-3 and C-7 of quercetin core is
between 500 and 20 µg/mL on PlGF/Flt-1; the active frs. fatal for the activity. To better investigate a structure-
D-F and H were then assayed on VEGF-A/Flt-1 at activity relationship we tested also kaempferol aglycon of
concentration of 100 and 20 µg/mL. Among the MeOH compounds 2, 4. Kaempferol was inactive in our test. The
fractions, Fr. H revealed the highest dose-dependent structures of quercetin and kaempferol are very similar
activity for PlGF/Flt-1 inhibition, provoking a reduction of except for the substituent at C-3’; the different activity
its Flt-1 binding to 20% at 500 µg/mL and to 60% at 100 observed for these compounds indicated that the presence
µg/mL, while at dose 100 µg/mL a 25% reduction of of OH group at C-3’ influence the resultant activity.
VEGF-A/Flt-1 interaction was observed.
Fractions obtained from C. moritzianus CHCl3 extract
Also D, E, F, I fractions exhibited a moderate inhibition were assayed on PlGF/Flt-1 and VEGF-A/Flt-1 interaction
for PlGF/Flt-1 complex, even if only Fr. D revealed to be by a competitive ELISA screening at concentration of 100
able to inhibit also hVEGF-A / Flt-1 complex, leading to a and 20 µg/mL.
binding reduction of 60% at 100 µg/mL (Figure 2). The
active fractions were studied in order to identify the Data in Figure 4 showed that the most active fraction was
compounds responsible for this inhibitory activity. Fr. AA which revealed a moderate activity for both the
growth factors causing a reduction of their Flt-1 binding to
Chromatographic and spectroscopic analyses of active 40% for PlGF and 60% for VEGF at dose 100 µg/mL.
Frs. D-F indicated the presence of flavonoidic derivatives
Antiangiogenic metabolites from Calycolpus moritzianus Natural Product Communications Vol. 6 (7) 2011 945

Figure 4: Inhibitory properties of chloroformic extract fractions were assayed on PlGF/Flt-1 interaction (A) and VEGF/Flt-1 interaction (B). The fractions were used at
100-20 µg/mL. As control a specific inhibiting peptide was used (CP). The white bar refers to ELISA experiment carried out without inhibitors. Each experiment was
performed three times and average values ± SD were reported.

Figure 5: Inhibitory activity of compounds 7-12 assayed at concentration of 100-20 µg/mL on PlGF/Flt-1 (A) and VEGF/Flt-1 (B). As control a specific inhibiting
peptide was used (CP). The white bar refers to ELISA experiment carried out without inhibitors. Each experiment was performed three times and average values ± SD
were reported.

Nonetheless BB showed a moderate inhibition for both Extraction and isolation: The air-dried powdered leaves
PlGF / Flt-1 and VEGF / Flt-1 interaction with a binding of C. moritzianus (890 g) were defatted with n-hexane and
reduction of about 40% at dose 100 µg/mL. extracted successively by exhaustive maceration (3 x 1 L,
for 48 h) with CHCl3, CHCl3-MeOH (9:1), and MeOH.
Chromatographic separation of AA and BB fractions The MeOH extract (5 g) was chromatographed over a
allowed to obtain the pure components: rosifoliol 7 [11], sephadex LH-20 column (100 x 5 cm) with MeOH as the
platanic acid 8 [12], oleanolic acid 9 [13], (-)-4,10-di-epi- eluent. A total of 110 fractions were collected (15 mL
5β,11-dihydroxyeudesmane 10 [14], 4,5-dioxoseco-γ- each) and combined according to TLC analysis [silica 60
eudesmol 11 [15], and ursolic acid 12 [13], all identified F254 gel-coated glass sheets with n-BuOH-AcOH-H2O
by means of 1D- and 2D-NMR spectroscopy, ESI-MS (60:15:25) and CHCl3-MeOH-H2O (40:9:1)] to give nine
analysis, and a comparison of their data with those pooled fractions (A-I). Fraction D (106 mg) was purified
reported in the literature. by RP-HPLC with a C18 μ-Bondapak column (30 cm x 7.8
mm, flow rate 2 mL/min) using MeOH-H2O (35:65) to
The isolated pure compounds were tested in dose obtain compound 1 (4.0 mg, tR = 20 min), and 2 (15.0 mg,
dependence manner on both PlGF/Flt-1 and hVEGF/Flt-1 tR = 26 min).
systems (Figure 5). Only compound 8 was moderately able
to inhibit PlGF/Flt-1 recognition; anyway the inhibition Fraction E (95 mg) was purified by RP-HPLC with a
activity showed by this compound cannot explain by itself C18 μ-Bondapak column (30 cm x 7.8 mm, flow rate 2
the activity of the original fraction . mL/min) using MeOH-H2O (35:65) to obtain compound 2
(5.0 mg, tR = 26 min). Fractions F (90 mg) was separately
On the basis of our results, we could hypothesize that the purified by RP-HPLC using MeOH-H2O (2:3) to give
inhibition activity of PlGF and VEGF interaction with Flt- compounds 3 (16 mg, tR = 10 min), 4 (6 mg, tR = 16 min),
1 receptor by the C. moritzianus CHCl3 extracts and and 5 (2 mg, tR = 20 min). Fraction H (20 mg) was
fractions may be due to the presence of a combination of identified as quercetin.
compounds acting synergistically or as vehicles enhancing
the biological activity. However, we cannot rule out that The CHCl3 extract (5.0 g) was submitted to silica gel flash
the activity of the extracts and fractions could be due to a column chromatography eluting with CHCl3 followed by
very minor compound not isolated. increasing concentrations of MeOH (between 1% and
70%). The following volumes of solvents were used: 4.2 L
Experimental of CHCl3, 1 L of CHCl3-MeOH (99:1), 4.3 L of CHCl3-
General experimental procedures: The instrumentation MeOH (49:1), 1 L of CHCl3-MeOH (95:5), 0.5 L of
used in this work is described in our previous paper [13]. CHCl3-MeOH (9:1), 0.5 L of CHCl3-MeOH (1:1), 0.5 L of
CHCl3-MeOH (3:7), and 0.3 L of MeOH. Fractions of 30
Plant material: The leaves of C. moritzianus were mL were collected and analyzed by TLC on silica 60 F254
collected in Venezuela in 2008 and identified by Ing. Juan gel-coated glass sheets eluting with CHCl3 or mixtures
Carmona of Herbarium (MERF), Facultad de Farmacia y CHCl3-MeOH, 99:1, 49:1, 95:5, 9:1, 4:1, and grouped into
Bioanalisis - Universidad de Los Andes, Merida; where a seven fractions (AA-GG). Fraction AA (95 mg) was
voucher specimen n.761 is deposited. subjected to RP-HPLC on a C18 μ-Bondapak column
946 Natural Product Communications Vol. 6 (7) 2011 Lepore et al.

(30 cm x 7.8 mm, flow rate 2.0 mL/min) with MeOH-H2O Plant extracts, fractions and compounds 1-13 dissolved in
(73:27) to yield compounds 7 (4 mg, tR = 10 min), 8 (10 DMSO (Sigma) were properly diluted and added to the
mg, tR = 16 min) and 9 (2 mg, tR = 34 min). Fractions BB wells pre-mixed with ligand. For dose-dependent
(70 mg) and was purified by RP-HPLC with MeOH-H2O experiments, concentration ranging between 20 and 500
(37:13) to give compounds 10 (3.5 mg, tR = 16 min), 11 (8 µg/mL were used.
mg, tR = 4 min), 12 (1.5 mg, tR = 26 min).
Acknowledgements - Authors are grateful to Ing. Forestal
ELISA-based assays: The ELISA based assay for plant Juan Carmona of Herbarium (MERF), Facultad de
extract, fractions and pure compounds screening was Farmacia y Bioanalisis - Universidad de Los Andes,
performed as described elsewhere [6]. Merida for the help in collecting the plant material.

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2011
NPC Natural Product Communications Vol. 6
No. 7
Chemical and Biological Activity of Leaf Extracts of 947 - 950

Chromolaena leivensis
Ruben D. Torrenegra G. and Oscar E. Rodríguez A.

Facultad de Ciencias y Tecnología, Universidad de Ciencias Aplicadadas y Ambientales U.D.C.A,


Bogota, Colombia

ruben.torrenegra@hotmail.com; rtorrenegra@udca.edu.co

Received: December 12th, 2010; Accepted: March 26th, 2011

The flavonoids 3,5-dihydroxy-7-methoxy-flavanone, 3,5-dihydroxy-7-methoxyflavone and 3,5,7-trihydroxy-6-methoxyflavone were isolated


from the leaves of C. leivensis. Preliminary observations in K562 cells (human erythroleukemia) using the trypan blue test, showed a 90%
viability at a concentration of 100 g/mL; however, further testing of the flavonoids at concentrations of 25, 50 and 100 g/mL showed
toxicity affecting the morphology of human erythroleukemia cells (K562) and human melanoma cells (A375). Induction of apoptosis was
produced by 3,5-dihydroxy-7-methoxyflavone at 72 hours after treatment with arrest in the G2 / M phase of the cell cycle. The A375 cells
treated with 50 µg/mL of 3,5-dihydroxy-7-methoxy-flavanone for 24, 48 and 72 hours, display effects on the behavior of the cell cycle. The
flavonoid 3,5-dihydroxy-7-methoxyflavone has activity on the mitochondrial membrane at concentrations of 25, 50 and 100 µg/mL, at time
intervals of 8 to 12 hours. The flavonoids 3,5-dihydroxy-7-methoxy-flavanone and 3,5-dihydroxy-7-methoxyflavone at a concentration of
25 g/mL increased the expression of costimulatory molecules corresponding to the phenotype presented by mature dendritic cells with
differentiation markers CD40, CD83, CD86 and HLA-DR. The two flavonoids at concentrations between 0.39 and 100 g/mL slightly
increased the proliferation of peripheral blood mononuclear cells in the presence and in the absence of phytohemagglutinin. These flavonoids
at concentrations of 50 and 100 g/mL slightly increased the proliferation of fibroblasts.

Keywords: Chromolaena leivensis, flavonoids, cytotoxicity, apoptosis.

Chromolaena species are invasive and cosmopolitan with Many studies on biological activity have been conducted
morphological diversity given by adaptations to different in Chromolaena odorata and a few in C. hirsuta, C.
environments and are considered weeds. This genus is moritziana C. perglabra, C. bullata and C tacotana.
constituted by 202 species of which only 13 have Among the tested biological activities the following are
undergone chemical studies, and only a few studies have highlighted: pesticide, insect repellent, antiprotozoal,
included biological activity. There are no known reports on insecticidal, trypanocidal, antibacterial, antifungal,
the species Chromolaena leivensis, C. perglabra, C. cytotoxic, antioxidant, mutagenic, proliferative agent of
tacotana, C. subscandens, C. opadoclinia, C. odorata, C. human keratinocytes and fibroblasts. Taleb et al. [6]
arnottiana, C. morii, C. Collina, C. connivens, C. evaluated the antiprotozoal effect of total extracts and
glaberrima, C. pseudoinsignis and C. chasleae. purified flavonoids from Chromolaena hirsuta and
Compounds such as sesquiterpenes, diterpenes, triterpenes, determined antiprotozoal activity against trypomastigotes
flavonoids, cyclic fatty acids, sesquiterpene lactones, of Trypanosoma cruzi and amastigotes of Leishmania
germacranolides, have been identified from Chromolaenas. amazonensis; the crude extracts significantly reduced
A sesquiterpene lactone was identified from the parasite viability and the flavonoids showed an
dichloromethane extract of Chromolaena opadoclina [1]. antiproliferative effect on these. Phan found that phenolic
5,3-dihydroxy-6, 7,4’-trimethoxyflavone, 5-hydroxy-6,7, compounds of Chromolaena odorata, p-hydroxybenzoic
3’,4’-tetramethoxyflavone, 5-hydroxy,6,7,3',4’,5’-penta- acid and p-coumaric acid, flavones, flavanones and
methoxyflavone and a common derived 3,4-dihydroxy- chalcones protect skin cells from oxidative damage and
acetophenone have been identified in Chromolaena repair skin conditions [7]. Bouda observed the effect of
arnottiana [2]. The heliangolide 4’-dihydrochromolaenid essential oils from leaves of Chromolaena odorata on the
and a sesquiterpene lactone were found in Chromolaena mortality of Sitophilus Curculionidae (Coleoptera) with a
glaberrima [3]. The acid 7α-acetoxy-trans-communic was LD50 of 6.78%[8]. Thang evaluated the antioxidant effect
identified In Chromolaena collina and in Chromolaena of extracts of Chromolaena odorata on human dermal
morii, germacrane D, squalene, flavonols and a fatty acid fibroblasts by measuring the protectant effect against
type prostaglandin were found [5b]. damage from hydrogen peroxide and hypoxanthine-
xanthine oxidase [9]. Phan showed that Eupolin extract
948 Natural Product Communications Vol. 6 (7) 2011 Torrenegra G. & Rodríguez A.

Table 1: Effect of the flavonoids on cell viability at 24h of treatment. Table 5: Percentage of viability of normal cells at several concentrations
of each flavonoid and control + Vincristine and DMSO.
CELL VIABILITY
Trypan blue MONONUCLEAR CELL 24H
FLAVONOID -g % TO 24 h 100 50 12,5 6,25
3,5-dihydroxy-7-methoxyflavanone-100 94.4 g/mL g/mL g/mL g/mL
3,5-dihydroxy-7-methoxyflavanone-50 100 Ethanol 23% 23% 22% 24%
3,5-dihydroxy-7-methoxyflavone-100 89 3,5-dihydroxy-7-methoxy
3,5-dihydroxy-7-methoxyflavone-50 100 flavanone 24% 28% 23% 23%
3,5-dihydroxy-7-methoxy
Table 2: Concentrations that affect cell morphology. flavone 13% 20% 21% 22%
Vincristine 26% 22% 24% 23%
EFFECT ON MORPHOLOGY DMSO 25% 23% 24% 25%
Direct microscopic observation
CELL-K562 CELL-K562 CELL-A375
[g/24 H] [g/48 H] [g/24H] Table 6: Percentage of viability of normal cells at several concentrations
3,5-dihydroxy-7- of each flavonoid and control + Vincristine and DMSO.
methoxy flavanone 25 25 100
3,5-dihydroxy-7- MONONUCLEAR CELL 24H
methoxy flavone 100 100 50 100 50 12,5 6,25
g/mL g/mL g/mL g/mL
Ethanol 23% 23% 22% 24%
Table 3: Percentage of cells in each phase of the cell cycle.
3,5-dihydroxy-7-methoxy
EFFECT ON CELL CYCLE, A375 CELLS flavanone 24% 28% 23% 23%
FLAVONOID - mg G0/G1 - S -% G2/M - SUB G1- 3,5-dihydroxy-7-methoxy
% % % flavone 13% 20% 21% 22%
3,5-dihydroxy-7-methoxy Vincristine 26% 22% 24% 23%
flavone-50 75.9 19.39 4.62 5.99 DMSO 25% 23% 24% 25%
CONTROL(-) DMSO 54.21 26.3 19.49 0.62
CONTROL (+) G2/M
VINC 8.63 16.27 75.10 2.29 Table 7: Percentage of viability of fibroblasts at several concentrations of
the flavonoids and control + Vincristine after 24h of treatment
Table 4: Percentage of depolarized cells at several concentrations of each FIBROBLASTS 24H
flavonoid after 8h and 12h of treatment. 100 25 12,5 6,25
g/mL g/mL g/mL g/mL
DEPOLARIZATION OF MEMBRANE MITOCHONDRIAL, JC-1 vincristine 26% 28% 26% 30%
FLAVONOID-g-time % DEPOLARIZED 3,5-dihydroxy-7-methoxy
3,5-dihydroxy-7-methoxyflavanone flavanone 40% 53% 45% 55%
25-8H 9.5 3,5-dihydroxy-7-methoxy
50-8H 10.1 flavone 41% 39% 40% 42%
100-8H 19.8
25-12H 9.5
50-12H 13.8 The species Chromolaena perglabra, C. tacotana, C.
100-12H 21.7 bullata, C. subscandens, C. leivensis and C. scabra are
3,5-dihydroxy-7-methoxy flavone
25-8H 18.4
found in the Cundiboyacense region of Colombia, and C.
50-8H 77.3 barranquillensis is found in the Atlantic coast., These
100-8H 95.4 species have not been studied at depth with respect to their
25-12H 32.1 biological activities, specifically as antiparasitic agents
50-12H 72.8
100-12H 97.4
against Chagas and Leishmania and their cytotoxic and
CONTROL(-) DMSO antitumor potential.
8H 19.6
12H 11.8 In this investigation we studied the production of
EtOH
8H 13.0
secondary metabolites in the leaves of C. leivensis and
12H 13.0 isolated the flavonoids 3,5-dihydroxy-7-methoxy-
flavanone, 3,5-dihydroxy-7-methoxyflavone and 3,5,7-
adhesion complex and fibronectin in human keratinocytes trihydroxy-6-methoxyflavone, the first two compounds
[10]. They found increased expression of integrin b1 and were tested for their activity on cancer cell lines. Using the
b4 induced by the extract at concentrations of 0.1 and 1 trypan blue test, it was observed in K562 cells
(erythroleukemia) viability percentages of 90% using a
g/mL, but the expression was reduced at higher doses of
concentration of 100 g/mL, which indicates that
Eupolin (10 to 150 g/mL). Other researchers [11] have
flavonoids at these concentrations are not cytotoxic. The
studied the proliferation of fibroblasts and endothelial cells
flavonoids tested at concentrations of 25, 50 and 100
treated with hydroethanolic leaves extracts of
Chromolaena odorata (Eupolin). The greatest growth of g/mL showed toxicity affecting the morphology of
fibroblasts and endothelial cells was found at human erythroleukemia cells (K562) and human
melanoma cells (A375). Induction of apoptosis was
concentrations of 10 g/mL and 100 g/mL of Eupolin
produced by the flavonoid 3,5-dihydroxy-7-methoxy-
extract, but it was found to be toxic at concentrations
flavone at 72 hours of treatment with arrest in G2/ M. In
exceeding 250 g/mL.
A375 cells treated with 50 g/mL of the flavonoids for 24,
Secondary metabolites in the leaves of C. leivensis Natural Product Communications Vol. 6 (7) 2011 949

48 and 72 hours, it was observed that the flavonoid 3,5- the incubation period, samples were analyzed in a flow
dihydroxy-7-methoxy-flavanone influences the behavior of cytometer, using Cell Quest Pro program and subsequent
the cell cycle. The flavonoid 3,5-dihydroxy-7-methoxy- analysis was performed by the Modfit program V. 2.0.
flavone have activity on mitochondrial membrane at (FACSCalibur, Beckton Dickinson). The calibration
concentrations of 25, 50 and 100 g/mL, at time intervals parameters were established, and the linearity of the laser
of 8 to 12 hours. It was also observed that the flavonoids was measured for further analysis with the program
3,5-dihydroxy-7-methoxy-flavanone and 3,5-dihydroxy-7- Modfit. The data are shown in Table 3.
methoxyflavone at a concentration of 25 g/mL increased
the expression of costimulatory molecules corresponding Mitochondrial membrane depolarization: The iodide
to the phenotype presented by mature dendritic cells with of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl
differentiation markers CD40, CD83, CD86 and HLA- carbocyamine (JC-1) is a lipophilic cationic compound
DR. The two flavonoids at concentrations between 0.39 sensitive to changes in mitochondrial membrane potential.
and 100 g/mL slightly increased the proliferation of In healthy cells the tagged mitochondria emits red
peripheral blood mononuclear cells in the presence and in fluorescence. The negative charge established by the intact
the absence of phytohemagglutinin. It was also determined mitochondrial membrane allows the JC-1, which has
that fibroblast proliferation increased slightly at delocalized positive charge to enter the mitochondrial
concentrations of 50 and 100 g/mL of these flavonoids. matrix where it accumulates. When it reaches a critical
concentration it forms J-aggregates that emit red
Experimental fluorescence. In apoptotic cells, the membrane potential of
mitochondria declines, and the JC-1 cannot accumulate
Materials were collected in the outskirts of Bogota,
within the organelle and remains in the cytosol as a
Colombia; a control sample was sent to the National
monomer emitting green fluorescence. The aggregate red
Herbarium of Colombia for identification and was
form has a maximum absorption / emission 585/590 nm,
determined to be Chromolaena leivensis (Hieron). King &
while for the green monomer is 510/527 nm. The test was
H. Rob. with the number COL-535 219 Colombian
performed using the cationic lipophilic fluorochrome JC-1,
National Herbarium. The extraction was carried out in
at concentration of 10 g/mL, from a diluted stock solution
Soxhlet with 95% ethanol (4L) with a yield of 31.04%.
in DMSO and kept at 4°C. The K562 cells (1X106) were
200 g of extract was mixed with silica gel (1:2) and
treated with flavonoids 3,5-dihydroxy-7-methoxy-
extracted solid - liquid with petroleum ether (3L), toluene
flavanone and 3,5-dihydroxy-7-methoxyflavone in 24 well
(2L), dichloromethane (2L), ethyl acetate (2L) and
plates and read at 4, 8 and 12 hours. The cells were then
methanol (3L), successively, with yields of 2.66% for
incubated for 10 minutes at 37°C and read immediately
petrol, 29.09% for Toluene, 15.68% for CH2Cl2, 14.66%
using a flow cytometer, using the Cell Quest Pro program
for AcOEt and 30.08% for MeOH. Five grams of the
(FACSCalibur, Beckton Dickinson). The data obtained are
toluene fraction were subjected to column chromatography
shown in Table 4.
with silica gel (100 g silica gel Merck Kieselgel), eluting
with petroleum ether, toluene and methanol in various Dendritic cells analysis: Peripheral Blood mononuclear
proportions. By fractional crystallization three flavonoids cells (PBMC) were obtained and washed in RPMI 1640
were isolated and identified as 3,5-dihydroxy-7- supplemented with 1% fetal bovine serum (FBS). The
methoxyflavanone, 3,5-dihydroxy-7-methoxyflavone [13] viability and cell numbers were assessed by trypan blue
and 3,5,7-trihydroxy-6-methoxyflavone.[14]. staining and Neubauer cell counting chamber, respectively.
The CD14+ cells were incubated in RPMI 1640 in the
Cell viability: Cell viability was measured using trypan
presence of 35 g/mL of interleukin (IL-4) and 50 g/mL
blue, a negatively charged chromophore that interacts with
growth factor granulocyte and monocyte GM-CSF (R&D
cell membranes whose integrity has been altered. Living
system) for 5 days of culture, verifying their morphology
cells exclude the dye while dead cells allow entry and are
by light microscopy. At day 5 of culture, DCs were treated
stained. The effect of flavonoids 3,5-dihydroxy-7-
with different concentrations (12, 25 and 50 g/mL) of
methoxy-flavanone and 3,5-dihydroxy-7-methoxyflavone
flavonoids 3,5-dihydroxy-7-methoxy-flavanone and 3,5-
on cell populations with concentrations less than or equal
dihydroxy-7-methoxyflavone, and 1 μg/mL of lipopoly-
to 100 g/mL were analysed at 24 and 48 hours. The data
saccharide (LPS) as a positive control for differentiation .
are listed in Table 1.
The expression of surface markers was assessed by flow
cytometry after 48 hours using antibodies against CD40,
Evaluation of cell cycle distribution: To demonstrate the
CD83, CD86 and HLADR. The data obtained showed
effect of flavonoids 3,5-dihydroxy-7-methoxy-flavanone
proliferation of dendritic cells.
and 3,5-dihydroxy-7-methoxyflavone on the cell cycle in
the A375 tumor cell line, cells were synchronized in G1 Evaluation of the effect of the flavonoids on the
phase by total withdrawal of fetal bovine serum for 3 viability of PBMNC and fibroblasts: The effect of
days. Once synchronized, the cells were grown in twelve- flavonoids 3,5-dihydroxy-7-methoxy-flavanone and 3,5-
well plates at a density of 400,000 cells/mL and incubated dihydroxy-7-methoxyflavone on cell viability of peripheral
in the presence and absence of the test compounds. After
950 Natural Product Communications Vol. 6 (7) 2011 Torrenegra G. & Rodríguez A.

blood mononuclear cells (PBMNC) cells was analyzed by mM MTT and incubated for 4 hours (37°C, 5% CO2, 95%
seeding the cells in 96-well plates at a density of 200,000 humidity) protected from light. After this time, the crystals
cells/well in 200 L of RPMI 1640 without phenol red of formazan resulting from the metabolism of MTT by
supplemented with 10% FBS and stimulated for 12 hours mitochondria of viable cells were dissolved with 100 L of
with the mitogen PHA (250 L/100 mL) and then placed SDS-HCl 0.01M. The plates were incubated again under
in contact with different concentrations of each flavonoid. the same conditions for 4 hours. The color intensity was
Cells treated with DMSO (vehicle) were used as a control. read by absorbance at 540 nm in an ELISA reader
Fibroblasts were analyzed in a similar manner. Briefly, (Labsystems Multiskan MCC/3340).
cells were seeded at a density of 15,000 cells/well in
200 L of medium in a 96-well plate and allowed to Acknowledgments - This work was supported by funds
adhere for 24 hours; different concentrations of the from Universidad de Ciencias Aplicadas y Ambientales,
flavonoids were added and incubated for 24 hours (37°C, UDCA. Thanks to Dr Fernando Echeverri,Universidad de
5% CO2, 95% humidity). After incubation with the Antioquia by NMR spectra and Dr. Victoria Ramsauer,
flavonoids, 110 L of medium was added to 10 L of 12 East Tennessee State University .

References
[1] El-Sayed NH, Misk IM, Whittemore AT, Mabry TJ. (1988) Sesquiterpene lactones from Chromolaena opadoclinia.
Phytochemistry, 27, 3312-3314.
[2] De Gutiérrez AN, Catalán AN, Díaz JG, Herz W. (1995) Sesquiterpene lactones, a labdane and other constituents of Urolepis
hecatantha and Chromolaena arnottiana. Phytochemistry, 39, 795-800.
[3] Ahmed AA, Whittemore AT, Mabry TJ. (1985) A heliangolide from Chromolaena glaberrima. Phytochemistry, 24, 605-606.
[4] Bohlmann F, Zdero C, Fiedler L, Robinson H, King RM. (1981) A labdane derivative from Chromolaena collina and a
p-hydroxyacetophenone derivative from Stomatanthes corumbensis. Phytochemistry, 20, 1141-1143.
[5] Bohlmann F, Gupta R, King K, Robinson HM. (1981) Prostaglandin-like fatty acid derivative from Chromolaena morii.
Phytochemistry, 20, 1417-1418.
[6] Taleb SH, Salvador MJ, Balanco JM, Albuquerque S, De Oliveira DC. (2004) Antiprotozoal effect of crude extracts and flavonoids
isolated from Chromolaena hirsuta (Asteraceae). Phytotherapy Research, 18, 250-254.
[7] Phan TT, Hughes MA, Cherry GW. (2001) Effects of an aqueous extract from the leaves of Chromolaena odorata (Eupolin) on the
proliferation of human keratinocytes and on their migration in an in vitro model of reepithelialization. Wound Repair and
Regeneration, 9, 305-313.
[8] Bouda H, Tapondjou LA, Fontem DA, Gumedzoe M. (2001) Effect of essential oils from leaves of Ageratum conyzoides, Lantana
camara and Chromolaena odorata on the mortality of Sitophilus zeamais (Coleoptera, Curculionidae). Journal of Stored Products
Research, 37, 103-109.
[9] Thang PT, Patrick S, Teik LS,Yung CS. (2001) Anti-oxidant effects of the extracts from the leaves of Chromolaena odorata on
human dermal fibroblasts and epidermal keratinocytes against hydrogen peroxide and hypoxanthine-xanthine oxidase induced
damage. Burns, 27, 319-327.
[10] Phan TT, Allen J, Hughes MA, Cherry G, Wojnarowska F. (2000) Upregulation of adhesion complex proteins and fibronectin by
human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin). European Journal of
Dermatology, 10, 522-527.
[11] Phan TT, Hughes MA, Cherry GW. (1998) Enhanced proliferation of fibroblasts and endothelial cells treated with an extract of the
leaves of Chromolaena odorata (Eupolin), an herbal remedy for treating wounds, Plastic & Reconstructive Surgery, 101, 756-765.
[12] Mabry TJ, Mabry H. (1975) The Flavonoids. Edited by Harborne JB, Chapman and Hall, 1204 p.
[13] Goel RN, Seshardri TR. (1958) New synthesis of tamaraxetin, alpinone and izalpinin. Proceeding of the Indian Academy of
Sciences section A, 47, 191-195.
[14] Asakawa J, Genjida F, Suga T. (1971) Four new flavonoids isolated from Alnus Seboldina. Bulletin of the Chemical Society of
Japan, 44, 297.
2011
NPC Natural Product Communications Vol. 6
No. 7
Citrus bergamia Juice: Phytochemical and Technological 951 - 955

Studies
Patrizia Picerno, Francesca Sansone, Teresa Mencherini, Lucia Prota, Rita Patrizia Aquino,
Luca Rastrelli and Maria Rosaria Lauro*

Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano,
Salerno, Italy

lauro@unisa.it

Received: December 10th, 2010; Accepted: March 25th, 2011

Fresh juice from bergamot (Citrus bergamia Risso) has been studied to evaluate the polyphenolic composition by HPLC-DAD analysis and
total polyphenols content by UV method. The main constituent, Naringin, has been selected as analytical and biological marker of the juice.
Juice has been loaded onto maltodextrin matrix by spray-drying. The produced maltodextrin/juice powder (BMP) showed neither significant
change in total polyphenols content nor decrease in antioxidant properties with respect to fresh juice. Moreover, BMP displayed high in vitro
dissolution rate of the bioactive constituents in water and in simulated biological fluids. BMP appears as promising functional raw material
for food, nutraceutical and pharmaceutical products. With this aim, a formulation study to develop tablets (BMT) for oral administration has
been also performed. The produced solid oral dosage form preserved high polyphenols content, showed complete disaggregation in few
minutes and satisfying dissolution rate of the bioactive constituents in simulated biological fluids.

Keywords: Citrus bergamia Risso, fresh juice, polyphenols content, Naringin, maltodextrin/juice powder, tablets, in vitro disaggregation and
dissolution tests.

Bergamot (Citrus bergamia Risso) is a natural hybrid fruit [7,8]. A convenient way to increase the shelf-life and to
derived from bitter orange and lemon. The plant grows improve the organoleptic characteristics of a plant
almost exclusively in the Reggio Calabria region (South derivative is to transform it into a stable dry powder form
Italy). Bergamot is used for production of essential oil [9a,9b]. Spray-drying is a micro-encapsulation technique
obtained from the peel, and its fruit juice is considered a appropriate for sensitive components such as polyphenols,
waste product of the industrial process [1a-1e]. Bergamot and commonly used in pharmaceutical and food industry
juice has drawn attention for its polyphenolic, mainly [10]. Food ingredients and additives in spray-dried powder
flavonoids content [2a-2c], being an attractive raw material form have reduced bulk weight and size, long-last
for the food and nutraceutical industry. It is well known biological stability, and are suitable for transportation and
that the consumption of polyphenol-rich products, mainly handling. Common carriers for spray-drying process
due to their antioxidant properties, is beneficial for human include carbohydrates, gums, semisynthetic cellulose
health [3a,3b]. Flavonoids from citrus fruits have many derivatives and synthetic polymers [11]. Currently,
health benefits including anticancer, antiviral, and anti- maltodextrins, water soluble modified starch derivatives,
inflammatory activities, as well as effects on capillary are used alone or in combination with other materials in
fragility, and inhibition activity on human platelet food and drug processing of plant extracts, aromatic
aggregation [4a,4b]. In recent studies [5a,5b], bergamot additives, carotenoids and vitamins [12a-12c].
juice has been shown to be effective in the prevention of Maltodextrins have multifaceted functions including
diet-induced hyperlipidemia. Moreover, it is able to bulking, caking resistance, film formation, binding of
enhance the antioxidant values of others industrial juices, flavour and fat as well as reduction of oxygen permeability
acting as synergistic compound for the synthetic additives of wall matrix. Moreover, as one of the administration
normally used [6]. problems of the bergamot juice is the bitter taste,
maltodextrins are also able to sweeten the final product.
Despite these beneficial effects, the unprocessed fresh
bergamot juice, showing penetrating smell and bitter taste, This paper reports on the evaluation of polyphenol
involves practical difficulties for an industrial use. components and antioxidant properties of fresh bergamot
Alterations of the functional and organoleptic properties of juice, as well as on the production and characterization of
polyphenols can take place during the storage period due powders obtained loading the fresh juice onto malto-
to constituents release and degradation/oxidative process dextrins as carrier (BMP) by spray-drying. Moreover, a
952 Natural Product Communications Vol. 6 (7) 2011 Picerno et al.

formulation study to develop tablets containing BMP for (GF) and 4.0 g/L in simulated intestinal fluid (IF),
oral administration has been performed. Characteristics of respectively. Sink conditions, which describe a dissolution
the tablets (BMT) were evaluated in term of disintegration system sufficiently dilute so that the dissolution process is
time and active compounds release in water and simulated not impeded by saturation of the solution, resulted 1.0 g/L.
biological fluids. The in vitro dissolution/release profiles of active
compounds from the BMP in each dissolution medium
Hand-squeezed crude bergamot juice was analyzed by (water, GF and IF) are reported in Figure 2. After 5
HPLC-DAD method. As shown in the chromatogram minutes a high amount (about 90%) of juice was
(Figure 1A) the eluted constituents were three flavanone dissolved/released both in water and simulated intestinal
neohesperidosides, Neoeriocitrin (1), Naringin (2), fluid; and about 60% in GF, in agreement with solubility
Neohesperedin (3), and a flavone neohesperidoside, results previously reported. The complete dissolution
Neodiosmin (4) (Figure 1B). Each compound was (about 100%) was achieved after 30 minutes in both water
identified by comparison of retention time, MS and UV and IF, and after 2 hours in GF (Figure 2). These results
spectra with those of standards. In agreement with are very interesting, because the high water solubility of
literature data [2a-2c], compounds (1-3), were found as the the powder, displaying high in vitro dissolution rate with
most abundant flavonoids in the bergamot juice (0.6 ± complete release of the active compounds in all dissolution
0.01, 0.6 ± 0.01, and 0.4 ± 0.01 mg/mL, respectively). To media. Formulation of BMP into tablets meets the
produce the powder form, an aqueous liquid feed, challenge to retain the original properties of powder during
containing both maltodextrins and bergamot juice, was compression. This was achieved by keeping low the
prepared and processed by spray-drying technique, as compression pressure and using the direct compression
described in the experimental section. The production procedure instead of wet granulation, thus avoiding
yield of Bergamot-Maltodextrin Powder (BMP) was very lengthy granulation steps and exposure to solvents used in
high (90%). The presence of maltodextrin, having high wet granulation [15]. BMP itself was used both as a
water solubility, significantly reduced apparent viscosity of directly compressible binder and as diluent. Bergamot
the feed dispersion favouring the atomization and drying juice is rich in sugars which can act as binder, and
of the liquid feed [12b]. Moreover, increasing temperature maltodextrins, used as carrier, which may also act as
during the spray-drying process, maltodextrins are able to diluent in the tablets preparation [15]. Moreover, CMC
induce the rapid formation of a glassy surface which was used as disintegrant and directly compressible filler,
allows air expansion inside particles, favouring the and magnesium stearate as a lubricant. The final
increase of particles diameter [12b]. For this reason, formulation of bergamot-maltodextrin tablets (BMT) is
smallest and lightest particles which are normally lost with reported in the experimental section. Each BMT resulted
the exhaust of the spray dryer are reduced, and the yield 8-mm tick (Figure 3) and their disintegration time (see
increases. On the other hand, a low viscosity liquid feed experimental section) was in about 15 minutes. Figure 3
led to a low retention of core material because of the delay shows the dissolution profiles of BMT in three different
in the formation of a semi-permeable layer by the internal dissolution media. In 5 minutes, about 20% and 30% of
components during drying [12b,13]. juice was dissolved in water and IF, respectively. In the
same time only 5% was dissolved in GF. 90% of
Polyphenol content of both unprocessed juice (actual dissolution was obtained in 90 minutes in IF, and in 150
polyphenol content, APCB) and BMP (APCBMP) was minutes, a total release was observed in all dissolution
determined by UV method (5.49 and 3.57%, respectively). media. Release of juice from the BMT resulted slower than
These values led to calculate the effectively loaded juice from the BMP, probably due to the enhancement of
(actual juice content, AJCBMP 32.7%) as described in the binding forces of sugars, contained in the juice, during the
experimental section. AJCBMP was reasonable with respect compression. Any how, this effect was balanced by the
to the theoretical juice content (TJC 50.0%). presence of CMC which promotes the disintegration of
Consequently, the loading efficiency (LE) value, tablets and enhancement of the active compounds
calculated as the ratio of AJC to TJC, was 65.4%. dissolution rate. In conclusion, the obtained water-soluble
powder BMP is able to preserve the polyphenols content
The functional stability of juice, before and after the spray- and antioxidant activity of unprocessed juice. Furthermore,
drying process, was evaluated as free-radical scavenging the spray-dried powder is suitable for the production of
activity using the DPPH test [14]. The antioxidant activity oral dosage tablets (BMT) by directly compression as well
of bergamot juice, expressed as EC50, (130 ± 5 and 140 ± as for manufacturing raw material functional food, and for
12 g/mL, respectively) was at the same level and pharmaceutical and food supplements products.
remained quite unaltered after the spray-drying process.
The process conditions used did not determine significant Experimental
loss of antioxidant activity. To evaluate the dissolution/
release profile of juice from the powder, its solubility in Chemicals: HPLC grade methanol (MeOH), formic acid
each dissolution medium was previously detected as (HCOOH) and the reagents used for the extractions were
described in the experimental section. Solubility of BMP purchased from Carlo Erba (Rodano, Italy). HPLC grade
resulted 4.0 g/L in water, 3.4 g/L in simulated gastric fluid water (18mΩ) was prepared using a Millipore Milli-Q
Polyphenol and antioxidant properties of fresh bergamot juice Natural Product Communications Vol. 6 (7) 2011 953

Figure 1: 1A ) HPLC-DAD chromatogram of C. bergamia juice. In increasing retention order: Neoeriocitrin (1), Naringin (2), Neohesperidin (3), Neodiosmin
(4); detection wavelength: 284 nm; 1B) compounds isolated from bergamot juice.

in vitro BMP Dissolution test


in vitro BMT Dissolution Test
wate r GF IF
water GF IF
100 100
80
80
% Dissolved

% Dissolved
60
60
40
40
20
20
0
0
0 15 30 45 60 75 90 105 120 135 150
Time (min) 0 15 30 45 60 75 90 105 120 135 150
Time (min)
Figure 2: in vitro BMP dissolution profiles. Figure 3: BMT picture and in vitro dissolution profiles.

purification system (Millipore Corp., Bedford, MA). juice (BMP) were extracted according to Pernice et al.
Neoeriocitrin, Naringin, Neohesperidin, Neodiosmin, and 2009 [6] with slight modifications. 2 mL of bergamot juice
Folin-Ciocalteau’s phenol reagent were provided from was extracted whit 10 mL methanol, agitated and sonicated
Sigma Chemical Co. (Milan, Italy). Maltodexstrins D.E. for 10 min. Juice was centrifuged for 10 min at 4000 rpm;
16, magnesium stearate and microcrystalline cellulose supernatant was collected, while the pellets was extracted a
from Acef, Italy. second time using the same procedure. Supernatants were
combined and centrifuged for 10 min at 2000 rpm. The
Instruments: HPLC analysis was carried out on an concentration of solid material was 20.4 mg/mL.
Agilent 1100 series system equipped with a Model G-1312
pump, and Rheodyne Model G-1322A loop (20 μl), and a HPLC-DAD analysis: A part of supernatant was
DAD G-1315 A detector. Peaks area were calculated with separated by HPLC using a 3.9 × 300 mm i.d. C18 μ-
an Agilent integrator. ESI-MS was performed on a Bondapack column. The mobile phase consisted in water
Finnigan LC-Q Deca instruments (Thermoquest, San Jose, (solvent A) containing 0.1% formic acid, and methanol
CA) equipped with Xcalibur software. To produce the (solvent B). The elution gradient was as follows: 0→5
juice-maltodexstrin powder a Mini Spray Dryer B-191 min, 15→30% B; 5→10 min, 30→35% B, 10→20 min,
Büchi (Laboratoriums-Tecnik, Flawil, Switzerland) was 35→50% B, 20→30 min, 50→75% B; 30→35 min,
used. Dissolution test was carried out by SOTAX AT 75→95% B; 35→40 min, 100% B. The flow rate was 1.0
Smart Apparatus (Basel, CH) on line with a mL min−1 with a DAD detector set at 284 nm. Elution
spectrophotometer (UV/Vis spectrometer Lambda 25, yielded four major compounds (Figure 1): Neoeriocitrin
Perkin Elmer Instruments, MA, USA). Balance Crystal (1, tR 12.6 min), Naringin (2, tR 15.1 min), Neohesperidin
100 CAL – Gibertini (max 110 g d=0,1 mg;+ 15°C/30°C). (3, tR 16.6 min), and Neodiosmin (4, tR 18.4 min), in
Mixer Galena Top (Ataena, Tecno-Pro srl, Italy). according to data reported in literature [2c]. Identification
Alternative compression apparatus GP1, Costamac srl, of constituents was carried out by comparison of their
Casatenovo (LC) Italy. retention times, UV and MS spectra data with those of
standard compounds, and confirmed by co-injections.
Plant material: Citrus bergamia fruits Risso were
collected in February 2009 from plants growing in Reggio Bergamot-powder (BMP) production by Spray drying:
Calabria, Italy. Bergamot juice was prepared by hand 200 g of maltodextrins (16 D.E.) were dissolved in 200 mL
squeezing fresh fruits, immediately after collection. It was of fresh bergamot juice, with a 1:1 polymer/juice weight
filtered through steel sieves of 1 mm and stored at -20°C ratio. The liquid feed was spray dried under the following
until required for our study. process conditions: inlet temperature 120°C; outlet
temperature 69-71°C; spray flow feed rate 5 mL/min;
Sample preparation for HPLC and UV analyses: nozzle diameter 0.5 mm; drying air flow 500 l/h, air
Unprocessed bergamot juice and processed spray dried pressure 6 atm, aspirator 100%. In order to keep
954 Natural Product Communications Vol. 6 (7) 2011 Picerno et al.

homogeneity, while feed was pumping into the spray between absorbance and concentration was verified in the
dryer, the suspension was gently stirred using a magnetic range 1 g/L to 5 g/L (R2>0.999).
stirring. Each preparation was carried out in triplicate.
Spray-dried bergamot-maltodextrin powder (BMP) was Bleaching of the Free-radical 1,1-Diphenyl-2-
collected and stored under vacuum for 48h at room picrylhydrazyl (DPPH Test): Free radical scavenging
temperature until the characterization. activity of unprocessed juice and BMP was determined
using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method
Quantitative HPLC analysis: HPLC equipment and [14]. Results were expressed as amount (g/mL) of
conditions were the same used for the qualitative analysis. antioxidant necessary to decrease the DPPH initial
Unprocessed juice and BMP were subjected to extraction concentration by 50% (EC50) ± standard deviation (SD).
as reported in Sample preparation. Reference standard Tocopherol (EC50 10.1±1.3g/mL) was used as a
solutions of Neoeriocitrin, Naringin and Neohesperedin positive control in the test.
were prepared at three concentration levels in the range
0.25-1.0 mg/mL. Standard curves were analyzed using the Tablets products: Ingredients and their relative amounts
linear least-squares regression equation derived from the used for the formulation of Bergamot-maltodextrin-tablet
peak area (R2>0.9999) corresponding to each compound. (BMT) were: BMP 1000 mg, magnesium stearate 10 mg,
Results were expressed as mg/mL of compound ± standard and microcrystalline cellulose (CMC) 5 mg. The
deviation. ingredients were mixed until uniformity. The resultant
mixture was compressed using round double concave
Total polyphenol content: Actual phenol content of punches of 10 mm diameter.
bergamot unprocessed juice (APCB) and BMP (APCBMP),
were determined by UV/Vis spectrometry at λ 284 nm. In vitro dissolution test: In vitro dissolution/release tests
Each analysis was made in triplicate. APC was expressed were carried out according with the Farmacopea Ufficiale
in percentage as total Naringin (N) equivalents (mg N/100 Italiana (F.U.I. XII, 2009) [17]. Release profile of BMP
mL juice). and BMT were determined in water, phosphate buffer at
pH 6.8 (simulated intestinal fluid without enzymes) and
Yield of the process and loading efficiency: Production hydrochloric acid buffer pH 1.2 (simulated gastric fluid
yield was gravimetrically determined and expressed as the without enzymes). Samples were analyzed
weight percentage of the final product compared to the spectrophotometrically at λmax 284 nm. Briefly, 1000 mg
total amount of the materials sprayed. of BMP or one BMT were placed in six dissolution vessels
containing 1000 mL of dissolution medium on a
Theoretical juice content (TJC) was calculated as dissolution test apparatus n.2: paddle, 100 rpm at
percentage of juice content compared to the initial total 37°C±0.5°C. All the dissolution tests were made in
content of all feed components before spray-drying. Actual triplicate; only the mean values are reported (standard
juice content (AJC), theoretical polyphenol content (TPC) deviations < 5%). Amount of juice dissolved was
and loading efficiency (LE) were calculated as reported in measured as Naringin equivalents. Results are graphically
the following formula: expressed as the dissolution rate (in percentage) with
AJC% = APCBMP / APCB x 100 respect to the time (in minutes).
TPC% = APCB x loaded juice/ feed components
LE% = AJC/TJC x 100
In vitro disintegration time: Test was carried out
BMP Solubility: Solubility of the powder was determined according with the Farmacopea Ufficiale Italiana (F.U.I.
in distilled water and in simulated biological fluids (gastric XII, 2009) [18]. A modified dissolution apparatus (paddle
fluid, pH 1.2, and intestinal fluid, pH 7.5 without enzymes) type) was used. The disintegration fluid was HCl pH 1.0
prepared according to USP 31 (2008) [16] at the conditions (1000 mL) at the temperature of 37±0.5°C with a stirring
reported elsewhere [8]. Concentration of juice in the media of 100 rpm. Six tablets were placed individually in six
was determined by UV/Vis spectrometry at λ 284 nm and sinkers and disintegration time was determined as the point
expressed as Naringin equivalents. Each analysis was at which the tablet disintegrated completely and passed
made in triplicate. Naringin calibration curves in the same through the screen of the sinker.
solvents were previously worked out. Proportionality

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volatile fraction of cold-pressed citrus peel oils. In G. Dugo & A. Di Giacomo (Eds.), Citrus. London: Taylor and Francis, 201-317;
(d) Figoli A, Donato L, Carnevale R, Tundis R, Statti GA, Menichini F, Drioli E. (2006) Bergamot essential oil extraction by
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[2] (a) Gattuso G, Barreca D, Caristi C, Gargiulli C, Bellocco E, Toscano G, Leuzzi U. (2006) Flavonoids glycosides in bergamot juice
(Citrus bergamia Risso). Journal of Agricultural and Food Chemistry, 54, 3929-3935; (b) Gattuso G, Barreca D, Caristi C,
Gargiulli C, Leuzzi U. (2007) Distribution of flavonoids and furocoumarins in juices from cultivars of Citrus bergamia Risso.
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Ficarra R. (2004) Study of the extraction procedure by experimental design and validation of a LC method for determination of
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345-346.
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2011
NPC Natural Product Communications Vol. 6
No. 7
Phenolic Derivatives from the Leaves of Martinella obovata 957 - 960

(Bignoniaceae)
Carolina Arevaloa*, Ines Ruiza, Anna Lisa Piccinellib, Luca Camponeb and Luca Rastrellib
a
Departamento de Control Químico, Facultad de Farmacia, Universidad Nacional Autonoma –
Tegucigalpa, Honduras
b
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte don Melillo,
84084 Fisciano (SA), Italy

anarevalohn23@yahoo.com

Received: December 15th, 2010; Accepted: March 16th, 2011

A new phenolic derivative, 4-methoxyphenol 1-O-β-D-apiofuranosyl-(1→6)-O--D-glucopyranoside (1), has been identified together with
uncommon 3,4-dimethoxyphenol 1-O-β-D-apiofuranosyl-(1→6)-O--D-glucopyranoside (2) and 3-hydroxy, 4-methoxyphenol 1-O-β-D-
apiofuranosyl-(1→6)-O--D-glucopyranoside (3) from the leaves of Martinella obovata (Kunth) Bureau & K. Schum., an Honduran species
used in folk medicine for the treatment of eyes diseases. Verbascoside, isoverbascoside, leucoceptoside A, vitexin, isovitexin, luteolin 8-C-β-
D-glucopiranoside and spireoside were also found. All structures were elucidated on the basis of mass spectrometry and 2D NMR techniques.

Keywords: Bignonaceae, Martinella obovata, Phenolic apiosides, 1D and 2D NMR.

The Bignoniaceae family includes about 120 genera and O O


R1

800 species, growing mainly in Africa, Central and South OH


O
OMe

America. Species of the Bignoniaceae are used for many OH


HO
O
purposes, such as horticulture, timber, dyes and medicine.
HO HO
OH

The best-known medicinal use of the Bignoniaceae is the 1; R1 = H

application of bark preparations of various species of


2; R1 = OMe
3; R1 = OH

Tabebuia as cancer cures [1]. However, members of the


Figure 1. Compounds 1-3 isolated from the leaves of Martinella obovata
family have been sparsely chemically investigated [2].

Martinella (Bignoniaceae) is a tropical genus consisting of The ESIMS in negative mode of compound 1 exhibited a
nine species. Root extracts of the Martinella quasi-molecular ion peak at m/z 417 [M-H]- and a high
iquitosensis vine, found in Amazonian lowland rainforests, resolution measurement indicated the molecular formula,
are used by indigenous peoples to treat various eye C18H26O11, in accordance with 13C NMR data. Major
ailments, including inflammation and conjunctivitis fragments at m/z 285 and 123 were assigned to the loss of
[3]. This medicinal use may be attributed, at least in part, a pentose unit (132 amu) and the successive loss of an
to the presence guanidine alkaloids which have been hexose unit (162 amu). The 1H NMR spectrum of 1
demonstrated to be modest antibiotics and micromolar exhibited a set of AAXX coupling system at  H 7.09
binders of several G-protein coupled receptors [4]. A (2H, d, J=8.5, H-2, H-6), and  H 7.01 (2H, d, J=8.5,
predominance of information regarding M. obovata use H-3, H-5), a methoxy signal at  H 3.85 (3H, s, OMe-3),
comes from Amazon Indian tribes of Peru, where this and two anomeric proton signals at  5.01 (1H, d, J=2.5
liana, uniformly called “yuquilla”, is often cultivated as the Hz, H-1'') and 4.78 (1H,d, J=8.0 Hz, H-1'). The 13C-NMR
preferred treatment for eye diseases. In general, the thick spectrum of 1 revealed 19 carbon signals, including one
fleshy root bark, with the rough outside part scraped off, is benzene ring carbon signal, a set of hexose carbon signals,
pounded and the resultant juice strained through cloth. One a set of pentose carbon signals, and a methoxy signal.
or two drops of this juice placed into the eyes is said to NMR data were in agreement with a 1,4-disubstitution of
have an immediate effect on inflammation. However, there benzene ring. The nature of the terminal sugar unit as
are no data in the literature concerning the possible β-D-apiofuranosyl was deduced by the following
pharmacological effects and the chemical constituents; this evidence: the 1H NMR spectrum indicated an anomeric
is the first chemical investigation of M. obovata leading to signal at δ 5.01 (H-1'', d, J = 2.0 Hz); in the 1D TOCSY
the isolation of 10 phenolic compounds. experiment, selective excitation of the signal at δ 5.01 led
958 Natural Product Communications Vol. 6 (7) 2011 Arevalo et al.

Table 1: 1H and 13C NMR (600 MHz) Data for Compound 1in CD3ODa. spectra indicated the presence of 1,2,4-trisubstituted
1 aromatic ring, with one methoxyl group as well as a -D-
Position δ 1H (JHH in Hz) δ13C
1 ----- 149.6
apiofuranosyl-(1→6)-O--D-glucopyranosyl unit. The
2 7.09 (d, 8.5) 116.8 spectroscopic data of compound 3 suggested the same
3 7.01 (d, 8.5) 114.4 skeleton as compound 2, but lacking a methoxyl group.
4 ----- 154.6
5 7.01 (d, 8.5) 114.4
The complete assignment was established by the
6 7.09 (d, 8.5) 116.8 resonance of C-3 () shifted upfield by 3.6 ppm and
Glu of C-4 (144.7) and C-2 () shifted downfield by
1' 4.78 (d, 8.0) 102.8
2' 3.28 (dd, 7.5, 9.5) 74.3 ca. 2.4 and 1.6 ppm with respect to dimethoxylated model
3' 3.43 (t, 9.5, 9.5) 78.3 (2). The 3-hydroxy, 4-methoxy substitution was also
4' 3.34 (t, 9.5, 9.5) 70.9 confirmed by HMBC experiments, the correlation peaks
5' 3.30 (m) 77.3
6' 3.70 (dd, 12.0, 3.5) 69.0 of H-2/C-6, C-4; H-6/C-2, C-4, H-5/ C-1, C-3; CH3O–/
3.56 (dd, 12.0, 5.0) C-4; H-1'/C-1 and H-1''/C-6' indicated that benzene ring
Api was substituted at C-1 with the apiofuranosyl-(1→6)-
1'' 5.01 (d, 2.0) 110.3
2'' 4.03 (d, 2.0) 77.4 glucopyranosyl unit and the methoxyl group was located
3'' ----- 80.5 at the C-4. Therefore, the structure of 3 was determined
4'' 3.82 (d, 10.0) 75.4 as 3-hydroxy, 4-methoxyphenol 1-O-β-D-apiofuranosyl-
4.05 (d, 10.0)
5'' 3.61 (2H, s) 66.2 (1→6)-O--D-glucopyranoside, reported previously only
OCH3 3.85 56.4 in the Indonesian medicinal plant Fagara rhetza
a
Chemical shift values are in ppm from TMS, and values in Hz are (Rutaceae) [7].
presented in parentheses. All signals were assigned by DQFCOSY,
HSQC and HMBC experiments.
The structures and molecular formulas of compounds
4-10 were determined from their ESIMS spectra, as well
to the the enhancement only of H-2'' (δ 4.03, d, J = 2.0
as from 1D and 2D 1H and 13C NMR data and by
Hz); and the multiplicity of H-2'' may be derived only
comparison of their NMR data with those in the
from the presence of a quaternary carbon at C-3''
literature. Compound 4 was identified as verbascoside by
characteristic of an apiofuranosyl structure. The 13C NMR
HPLC comparison with authentic standard and according
spectrum gave 11 carbon signals for the sugar moiety, of
to its 1H, 13C NMR, and ESI-MS data [8]. The structure
which three methylenes were ascribable to C-4'' (δ 75.4)
of isoverbascoside (5) was confirmed using 1H and
and C-5'' (δ 66.2) of an apiofuranosyl unit and to C-6' 13
C NMR. The 1H NMR spectrum of isoverbascoside was
(δ 69.0) of a glucopyranosyl unit, respectively. Analysis
similar to that of verbascoside, except for differences in
of the correlated 13C NMR signals in the HSQC spectrum
and of the resonances of the quaternary carbon signal the chemical shifts of H-4' (verbascoside,  4.81;
(δ 80.5, C-3'') matched well with a terminal β-D- isoverbascoside,  3.41) and 2H-6 (verbascoside,  3.63
apiofuranosyl linked to an inner β-D-glucopyranosyl. C-6' and 3.84; isoverbascoside,  4.34 and 4.50) in their
of the glucopyranosyl unit was shifted downfield glucosyl moiety. The 13C NMR chemical shifts of
(β-effect) demonstrating the (1→6) linkage between the isoverbascoside were close to those of verbascoside,
apiosyl and glucosyl units. The interglycosidic linkage but slight differences were observed in the shifts at
was also confirmed unambiguously to be at C-6’’ based C-3', C-4', and C-6' (verbascoside,  81.66, 70.69, 62.49;
on the HMBC cross-peak, between H-1'' and C-6'. isoverbascoside,  84.45, 70.94, 65.20) [9]. Compound 6
Correlations due to long-range HMBC couplings were showed ESI-MS and 1H and 13C NMR data
also observed between H-1' and C-1.Therefore, the superimposable with those reported in the literature for
structure of 1 was determined as 4-methoxyphenol 1-O-β- leucoceptoside A [10]. Compounds 7-9 were identified as
D-apiofuranosyl-(1→6)-O--D-glucopyranoside (1). the flavones vitexin, isovitexin and luteolin 8-C-β-D-
glucopiranoside, compound 10 as the flavonol spireoside
Comparison with the NMR spectral showed that the on the basis of their spectroscopic data and specifically
NMR signals in 2 were similar to those of 1, except for by comparison of their NMR data with those in the
the presence of an extra O-methyl, suggesting that 2 was literature [11].
a 1,3,4- trisubstituted benzene with two methoxyl groups
and one apiofuranosyl(1→6)-glucopyranosyl group. The Experimental
location of these groups was verified by HMBC and NOE General Experimental Procedure: A Bruker DRX-600
spectra. Therefore, compound 2 was concluded to be NMR spectrometer, operating at 599.19 MHz for 1H and at
3,4-dimethoxyphenol 1-O-β-D-apiofuranosyl-(1→6)-O-- 150.86 MHz for 13C, was used for NMR experiments;
D-glucopyranoside reported previously only in two chemical shifts are expressed in (parts per million)
species Symplocos caudata (Symplocaceae) [5] and referring to the solvent peaks  H 3.34 and  C 49.0 for
Tabebuia impetiginosa (Bignoniaceae) [6]. CD3OD; coupling constants, J, are in Hertz. DEPT, 13C,
DQF-COSY, HSQC, HMBC and NOESY NMR
Compound 3 has the molecular formula C18H26O12, as experiments were carried out using the conventional pulse
deduced from ESIMS analysis. The 1H and 13C NMR sequences as described in the literature. Electrospray
Phenolic compounds from Martinella obovata Natural Product Communications Vol. 6 (7) 2011 959

ionization mass spectrometry (ESIMS) was performed 4-methoxyphenol 1-O-β-D-apiofuranosyl-(1→6)-O--D-


using a Finnigan LCQ Deca instrument from Thermo glucopyranoside (1)
Electron (San Jose, CA) equipped with Xcalibur software. White amorphous solid.
Instrumental parameters were tuned for each investigated [α]D: -58.9 (c 0.20, MeOH).
compound: capillary voltage was set at 3 V, the spray UV/Vis λmax (MeOH) nm (log ε): 202 (4.42), 223 (3.85),
voltage at 5.10 kV and a capillary temperature of 220°C 279 (3.42).
1
and the tube lens offset at - 60 V was employed; specific H and 13C NMR (600 MHz, CD3OH): see Table 1
collision energies were chosen at each fragmentation step ESI-MS m/z 417 [M-H]-, m/z 285 [M-132]- and m/z 123
for all the investigated compounds, and the value ranged [M-132-162]-
from 15-33% of the instrument maximum. Data were HREIMS m/z 418.3560 (calcd for C18H26O11, 418.6570).
acquired in the MS1 scanning mode (m/z 150-700). All
compounds were dissolved in MeOH : H2O (1:1) and 3,4-dimethoxyphenol 1-O-β-D-apiofuranosyl-(1→6)-O-
infused in the ESI source by using a syringe pump; the -D-glucopyranoside (2)
flow rate was 5 L/min. Exact masses were measured by a 1
H and 13C NMR data were consistent with those
Q-TOF premier (Waters, Manifold, MA, USA) instrument. previously reported [4].
Chromatography was performed over Sephadex LH-20 ESI-MS m/z 447 [M-H]-, m/z 315 [M-132]- and m/z 153
(Pharmacia, Uppsala, Sweden) employing MeOH as [M-132-162]-
solvent. Column chromatography was carried out HREIMS m/z 448.2560 (calcd for C19H28O12, 448.4430).
employing Silica gel RP18 (0.040–0.063 mm; Carlo Erba)
and MeOH:H2O gradients. HPLC separations were 3-hydroxy, 4-methoxyphenol 1-O-β-D-apiofuranosyl-(1-
performed on a Waters 590 series pumping system >6)-O--D-glucopyranoside (3)
equipped with a Waters R401 refractive index detector and 1
H and 13C NMR data were consistent with those
a Kromasil C18 column (250 x 10 mm i.d., 10m, previously reported [7]
Phenomenex). HPLC-grade methanol was purchased from ESI-MS m/z 433 [M-H]-, m/z 301 [M-132]- and m/z 139
Sigma Aldrich (Milano, Italy). HPLC-grade water (18 [M-132-162]-
mΩ) was prepared by a Milli-Q50 purification system HREIMS m/z 434.3177 (calcd for C18H26O12, 434.7100).
(Millipore Corp., Bedford, MA). TLC analysis was
performed with Macherey-Nagel precoated silica gel 60 Verbascoside (4)
1
F254 plates. H and 13C NMR data were consistent with those
previously reported [8]. ESI-MS m/z 621 [M-H]-.
Plant Material: The leaves of M. obovata (Kunth) Bureau
& K. Schum. were collected in Pico Bonito, Francisco Isoverbascoside (5)
1
Morazan, Honduras, in August 2005. The plant was H and 13C NMR data were consistent with those
identified by Dr. Cirilo Nelson. A voucher specimen was previously reported [9]. ESI-MS m/z 621 [M-H]-.
deposited in the herbarium of the Botanical Department of
the Universidad Nacional Autonoma de Honduras, Leucosceptoside A (6): 1H and 13C NMR data were
Tegucigalpa, Honduras, (Voucher No. 314). consistent with those previously reported [10]. ESI-MS
m/z 635 [M-H]-.
Extraction and Isolation Procedure of Compounds 1-
10: Dried and powdered leaves (1 kg) of M. obovata were Vitexin (7)
1
extracted for a week, three times, at room temperature H and 13C NMR data were consistent with those
using solvents of increasing polarity; namely, petroleum previously reported [11]. ESI-MS m/z 431 [M-H]-.
ether, chloroform, and methanol. Part (3 g) of MeOH
extract was chromatographed on a Sephadex LH-20 Isovitexin (8)
1
column (100 cm x 5.0 cm) using CH3OH as mobile phase H and 13C NMR data were consistent with those
and a flow rate of 1 mL/min to furnish 6 fractions (I-VI). previously reported [11]. ESI-MS m/z 431 [M-H]-.
Fraction II and III (258.4 mg) were purified by RP-HPLC
(40% CH3OH) to give 2 (9.5 mg) and 3 (4.9 mg) and 1 Luteolin 8-C--D-glucopiranoside (9)
1
(9.2 mg). Fr. IV (184.1 mg) was purified by RP-HPLC H and 13C NMR data were consistent with those
(35% CH3OH) to give 4 (16.8 mg), 5 (6.4 mg) and 6 (3.8 previously reported [11]. ESI-MS m/z 447 [M-H]-.
mg). Finally Fr. V and VI containing flavones were
purified with 70:30 MeOH-H2O to yield compounds 7 Spireoside (10)
1
(15.1 mg), 8 (12.2 mg), 9 (6.1 mg) and 10 (7.2 mg). H and 13C NMR data were consistent with those
previously reported [11]. ESI-MS m/z 463 [M-H]-.

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[4] Witherup KM, Ransom RW, Graham AC, Bernard AM, Salvatore MJ, Lumma WC, Anderson PS, Pitzenberger SM, Varga SL.
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Bulletin, 53, 110-113
[6] Warashina T, Nagatani Y, Noro T. (2006) Constituents from the Bark of Tabebuia impetiginosa. Chemical & Pharmaceutical
Bulletin, 54, 14-20.
[7] Shibuya H, Takeda Y, Zhang RS, Tanitame A, Tsai YL, Kitagawa I. (1992) Indonesian medicinal plants. IV. On the constituents of
the bark of Fagara rhetza (Rutaceae). (2). Lignan glycosides and two apioglucosides. Chemical & Pharmaceutical Bulletin, 40,
2639-2646.
[8] Sticher O, Lahloub MF (1982) Phenolic glycosides of Paulownia tomentosa bark. Planta Medica, 46, 145-148.
[9] Kawada T, Asano R, Makino K, Sakuno T. (2002) Synthesis of isoacteoside, a dihydroxyphenylethyl glycoside. Journal of Wood
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glycosides from Leucoseptrum japonicum (Miq.) Kitamura et Murata. Chemical & Pharmaceutical Bulletin, 30, 2732-2737.
[11] (a) Agrawal PK. (1989) Carbon-13 NMR of Flavonoids. Elsevier, London; (b) Harborne JB. (1994) The flavonoids: Advances in
Research since 1986. Chapman & Hall, New York.
2011
NPC Natural Product Communications Vol. 6
No. 7
Phenolic Chemical Composition of Petroselinum crispum 961 - 964
Extract and Its Effect on Haemostasis
Douglas S. A. Chavesa#, Flávia S. Frattanib, Mariane Assafimb, Ana Paula de Almeidac,d,e,
Russolina B. Zingalib and Sônia S. Costaa*
a
Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, 21 941-902,
Rio de Janeiro, RJ, Brazil
b
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 21 941-902, Rio de Janeiro,
RJ, Brazil
c
Departamento de Ciências Químicas, Laboratório de Química Orgânica e Farmacêutica,
Faculdade de Farmácia, Universidade do Porto, 4050-047, Porto, Portugal
d
Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), 4050-047, Porto, Portugal
e
Laboratório de Estudo Químico e Farmacológico de Produtos Naturais, Universidade Severino Sombra,
27 700-000, Vassouras, RJ, Brazil
# Present address: Instituto de Ciências Exatas, Departamento de Química, Universidade Federal
Rural do Rio de Janeiro, 23890-000, Seropédica, RJ, Brazil

sscbh@terra.com.br; sscosta@nppn.ufrj.br

Received: November 11th, 2010; Accepted: March 16th, 2011

From the aqueous extract (Pc) of Petroselinum crispum (Mill) flat leaves specimens were isolated and identified the flavonoids apigenin (1),
apigenin-7-O-glucoside or cosmosiin (2), apigenin-7-O-apiosyl-(1→2)-O-glucoside or apiin (3) and the coumarin 2’’,3’’-dihydroxy-
furanocoumarin or oxypeucedanin hydrate (4). The inhibitory activity toward clotting formation and platelet aggregation was assessed for Pc
flavonoids (1) and (2), and the coumarin (4). Pc showed no inhibition on clotting activity when compared with the control. On the other
hand, a strong antiplatelet aggregation activity was observed for Pc (IC50 = 1.81 mg/mL), apigenin (IC50 = 0.036 mg/mL) and cosmosiin
(IC50 = 0.18 mg/mL). In all cases ADP was used as inductor of platelet aggregation. Our results showed that Pc, apigenin and cosmosiin
interfere on haemostasis inhibiting platelet aggregation. To the best of our knowledge this is the first report for the cosmosiin antiplatelet
aggregation in vitro activity.

Keywords: Petroselinum crispum, parsley, flavonoids, cosmosiin, cardiovascular disease, haemostasis, platelet aggregation.

The species Petroselinum crispum, known as parsley, is an Aspirin, an antiplatelet agent and warfarin, an oral
aromatic herb from Apiaceae family that has been anticoagulant were developed from secondary metabolites.
employed in food, pharmaceutical, perfume and cosmetic However, aspirin consumption can increase the risk of
industries [1]. Widespread in all continents, parsley may gastrointestinal bleeding and other adverse effects [16].
be one of the oldest herbs used as condiment in food. Antiplatelet and anticoagulant drugs are used to treat
Previous studies on the chemical composition of parsley cardiovascular diseases and strokes preventing or slowing
have revealed the presence of flavonoids [2-5], coumarins down blood clots formation and enlargement of existing
[6-8] and terpenes [9,10]. In popular medicine, parsley is blood clots. [17].
used to treat various illnesses such as Alzheimer’s disease,
thrombosis and strokes [11,12]. In Morocco and Brazil, According to World Health Organization (WHO), in the
parsley is widely employed against cardiovascular diseases next 20 years there will be 24 million deaths from
[12-14]. cardiovascular diseases [18]. The current spending with
antithrombotic treatment is extremely high [19]. Many
The ethnopharmacological knowledge is useful to identify antithrombotic substances from synthetic or semi-synthetic
potential therapeutic targets from medicinal plants. origin or derived from natural products are currently under
Substances from vegetal kingdom, which have already clinical evaluation (phases I, II, III or IV). About 450
contributed with several compounds in prophylaxis and substances are considered promising candidates as new
treatment of a large variety of pathologies, have been antithrombotic drugs in the Stroke Trials Registry [20].
investigated for their potential as antithrombotic agents
[15,16].
962 Natural Product Communications Vol. 6 (7) 2011 Chaves et al.

OH OH

RO O O
A possible explanation for the difference in these results
OH should be due to the different extraction procedures
H employed in both studies. We prepared a decoction from
OH O O O O
fresh leaves at 10% (w/v), while Mekhfi et al. (2004)
1-3 4
prepared an infusion at 5.5% (w/v) without mention if
Compounds R Rt UV [M-H+]
fresh or dried aerial parts were used. The variety of P.
min λ max (nm) m/z crispum was not mentioned by those authors, while we
1 H 13.98 256; 336 269.18 used the flat leaf specimens. Furthermore, other factors
2 β-D-Glc 12.91 256; 337 433.26 such as cultivation conditions, sunlight exposure and
3 β-D-Api (1→2)-O-Glc 21.52 268; 339 563.30 season may lead to differences in the production of
4 ------- 33.78 249; 265; 315 305.20 bioactive secondary metabolites [25].
Figure 1: Compounds identified in the aqueous extract of Petroselinum
crispum (Pc) based on HPLC-DAD, ESI and NMR analyses.

This study led to the isolation of known 5,7,4’-trihydroxy-


flavone (apigenin), apigenin 7-O-glucoside (cosmosiin),
apigenin 7-O-apiosyl-(1→2)-O-glucoside (apiin)
and 2’’,3’’-dihydroxyfuranocoumarin (oxypeucedanin
hydrate) identified according to reported NMR data
[21,22]. These flavones and the coumarin derivative were
previously described for this species [4,6]. Two other Figure 2: Inhibitory effect of Petroselinum crispum (Pc) aqueous extract
on the platelet aggregation in vitro induced by ADP (5 µM) using human
flavones diosmetin apiosyl-glucoside and diosmetin platelet-rich plasma (n = 3).
apiosyl-glucoside isomer were identified with basis on
HPLC-DAD chromatogram and comparison with literature
data [4]. Apiin is the most abundant flavonoid in parsley,
while apigenin is the minor component as reported
previously in the literature [3].

In experiments assessing the intrinsic pathway (aPTT), and


the extrinsic pathway (PT) of coagulation, Pc extract,
apigenin, cosmosiin and oxypeucedanin hydrate did not
show any significant activity, since the clotting time was Figure 3: Inhibitory effect of apigenin on the platelet aggregation in vitro
not significantly increased. Figures 2, 3 and 4 show the induced by ADP (5 µM) using human platelet-rich plasma (n = 3).
antiplatelet aggregation effect observed for Pc, apigenin
and cosmosiin, respectively. A strong antiplatelet
aggregation activity was observed for Pc (IC50 = 1.81
mg/mL), apigenin (IC50 = 0.036 mg/mL) and cosmosiin
(IC50 = 0.18 mg/mL).

Our results confirm the already known antiplatelet activity


reported for Pc and apigenin [12,23]. Cosmosiin exhibited
a significant antiplatelet activity, although less active than
apigenin. We can deduce from our findings that the
Figure 4: Inhibitory effect of cosmosiin on the platelet aggregation in
presence of glycosylation decreases the activity of vitro induced by ADP (5 µM) using human platelet-rich plasma (n = 3).
cosmosiin, since the apigenin skeleton is common to both
structures [24]. Flavonoids and other phenolic substances are able to
interfere in the platelet system. Apigenin was shown to
Studies on the medicinal species P. crispum (Pc) showed block the inducer collagen and ADP in platelet-rich plasma
that its aerial parts aqueous extract was able to inhibit [24,26]. A diet rich in phenolic compounds may favorably
platelet activity induced by ADP [13]. In these studies, a contribute for reducing risks of cardiovascular diseases
crude extract at 10 mg/mL inhibited the platelet through several mechanisms. Several studies on the
aggregation by 78%. In our study we observed that Pc cardiovascular protective effect of flavonoids have been
leaves extract significantly inhibited (IC50 = 1.81 mg/mL) reported, suggesting that both apigenin and luteolin may
the platelet aggregation induced by ADP in human act as competitors with the receptor of thromboxane A2
platelet–rich plasma. Moreover, at concentrations 2.6 times (TXA2), an inducer of platelet aggregation [27].
lower (3.80 mg/mL) than that used by Mekhfi et al. (2004)
we obtained a higher platelet aggregation inhibition We can observe that some flavonoids, particularly
(94.4%) [13]. flavones, may be related to the in vitro inhibitory effect of
Phenolic composition of P. crispum and haemostasis Natural Product Communications Vol. 6 (7) 2011 963

platelet aggregation induced by ADP [12,13]. Our results the extract was filtered (Whatman filter paper Nr.1), frozen
showed that Pc extract, apigenin and cosmosiin interfere at -20°C and lyophilized (1.8 g). After its re-suspension in
on haemostasis inhibiting platelet aggregation. To the best water, the resulting solution was partitioned successively
of our knowledge this is the first report for the cosmosiin with ethyl acetate (3 x 300 mL) and n-butanol (3 x 300
in vitro-antiplatelet aggregation activity. The study of apiin mL). The ethyl acetate fraction (301.0 mg) was
and the coumarin oxypeucedanin hydrate effect on the chromatographed over Sephadex LH-20 (23 x 0.7 cm;
coagulation process is undergoing in our laboratories. ethanol) affording three fractions. The second fraction
(64.8 mg) showed a yellow crystalline solid (22.3 mg) that
Experimental was separated by centrifugation and identified as apigenin
(1). The third fraction was purified on an RP-2 column (30
Chemical
x 1.2 cm; H2O/EtOH), followed by a Sephadex LH-20
General: Melting points were determined using a Koppler chromatography (23 x 0.7 cm; MeOH/H2O 1:1), affording
melting point apparatus. Optical rotations were measured oxypeucedanin hydrate (4) as a brown powder (15.4 mg).
on a Jasco P-2000 digital polarimeter. All 1D and 2D
experiments were performed on a Varian 400 MHz
Biological
spectrometer. The NMR spectra were recorded in DMSO-
In vitro determination of activated partial thromboplastin
d6. ESI-MS spectra were recorded on a tandem – triple
time (aPTT) and prothrombin time (PT): Blood samples
quadrupole m/z 30-3000. HPLC separation was performed
were centrifuged (2000 x g, 10 min), and the platelet-poor
using a Shimadzu liquid chromatograph LC-10AD
plasma was stored at -20°C until use). aPTT and PT were
equipped with an UV SPD-10A wavelength detector.
measured on an Amelung KC4A coagulometer as follows.
The reversed-phase column used was Merck C18 (5 µm,
For aPTT tests, cephalin plus kaolin (aPTT reagent,
250 mm, 2.5 mm) with mobile phase consisted of water
BioMériaux, RJ, Brazil) were incubated for 1 min with 50
containing phosphoric acid 0.01% (eluent A) and methanol
µL of pre-warmed plasma (37°C) and P. crispum (Pc),
(eluent B). The samples were run for 44 minutes at
apigenin, cosmosiin and oxypeucedanin hydrate at various
1 mL/min and absorbance was monitored between
concentrations (suspension in PBS buffer). The reaction
200 – 500 nm. The gradient used was 0 – 5 min (100 –
was started by addition of 100 µL of pre-warmed CaCl2
65% A), 5 – 15 min (65 – 55% A), 15 – 25 min (55 – 52%
(25 mM).
A), 25 – 35 min (52 – 45% A), 35 – 40 min (45 – 20% A),
40 – 42 min (20 – 0% A) and 42 – 44 min (0 – 100% A).
For PT tests, 50 µL of pre-warmed plasma was incubated
Thin layer chromatography (TLC) was performed on silica
with Pc extract, apigenin, cosmosiin and oxypeucedanin
gel 60 F254 (Merck) eluted with n-butanol/acetic acid/water
hydrate at various concentrations (suspension in PBS
(BAW) 8:1:1, visualized under UV light (254 and 365 nm)
buffer) for 2 min (37°C) and reaction was started by
and developed with ceric sulfate solution for flavonoids.
addition of 100 µL of pre-warmed thromboplastin with
Coumarin was detected using 5% potassium hydroxide
calcium (PT reagent, BioMériaux, RJ, Brazil). Apiin was
solution in ethanol.
not evaluated since it is insoluble in water.
Plant material: Leaves from Petroselinum crispum (Mill.) Platelet aggregation assays: Human blood was collected
Nym.ex A.W. Hill (flat leaf specimens) were collected out in EDTA 0.2 M (9:1 v/v). Platelet-rich plasma (PRP) was
of blooming season from specimens grown in an prepared by centrifugation (500 x g, 10 min) at room
experimental garden at Severino Sombra University temperature. The platelet-poor plasma (PPP) was prepared
(Vassouras, RJ, Brazil). A voucher specimen (RFA – by centrifugation of the PRP (2000 x g, 10 min) at room
31241) was classified by Dr. Ricardo C. Vieira and temperature). In some cases, experiments were performed
deposited in the herbarium of the Institute of Biology, using washed platelets as described [28]. Platelet
UFRJ, Brazil. aggregation was monitored by the turbidimetric method on
a Chrono-Log aggregometer. PRP (400 µL) was incubated
Extraction and isolation: Fresh leaves (160 g) were (37°C, 1 min) with continuous stirring at 900 rpm. Platelet
triturated using a food processor and extracted with aggregation was induced by ADP (2-10 mM). P. crispum
distilled water (10% w/v) by decoction (10 minutes). After extract (Pc), apigenin, cosmosiin and oxypeucedanin
the extract filtration, a spontaneous precipitation at room hydrate at various concentrations (suspension in PBS
temperature yielded a solid that was separated by buffer) or vehicle (0.5% DMSO v/v) was added to PRP
centrifugation. This precipitate (1.6 g) was re-suspended in samples 1 min before addition of the agonist.
methanol and chromatographed over Sephadex LH-20
(30 x 1.5 cm; MeOH) yielding 2 fractions: a minor Supplementary data: Compounds from Petroselinum
methanol-soluble fraction (396.2 mg) and a methanol- crispum (Mill.) Nym.ex A.W. Hill, an aromatic herb
insoluble one (1.2 g). Each fraction was purified over popularly known as parsley.
Sephadex LH-20 (23 x 0.7 cm; MeOH/H2O 1:1) affording
cosmosiin (2), for the soluble fraction, as a yellow powder Acknowledgments: Authors gratefully acknowledge
(23.4 mg) and apiin (3), for the insoluble fraction as a CNPq and CAPES for financial support. We also thank
white powder (676.8 mg). After the precipitate separation
964 Natural Product Communications Vol. 6 (7) 2011 Chaves et al.

Dr. R. C. Vieira (IB, UFRJ, Brazil) and Dr. Jean-Pierre thank CEQUIMED (FCT, I&D 4040) for allowing Ana
Férézou (ICMMO, Université Paris-Sud, France). We Paula de Almeida to collaborate in this study.

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2011
NPC Natural Product Communications Vol. 6
No. 7
Bioactivities of Chuquiraga straminea Sandwith 965 - 968

María Elena Mendiondoa, Berta E. Juáreza, Catiana Zampinia,b, María Inés Islaa,b and
Roxana Ordoñeza,b,*
a
Facultad de Ciencias Naturales e Instituto Miguel Lillo.UNT. Fundación Miguel Lillo. CONICET
Miguel Lillo 205/251. (4000).San Miguel de Tucumán. Tucumán. Argentina
b
INQUINOA. CONICET. Facultad de Bioquímica, Química y Farmacia. UNT. Ayacucho 471.
San Miguel de Tucumán. Tucumán. Argentina

rmordoniez@fbqf.unt.edu.ar

Received: December 10th, 2010; Accepted: March 16th, 2011

Methanolic extracts of Chuquiraga straminea Sandwith, subfamily Barnadesioideae (Asteraceae) showed the presence of quercetin-3-O-
glucoside, quercetin-3-O-rutinoside, kaempferol, kaempferol-3-O-glucoside and kaempferol-3-O-rutinoside. Antioxidant and antimicrobial
activity was determined. The total extracts showed antioxidant activity by DPPH and ABTS method (SC50 14.5 to 34.9 µg/mL). A
significantly positive correlation was observed between the antioxidant activity and the total phenolics (R2>0.93). The extracts were active
against ten methicillin resistant and sensitive Staphylococcus aureus strains isolated from nosocomial infection (MIC values between 200 to
800 μg/mL). These preliminary studies are highly interesting as they open new ways for further applications in the treatment of infections by
methicillin resistant S. aureus.

Keywords: Flavonoids, Chuquiraga straminea, Antioxidant activity, Antimicrobial activity.

From an estimated 250,000 higher plants in the world only has long been considered a major problem of Public
5-15% have been studied for a potential therapeutic value. Health. The aim of this study was to investigate
The Argentinian flora offers great possibilities for the antioxidant and antimicrobial activities of C. straminea
discovery of new compounds with medicinal uses. The extracts obtained from aerial parts and flowers.
genus Chuquiraga is represented in Argentina by 15
species distributed in arid regions between the Andes and Extracts were analyzed for their contents of phenolic
Patagonia. Previous reports indicated that the flavonoids compounds (total phenols, flavonoids). A relationship
identified in the species of Chuquiraga genus are between antimicrobial activity, antioxidant activities and
identical, so the compounds are useful as phylogenetic the content of phenolic compounds was evaluated.
micromolecular markers in the genus [1]. Chuquiraga
straminea is a medium xerophytic shrub. Its distribution is The content of total phenols and flavonoids of flower and
in southern Argentina, from northwestern Chubut province aerial parts extracts from C. straminea are given in Table
to eastern Neuquen province. It inhabits in Patagonian 1. The amount of total phenolic compounds extracted
phytogeographic province between 600 and 1000 meters ranged from 1.56 to 2.74 mg gallic acid equivalent
above sea level (masl). This species has been employed as (GAE)/mL extract. The C. straminea aerial parts extracts
traditional medicine by native people, either as building contained the highest amounts of phenolic compounds.
and crafts material and forage [2].
Table 1: Phenolic compounds of C. straminea extracts.
It is reported that phenolic compounds from herbs are Alcoholic Phenolic compounds Flavonoids Compounds
active against many human pathogenic bacteria and fungi Extracts (mg GAE/mL) (mg QE /mL) isolated
quercetin-3-O-glucoside,
and have antioxidant activity [3-5]. Recently, there has Flowers 1.56±0.04 0.12±0.01 quercetin-3-O-rutinoside,
been considerable interest in the use of such antioxidants kaempferol, kaempferol-
and antimicrobial compounds from natural sources, not 3-O-glucoside and
Aerial 2.74±0.08 0.14±0.01 kaempferol-3-O-
only in pharmaceutical industry but also for the rutinoside
Parts
preservation of foods and improving the shelf life of food
products, for increasing the stability of fats and oils and to
control the plant diseases of microbial origin. Kaempferol, quercetin-3-O-glucoside, quercetin-3-O-
Staphylococcus aureus is a common pathogen associated rutinoside, kaempferol-3-O-glucoside and kaempferol-3-
with serious community and hospital acquired diseases and O-rutinoside were identified in the extracts. The flavonoid
966 Natural Product Communications Vol. 6 (7) 2011 Mendiondo et al.

A B

Figure 1: Residual scavenging activity (% RSA) of samples on DPPH (A) and ABTS (B) radicals at a phenolic compounds concentration until of 50 µgGAE/mL. (■) C.
straminea flower extract (SC50DPPH=34.9±1.2 and SC50ABTS= 31.0±1.1 µgGAE/mL) and (♦) C. straminea aerial parts extract (SC50 DPPH= 18.0±0.7µg and
SC50ABTS= 14.5±0.8µg GAE/mL).

glycosides are comparable with those previously isolated, Table 2: Antibacterial activities of C. straminea extracts against sensitive
and antibiotic resistant Staphylococcus aureus strains.
by us from other argentine species belonging to
Chuquiraga genus, [1,6]. C. straminea extracts
Strain Phenotype MIC/MBC (μgGAE/mL)
Antioxidant activity: Free radical species play a critical Flowers Aerial parts
role in cardiovascular and inflammatory diseases as well as MRSA F2 Metr Oxar Genr 400/400 800/>800
in neurodegenerative disorders, cancer and aging. The C. MRSA F7 Metr Oxar GenrVans 400/800 800/>800
straminea extracts were effective as ABTS [2,2`-azinobis MRSA F31 Metr Oxar GenrVans 800/>800 800/>800
(3-ethylbenzothiazoline-6 sulfonic acid) diammonium salt] MSSA F13 Mets Oxas GensVans 200/400 800/>800
MSSA F16 Mets Oxas GensVans 400/400 400/>800
and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-
MSSA F24 Mets Oxas GensVans 400/400 400/>800
scavengers. The results obtained are represented in Figure MRSCN F22 Metr Oxar Genr Vans 400/800 800/>800
1 (SC50 values denote the sample concentration required to MRSCN F27 Metr Oxar Genr Vans 800/>800 800/>800
scavenge 50% ABTS or DPPH free radicals). Aerial parts MSSCN F29 Mets Vans 400/800 400/>800
extracts were the most effective radical-scavengers with MSSCN F30 Mets Vans 400/800 800/>800
SC50 of 18.0±0.7 and 14.5±0.79 µg GAE/mL for DPPH ATCC 29213 Control strain 400/400 800/800
and ABTS respectively, flowers extracts result also active MRSA: methicillin resistant Staphylococcus aureus, MSSA: methicillin
with SC50 of 34.5±1.2 and 31.0±1.1 µg GAE/mL for DPPH sensitive Staphylococcus aureus, MRSCN: methicillin resistant
Staphylococcus coagulase negative, MSSCN: methicillin sensitive
and ABTS radicals, respectively. Staphylococcus coagulase negative, ATCC: American Type Culture
Collection. r and s, resistance or susceptibility to antibiotics. Met,
A significantly positive correlation was observed between methicillin; Oxa, oxacillin; Gen, gentamicin; Van, vancomycin. MIC was
defined as the lowest concentration of extract that had restricted growth to
the antioxidant potential determined by ABTS and DPPH a level <0.05 at 550nm. MBC (minimal bactericidal concentration) was
assays, and the total phenolics (R2>0.93).Contact defined as the lowest extract concentration at which 99.9% of the bacteria
autography, indicated that more than one compounds with have been killed.
antioxidant activity in all crude extracts. The flavonoids
could be the more active metabolites in this plant specie. differences between minimal inhibitory concentration
(MIC) values with respect to the control strain were not
Antimicrobial activity: The antibiotic resistant clinical S. significant. Similar behavior was observed for methicillin
aureus strains assayed in this work were isolated from sensitive and resistant Staphylococcus coagulase negative
human infections from a local hospital. (MSSCN and MRCN, respectively). All nosocomial
strains were sensitive to extracts and vancomycin, a
By means of bioautographic assay was qualitatively glycopeptidic antibiotic. In most cases the extracts showed
demonstrated that several phenolic compounds in the bactericidal effect.
methanolic extracts were active against methicillin
resistant and sensitive S. aureus strains. Table 2 show the The reported data in the present work for S. aureus was
antimicrobial activity of C. straminea methanolic extracts similar to those obtained for other Chuquiraga species that
against ten S. aureus antibiotic resistant and sensitive grow in arid regions of Argentine [5].
strains.
Up to the present, there are limited reports about the
The two methanolic extracts of C. straminea were active bioactivities of C. straminea. The ethnobotanical data
against all methicillin resistant and sensitive S. aureus indicated that this species was used by mapuches and
strains (MRSA and MSSA, respectively) and the observed tehuelches as the main therapeutic tool in traditional
Bioactivity of Chuquiraga straminea Natural Product Communications Vol. 6 (7) 2011 967

medicine as antiinflammatory and antiseptic on skin The ABTS method: Antioxidant capacity assay was carried
infection [2]. According with our results the C. straminea out by the improved ABTS method as described Re [11].
extracts could serve as good candidates for the ABTS•+ radical cation was generated by reacting 7 mM
development of new antimicrobial and antioxidant agents ABTS and 2.45 mM potassium persulfate after incubation
and/or standardized phytomedicines. at room temperature (23 ºC) in the dark for 16 h. ABTS•+
solution (1mL; absorbance of 0.7 ± 0.02 at 734 nm) was
Experimental added to 5-50 μg GAE of each tested sample and mixed
Plant material: The plant was collected in the province of thoroughly. The reactive mixture was allowed to stand at
Neuquén, Department Collan Cura. Grow in patches on the room temperature and the absorbance was recorded at 734
slope of Piñon Cerrito, on rocky ground. A voucher nm, 1 min. after initial mixing and up to 6 min. Results
specimens is deposited at the Fundación Miguel Lillo were expressed in terms of percentage (%) of radical
Herbarium (LIL 605812). scavenging activity (RSA) at 6 minute and SC50 values
denoted the sample concentration required to scavenge
Preparation of plant extracts: The aerial parts (flowers and 50% ABTS free radicals.
leaves) of C. straminea were dried and extracted with
methanol 80%. The solvent was evaporated at reduced Autographic assay: For rapid visualization of antiradical
pressure and then, resuspended in DMSO activity, 5 μg of extracts were applied on silica gel 60 F254
(dimethylsulfoxide) for avoid the extraction solvent TLC plates. Mixtures of chloroform-ethyl acetate (80:20;
interference in the biological screening. v/v) was used as mobile phase. Then, the plates were dried
overnight and covered with 3 mL of soft medium (agar
Determination of total phenolics compounds (TPC) and 0.9%) containing 1mL ABTS•+ (7 mM ABTS and 2.45
flavonoids: TPC was determined using the Folin- mM potassium persulfate) or DPPH (1mg/mL). Plates
Ciocalteau method [7] and gallic acid was used as were incubated at room temperature during 1 minute in the
standard. The results were expressed as mg GAE/mL dark. Active samples appeared as light spots against a
extract. Flavonoid content was determined according to green-blue or purple background for ABTS or DPPH
Woisky and Salatino [8] and quercetin was used as assay, respectively [12].
standard. The results were expressed as mg QE/mL
extract. Microorganism: The microorganisms used in this study
consisted of ten Staphylococcus aureus strains recovered
Compounds identification: The extracts were from clinical samples obtained from the Hospital Nicolás
chromatographed bidimensionally according to Mabry [9] Avellaneda, San Miguel de Tucumán, Tucumán,
using TBA (tert-butanol-acetic acid-water 3:1:1) and 15% Argentina: methicillin resistant S. aureus (MRSA) (n=3),
AcOH as development solvents. Eluted spots were methicillin sensitive S. aureus (MSSA) (n=3), methicillin
analyzed by paper and thin layer chromatography in resistant S. coagulase negative (MRSCN) (n=2) and
different solvent systems. The plates were observed under methicillin sensitive S. coagulase negative (MSSCN)
ultraviolet light, in the absence and presence of ammonia (n=2). A reference strain was included in the study: S.
and natural product reagent. Spectral data with shift aureus ATCC 29213.
reagents, NaOMe (sodium methoxide), AlCl3 (aluminum
trichloride), HCl (hydrochloric acid), NaOAc (sodium Antimicrobial activity
acetate), H3BO3 (boric acid) were used. Bioautographic assays: Extracts (25 μg) were seeded on
TLC plates and the components were separated using
Free radical scavenging activity chloroform-ethyl acetate (80:20; v/v) as development
The DPPH method: The reduction capability of extracts solvents. Then, the plates were dried overnight in a sterile
was measured by DPPH method according to Zampini et al room and were covered with 3 mL of soft medium (BHI
[10]. DPPH solution (1.5 mL of 300 μM in 96% ethanol) with 0.6% agar) containing 1 x 105 colony forming units
was incubated with the samples (5-50 μg GAE). The (CFU) of S. aureus (F7) incubated at 35C for 20 h. Next,
reaction mixture was shaken and incubated during 20 min. the plates were sprayed with a 2.5 mg/mL MTT solution
at room temperature. Then, absorbance was measured at (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) in
515 nm. PBS (10 mM sodium phosphate buffer, pH 7, with 0.15 M
NaCl) to determine cellular viability. Plates were
The percentage (%) of radical scavenging activity (RSA) incubated at 35C for 1h in the dark for color development
was calculated using the following equation: [13].
RSA % = [(A0 - As)/A0] x 100.
Where A0 is the absorbance of the control and As is the Broth microdilution susceptibility assay: This assay was
absorbance of the samples at 515 nm. SC50 values denoted performed in sterile 96-well microplates. The extracts were
the sample concentration required to scavenge 50% DPPH transferred to each microplate well in order to obtain two-
free radicals. fold serial dilutions of the original extract (25 to 800
µg/mL). The inoculum (100 µL) containing 5×105 CFU
968 Natural Product Communications Vol. 6 (7) 2011 Mendiondo et al.

was added to each well. A number of wells were reserved MBC was defined as the lowest extract concentration at
in each plate for sterility control (no inocula added), which 99.9% of the bacteria have been killed.
inocula viability (no extract added), and solvent effect
(DMSO) [14]. MIC values were also determined for different commercial
antibiotics. Resistance was defined for each case:
Plates were aerobically incubated at 35°C. After methicillin (Met, MIC > 16 μg/mL), oxacillin (Oxa, MIC >
incubation for 16-20 h, bacterial growth was indicated 16 μg/mL), gentamycin (Gen, MIC > 100 μg/mL) and
by the presence of turbidity and a pellet on the well vancomycin (Van, MIC > 6 μg/mL) for S aureus strains.
bottom. A cytotoxicity assay was also carried out. All experiments were carried out in triplicate.
After the broth microdilution susceptibility assay, 20 L
of methylthiazolyltetrazolium chloride solution (MTT) Statistical analysis: Data are represented as mean ±
(12 mg/mL in PBS) was added to the wells and incubated standard deviation. The statistical tests were carried out by
for 1 h. Cellular viability was determined by absorbance at analysis of variance (one-way ANOVA) and the post-test
550 nm. of Turkey, using a probability level of less than 5% (p <
0.05).
MIC was defined as the lowest concentration of extract
that had restricted growth to a level <0.05 at 550nm (no Acknowledgments - This research was partially supported
macroscopically visible growth). by Grants from Consejo de Investigación de la
Universidad Nacional de Tucumán (CIUNT, Tucumán,
To confirm MIC and to establish MBC, 10 µL of each Argentina) and Consejo Nacional de Investigaciones
culture medium was removed from each well with no Científicas y Técnicas (CONICET; Buenos Aires,
visible growth and inoculated in Müller Hinton Agar Argentina).
plates. After 16-20 h of aerobic incubation at 35°C, the
number of surviving organisms was determined.

References
[1] Mendiondo ME, Juarez BE, Seeligmann P. (2000) Flavonoid profiles of some Argentine species of Chuquiraga (Asteraceae).
Biochemical Systematics and Ecology, 28, 283-285.
[2] González S, Morales S (2004) Plantas medicinales utilizadas en comunidades rurales del Chubut, Patagonia-Argentina. Boletín
Latinoamericano de Plantas Medicinales y Aromáticas, 3, 58-62.
[3] Arias ME, Gómez, JD, Vattuone MA, Isla MI. (2004) Antibacterial activity of ethanolic and aqueous extract of Acacia aroma Gill
ex Hook. Life Science, 75, 191-202.
[4] Zampini IC, Vattuone M, Isla MI. (2005) Antibacterial activity against antibiotic-resistant Gram negative human pathogenic
bacteria of hydroxychalcone isolated from Zuccagnia punctata Cav. Journal of Ethnopharmacology, 102, 450-456.
[5] Zampini IC, Cuello S, Alberto MR, Ordoñez RM, D’ Almeida R, Solorzano E, Isla MI. (2009) Antimicrobial activity of selected
plant species from “the Argentine Puna” against sensitive and multi-resistant bacteria. Journal of Ethnopharmacology, 124,
499–505.
[6] Juárez BE, Mendiondo ME. (2002) Flavonoid Chemistry of Chuquiraga (Asteraceae). Biochemical Systematics and Ecology, 30,
371-373.
[7] Singleton VL, Orthofer R, Lamuela-Raventos RM. (1999) Analysis of total phenols and other oxidation substrates and antioxidants
by means of Folin Ciocalteu reagent. Methods in Enzymology, 299, 152-178.
[8] Woisky R, Salatino A (1998) Analysis of propolis: some parameters and procedures for chemical quality control. Journal
Apiculture Research, 37, 99-105.
[9] Mabry TJ, Markham KR, Thomas MB. (1970) In The systematic identificaction of flavonoids. Springer-Verlag, New York, Chapter 5.
[10] Zampini IC, Meson Gana J, Ordoñez RM, Sayago JE, Nieva Moreno MI, Isla MI. (2008) Antioxidant and xanthine oxidase
inhibitory activities of plan species from the Argentine Puna (Antofagasta, Catamarca). Recent Progress in Medicinal Plants, 21,
95-110
[11] Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice Evans C. (1999) Antioxidant activity applying an improvement ABTS
radical cation decoloration assay. Free Radical Biology and Medicine, 26, 1231-1237.
[12] Zampini I, Ordoñez R, Isla MI. (2010) Autographic assay for the rapid detection of antioxidant capacity of liquid and semisolid
pharmaceutical formulations using ABTS●+immobilized by gel entrapment. AAPSPharm Sci Technology, 11, 1159-1163.
[13] Nieva Moreno MI, Isla MI, Cudmani NG, Vattuone MA, Sampietro AR. (1999) Screening of antibacterial activity of Amaicha del
Valle (Tucumán, Argentina) propolis. Journal of Ethnopharmacology, 68, 97-102.
[14] CLSI (Clinical and Laboratory Standards Institute, formerly National Committee for Clinical and Laboratory Standards, NCCLS).
(2006) Methods M27-A2 and M-38A, 2nd ed.; Wayne, PA. Ed.; Vol. 22 (15), 1-29; (16), 1-27.
2011
NPC Natural Product Communications Vol. 6
No. 7
Free Radical Scavenging Activity, Determination of 969 - 972
Phenolic Compounds and HPLC-DAD/ESI-MS Profile of
Campomanesia adamantium Leaves
Aislan C.R.F. Pascoala, Carlos Augusto Ehrenfriedb, Marcos N. Eberlinc,
Maria Élida Alves Stefanellob and Marcos José Salvadora,*
a
Pharmacy School, Department of Plant Biology, Institute of Biology,
State University of Campinas (UNICAMP), Campinas, SP, 13083-970, Brazil
b
Departament of Chemistry, Federal University of Paraná (UFPR), Curitiba, PR, 81531-990, Brazil
c
Thomson Mass Spectrometry Laboratory, Institute of Chemistry, State University of Campinas
(UNICAMP), Campinas, SP, 13083-970, Brazil

marcosjs@unicamp.br

Received: December 10th, 2010; Accepted: March 16th, 2011

Numerous diseases are induced by free radicals via lipid peroxidation, protein peroxidation and DNA damage. It has been known that a
variety of plant extracts have antioxidant activity to scavenge free radicals. Campomanesia adamantium (Myrtaceae) is a small tree with
edible fruit, commonly known as “guavira” or “guabiroba-branca” that has been used in popular medicine as depurative anti-diarrhoeic, anti-
inflammatory, anti-rheumatic and to liver diseases. In this study, the antiradical activities of ethanol crude extract of the leaves from C.
adamantium and the ethyl acetate and butanol fractions obtained by partition, were determined using DPPH (2,2-Diphenyl-1-picrylhydrazyl
radical) and ORAC-FL (Oxygen Radical Absorbance Capacity) assays. The total phenol content in the samples was estimated by Folin
Ciocalteau method (FCR). In an initial evaluation the ethanolic extract and the fractions ethyl acetate and butanol have shown levels of
phenolic compounds between 15- 74 mg GAE/g in FCR assay, showed DPPH free-radical scavenging activity with SC50 in the range of 7.77-
13.35 µg/mL and demonstrated antioxidant capacity between 2648-3502 µmol TE/g of extract and fractions in the ORAC-FL assay. HPLC-
DAD and ESI-MS analysis revealed were that the extract of the leaves of C. adamantium studied appears to contain flavonoids as major
constituents, including isoquercetrin and quercetin that exhibit proven antioxidant activity.

Keywords: radical scavenger, DPPH, Myrtaceae, Campomanesia adamantium, HPLC- DAD/ESI-MS.

The organic and inorganic molecules and atoms that from medicinal plants, acquire great pharmacological
contain one or more unpaired electrons, with independent importance and the research on these classes of
existence, can be classified as free radicals [1]. Free compounds has been increased in the last years [6-8].
radicals are responsible for lipid peroxydation occurred
during production and storage of nutrients [2], and are The genus Campomanesia (Myrtaceae) comprises around
directly involved in some cancers, cardiovascular 30 species of shrubs or small trees, aromatic, distributed
disorders, diabetes [3], Alzheimer's disease, atherosclerosis mainly in tropical and subtropical South America [9]. Most
and others human pathologies [4]. The radicals O2• and species produce edible fruits that are widely used to make
their reduction products, H2O2, and especially the radical liqueur, juices and jellies [10]. Several species are
OH•, are some of those responsible for cell damage by considered medicinal and have been used in folk medicine
promoting lipid peroxidation, with damage to mainly against digestive problems and diarrhea [11].
mitochondria, lysosomes and cell membrane itself, leading Campomanesia adamantium Camb. is a small tree, known
to cell death. In animal, different biochemical routes as “guavira” or “guabiroba-branca”, largely spread in
involve free radicals formation, but in these cases defense Brazil. It can be found growing wild in the Midwest,
mechanisms against the oxidative process propagation are Southeast and South regions of Brazil, and frequently is
also involved. These mechanisms do not show a constant cultivated in home gardens for its fruits [12]. Its leaves and
efficacy [3]. However, exogenous antioxidant compounds fruits have been used against rheumatism, liver and urinary
act as an auxiliary function in this defense processes. diseases [13]. Previous phytochemical studies in
Antioxidants block the free radicals formation through Campomanesia have reported the identification of
different ways and establish important control function in quercetin, myricetin and rutin in C. xanthocarpa [14] and
some oxidative stress diseases [4] and in food conservation β-triketone type compounds, named champanones in C.
[5]. Thus, new natural antioxidants, mainly those isolated lineatifolia [15]. Recently it was reported the isolation of
970 Natural Product Communications Vol. 6 (7) 2011 Pascoal et al.

flavonoids in C. adamantium [16,17] as well as the analyzed by ESI (-)-MS [20-21]. The analysis by ESI(-)-
antioxidant activity of extracts and fractions [17,18]. MS showed that major constituents in the samples of
However, these studies were carried on specimens growing C. adamantium leaves, including the crude ethanol extract
in Midwest region that can have a chemical profile and TLC yellow spot sample, were coincided with the
different from those growing in other regions of country. mass of the chalcones 2’,4’-dihydroxy-6’-methoxy-
These facts prompted us to investigate the antioxidant chalcone, 2’,4’-dihydroxy-5’-methyl-6’- methoxychalcone
capacity of the ethanolic extract and fractions of C. and, 2’,4’-dihydroxy-3’,5’-dimethyl-6’-methoxychalcone
adamantium from South region of Brazil and, characterize together with the flavonols isoquercitrin, quercitrin,
the major constituents responsible for antioxidant activity. quercetin, and myricetin (Table 2, Figure 1). Structural
analysis of single ions in the mass spectra from extract and
The samples analyzed in the present study showed a total fractions were performed by ESI-MS/MS. The compounds
phenol content in the range of 15.78 – 74.83 mg GAE/g were identified by comparison of their ESI-MS/MS
extract (Table 1). Phenolic compounds are recognized as fragmentation spectra with fragmentation spectra of the
one of most important class responsible for antioxidant authentic standard samples (compounds 1, 5, 6 and 7) and
capacity in plants [19]. with literature data [21].

Ethanol extract, ethyl acetate and butanol fractions To confirm the presence of the flavonoid isoquercitrin
exhibited antioxidant activity concentration-dependent in were made the HPLC-UV/DAD analysis of standard
DPPH assays, with SC50 varying from 7.77 to 13.35 isoquercitrin and of the crude extract. We noted the
µg/mL. The highest antioxidant activity was exhibited by formation of a peak with a retention time coincident with
butanol fraction. In ORAC-FL kinetic assay, based on the same pattern and absorption peaks. Furthermore, the
hydrogen transfer mechanism, the extracts showed identity of major constituent isoquercitrin was also
antioxidant capacity between 2648 and 3502 µM of confirmed through co-elution with authentic standard
TROLOX equivalent per gram of extract (Table 1) In sample. These results are enough to confirm that one of the
comparison with previous studies [16,18], our extracts and major constituents and the responsible for antioxidant
fractions showed higher antioxidant activity and there were activity is the flavonoid isoquercitrin, since that the same
chemical differences between C. adamantium leaves mass was also found in the analysis of TLC spot yellow
analyzed in this study of South region of Brazil and C. sample that was reveled with solution of DPPH.
adamantium growing in Midwest region of Brazil [16-18].
The chalcones were previously reported in C. adamantium Thus, the results of the present study suggest that the
growing in Midwest region of Brazil [16], while the antioxidant capacity of C. adamantium is correlated to the
flavonols isoquercetrin, myricetin, quercitrin and quercetin content of flavonoids, including isoquercitrin, which is
are being reported for the first time in this plant. Myricetin present in the crude ethanolic extract and TLC spot yellow
had been already identified in C. xanthocarpa [14]. sample. Moreover, this activity presents a positive
correlation with the total phenolic soluble content
Table 1: Total phenol content and antioxidant capacity by the DPPH and
ORAC assays of ethanol extract of Campomanesia adamantium leaves
measured by FCR assay. However, further investigations
and its fractions ethyl acetate (EtOAc) and butanolic (BuOH). are necessary to confirm if this plant and its constituents
represent a source of powerful antioxidant products useful
Samples Phenol contenta DPPH assay, ORAC a in vivo.
(mg of GAE/g)b SC50a (µg/mL)c (µmol TE/g) d
Ethanol extract 35.04(5.48) 13.00 (5.03) 2648 (1.77) d
EtOAc fraction 74.83(10.77) 13.35 (16.85) 3150 (6.66) d Experimental
BuOH fraction 15.78(15.29) 7.77 (5.00) 3502 (5.71) d Plant Material: The leaves of Campomanesia
Quercetin* - 12.80 (2.00) 5.62 (0.89) e
Isoquercitrin* - - 5.21 (1.60) e
adamantium were collected from wild specimen growing
Trolox* - 2.55 (1.40) - in Curitiba, Paraná State, Brazil (25o25’48’’ S, 49o16’15’’
*Experimental positive controls. W) at 934 m of altitude. The plant was identified by Dr.
-: not evaluated. Armando Carlos Cervi, which deposited a voucher
a
b
Mean (%RSD, relative standard deviation) of triplicate assays. specimen at the herbarium of UFPR (UPCB 60503).
Total phenolics data expressed as milligrams of gallic acid equivalents per
gram (mg of GAE/g) of extract or fractions.
c
DPPH assay data expressed as SC50 (concentration that inhibited 50% of the Extracts preparation: The powder was subjected to the
DPPH radical) in micrograms per milliliters (µg/mL).
d
process of maceration with ethanol at a ratio of powder /
ORAC data expressed as micromol of Trolox equivalents per gram (µmol of
TE/g) of extract or fractions. solvent of 1:5 (weight / volume). The ethanolic crude
e
ORAC data expressed as relative Trolox equivalent, mean (%RSD, relative extract was suspended in methanol/water (9:1, v/v) and
standard deviation) of triplicate assays fractionated by liquid-liquid extraction with hexane and
ethyl acetate. The hydroalcoholic phase remaining was
The ESI-MS technique has been applied in the analyze of partitioned with n-butanol and water to afford an n-
several complex matrix, such as wine, oil, beer and butanol-soluble portion. This procedure yielded the
extracts from natural sources. In this study, the samples fractions of hexane (Hex), ethyl acetate (EtOAc) and
presented high content of phenolic compounds and were butanol (BuOH).
Antioxidant activity and HPLC/ESI-MS profile of C. adamantium Natural Product Communications Vol. 6 (7) 2011 971
OH

OH
R3 CH3

HO O HO O
R1

OR2
R4
OH O
OH O

1 Isoquercitrin R1=H, R2=glucose


5 2’,4’-dihydroxy-6’-methoxychalcone R3=R4=H
2 Quercitrin R1=H, R2=rhamnose
6 2’,4’-dihydroxy-5’-methyl-6’-methoxychalcone R3=CH3, R4=H
3 Myricetin R1=OH, R2=H
7 2’,4’-dihydroxy-3’,5’-dimethyl-6’-methoxychalcone R3=R4=CH3
4 Quercetin R1=R2=H

Figure 1: Structure of the compounds identified in ethanol extract from the leaves of Campomanesia adamantium of the South of Brazil and its fractions

Table 2: Compounds identified in ethanol extract from the leaves of Campomanesia adamantium and its fractions using ESI(-)-MS/MS.

ESI-MS ions (m/z)


Compounds Campomanesia adamantium samples Deprotonated ions [M-H]- m/z MS/MS ions m/z
Ethanol EtOAc BuOH TLC
extract fraction fraction yellow spot
1 + + + + 463 25 eV: 463→301, 255, 151
2 + + - + 447 25 eV: 447→301
3 + - - + 317 25 eV: 317→287
4 + + + - 301 25 eV: 301→271, 255
5 + + - + 269 25 eV: 269→253, 226, 198, 184, 177, 165, 150, 139,
122, 108, 97, 94, 65
6 + + - + 283 25 eV: 283→268, 240, 198, 179, 164, 136, 108, 79
7 + + - + 297 25 eV: 297→282, 254, 191, 178, 163, 150, 134, 122
+: detected; -: not detected

Radical scavenging activity by DPPH assay: The extract or fraction (mg of GAE/g). The analyses were
antiradical activity of extract and fractions of EtOAc and performed in triplicate.
BuOH were determined using the stable 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH) [22]. Fifty microliters of a Separation and analysis of antioxidants on thin layer
250 μM DPPH solution in ethanol was added to a range of chromatography: The sample was applied on TLC and
solutions of different concentrations (seven serial 3-fold eluted with BAW (butanol: acetic acid: water), after that
dilutions to give a final range of 100 to 1.6 μg mL-1) of we sprayed with a solution 500 µg/mL of DPPH in
extracts to be tested in ethanol (10μL). Absorbance at 517 ethanol. After solvent evaporation (about 5 minutes), the
nm was determined 20 min after the addition of each of the potential anti-free radical was verified by the appearance
compounds tested, and the percentage of activity was of yellow spots on violet background, according to the
calculated. Quercetin and Trolox were used as positive described in the literature [25]. The yellow spot was
controls. The antioxidant activity was expressed as the scraped, dissolved in methanol, filtered and this was called
SC50 value in μg mL-1. All samples were tested in triplicate. TLC yellow spot sample.

Evaluation of antioxidant capacity by ORAC assay: HPLC-UV/DAD/ESI-MS profile: The TLC yellow spot
The antioxidant capacity of the ethanolic extract and sample, crude extracts and fractions of C. adamantium
AcOEt and BuOH fractions were assessed through the leaves were diluted in a solution containing 50% (v/v)
oxygen radical absorbance capacity (ORAC) [23]. In this methanol (chromatographic grade), 50% (v/v) deionized
assay, measures the antioxidant scavenging activity against water and, 0.5% of ammonium hydroxide (Merck,
peroxyl radicals using fluorescein as the fluorescent probe. Darmstadt, Germany). In the fingerprinting ESI-MS
ORAC assays were carried out on a Synergy-2 multi- analysis, the general conditions were: source temperature
detection microplate reader system. The temperature of the of 100 oC, capillary voltage of 3.0 kV and cone voltage of
incubator was set at 37 °C. The data were expressed as 30 V. For measurements were performed according to the
micromoles of Trolox equivalents (TE) per gram of extract described in the literature [20,21]. Structural analysis of
or fraction on dry basis (μmol of TE/g) and as relative single ions in the mass spectra from extract and fractions
Trolox equivalent for pure compounds. The analyses were were performed by ESI-MS/MS. The ion with the m/z of
performed in triplicate. interest was selected and submitted to 25 eV collisions
with argon in the collision quadrupole. The compounds
Quantitative determination of total soluble phenols: were identified by comparison of their ESI-MS/MS
The dried ethanolic extract and its EtOAc and BuOH fragmentation spectra.
fractions, dissolved in ethanol, were analyzed for their total
soluble phenolic content according to the Folin-Ciocalteau HPLC analyses were conducted using a RP-18 column
colorimetric method [24]. The results were expressed as (Lichrospher“, 5 μm, 225\4.6 mm, Merck). The mobile
milligrams of gallic acid equivalents (GAE) per gram of phase consisted of a linear gradient combining solvent A
972 Natural Product Communications Vol. 6 (7) 2011 Pascoal et al.

(acetonitrile) and solvent B (water/acetic acid, 99:1, v/v, Statistical analysis: Data are reported as mean (%RSD,
pH 2.88) as follows: 15% A (15 min), 15-40% A (5 min), relative standard deviation) of triplicate determinations.
40-60% A (5 min), 60-100% A (5 min), 100-15% A (5 The statistical analyses were carried out using the
min), 15% A (5 min). The analyses were carried out in Microsoft Excel 2002 software package (Microsoft Corp.,
triplicate at a flow rate of 0.8 mL/ min and an injection Redmond, WA)
volume of 20 μL. UV-DAD detector was set to record
between 200 and 600 nm, and the UV chromatograms Acknowledgments - The authors are grateful to Dr.
were measured at 254 and 350 nm. The samples were Armando C. Cervi (UFPR) for plant identification and to
crude extract and isoquercitrin standard sample at FAPESP, CNPq and FAEPEX-UNICAMP for financial
1mg/mL. support.

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2011
NPC Natural Product Communications Vol. 6
No. 7
Activity of Cuban Propolis Extracts on Leishmania 973 - 976

amazonensis and Trichomonas vaginalis


Lianet Monzote Fidalgo*, Idalia Sariego Ramosa, Marley García Parraa, Osmany Cuesta-Rubiob,
Ingrid Márquez Hernándezb, Mercedes Campo Fernándezb, Anna Lisa Piccinellic and Luca Rastrellic
a
Parasitology Department, Institute of Tropical Medicine “Pedro Kourí”, Havana City, Cuba
b
Department of Pharmacy, Institute of Pharmacy and Food, University of Havana, Cuba
c
Department of Pharmaceutical Sciences, Via Ponte Don Melillo 84135, Fisciano, Salerno, Italy

monzote@ipk.sld.cu

Received: December 10th, 2010; Accepted: March 16th, 2011

In this paper we analyzed the antiprotozoal effects of eighteen Cuban propolis extracts (brown, red and yellow type) collected in different
geographic areas, using Leishmania amazonensis (as a model of intracellular protozoa) and Trichomonas vaginalis (as a model of
extracellular protozoa). All evaluated propolis extracts caused inhibitory effect on intracellular amastigotes of L. amazonensis. However,
cytotoxicity on peritoneal macrophages from BALB/c mice was observed. Only five samples decreased the viability of T. vaginalis
trophozoites at concentrations lower than 10 g/mL. No correlation between the type of propolis and antiprotozoal activity was found. Cuban
propolis extracts demonstrated activity against both intracellular and extracellular protozoa model, as well as the potentialities of propolis as
a natural source to obtain new antiprotozoal agents.

Keywords: Propolis, antiprotozoal, Leishmania amazonensis, Trichomonas vaginalis.

Propolis is a resinous hive product that honey bees produce In the present work, we analyzed the antiprotozoal effects
employing parts of plants as buds, leaves, exudates or of brown, red and yellow Cuban propolis extracts collected
resins, and beeswax. Its chemical composition is highly in different geographic areas, using Leishmania
variable and depends greatly according to the plants found amazonensis (as a model of intracellular protozoa) and
around the hive [1a]. Cuban propolis has shown significant Trichomonas vaginalis (as a model of extracellular
differences in its chemical composition with respect to protozoa. Leishmania are protozoa that cause leishmaniasis
propolis from temperate zone. They also vary significantly [5a]. The disease is endemic in 88 countries throughout
among themselves and can be clearly divided into three Latin America, Africa, Asia and Southern Europe.
groups: brown Cuban propolis type (BCP type) has shown Approximately 350 million people are thought to be at risk
to be rich in polyisoprenylated benzophenones, red type with a worldwide prevalence of 12 million and annual
(RCP type), containing isoflavonoids as the main incidence of 2 million new cases [5b].
constituents, and yellow type (YCP type) with a variety of
triterpenoids as the major chemical components [1b]. All propolis samples caused inhibition growth against
L. amazonensis, with IC50 < 27 µg/mL; although a high
Diverse pharmacological activities of propolis have been toxicity on peritoneal macrophage from BALB/c mice was
explored, such as: anti-inflammatory and anti-tumoral observed (Table 1). The better selectivity was showed by
effect [2a]. Up to now, antimicrobial properties have been BCP-1, a propolis sample that exhibited a high content of
widely investigated; including antibacterial [2b], antiviral nemorosone [1b]. However, other BCP samples containing
[2c] and antifungal activity [2d]. Antiparasitic activities also high content of this compound exhibited higher values
have also been reported against Trypanosoma cruzi [2e] of IC50. BCP-16 and RCP-29 showed unspecific action. In
and Giardia duodenalis [2f]. However, only a few studies general, RCP was the most active (IC50 average of 13.9
have been carried out for the antileishmanial [3a-3c] and µg/mL) and the most cytotoxic (CC50 average of 37.2
antitrichomonocidal activity [4a]. In Cuba, propolis has µg/mL). There are a number of studies documenting the
displayed therapeutic potentialities as antipsoriatic, anti- antiprotozoal activity of flavonoids [6a-6e]. These
inflamatory, analgesic [4b], antibacterial [4c] and compounds have shown activity against Plasmodium
antitumoral [4d]. Scarce reports can be found about its falciparum, Leishmania spp., Trypanosoma cruzi or
antiparasitic activity. Thus, its biological potentiality has Giardia intestinalis. Four flavonoids detected in RCP
not been explored totally. including biochanin A, 3,8-dihydroxy-9-methoxy-
pterocarpan, formononetin, and liquiritigenin have shown
974 Natural Product Communications Vol. 6 (7) 2011 Monzote et al.

Table 1: Antileishmanial activity and cytotoxicity of Cuban propolis extracts. Table 2: Activity of Cuban propolis extracts on T. vaginalis.
Propolis extracts IC50† CC50§ SI ƒ
Propolis extracts IC50† (µg/mL) ± SD‡
(µg/mL) ± SD‡ (µg/mL) ± SD‡ BCP-1 6.2 ± 0.3
BCP-1 7.8 ± 0.9 51.8 ± 4.5 7 BCP-4 > 200
BCP-4 23.4 ± 0.9 70.0 ± 4.4 3 BCP-5 > 200
BCP-5 21.2 ± 2.4 52.0 ± 0.3 2 BCP-16 > 200
BCP-16 33.9 ± 1.7 43.8 ± 0.7 1 BCP-17 > 200
BCP-17 22.5 ± 0.7 47.3 ± 0.8 2 RCP-9 > 200
RCP-9 12.5 ± 1.8 25.0 ± 1.5 2 RCP-29 8.9 ± 0.1
RCP-29 17.8 ± 1.5 23.8 ± 2.3 1 RCP-35 > 200
RCP-35 14.6 ± 0.7 50.1 ± 2.2 3 RCP-37 > 200
RCP-37 11.8 ± 1.5 47.7 ± 2.2 4 RCP-45 > 200
RCP-45 17.2 ± 2.7 51.2 ± 0.1 3 RCP-72 > 200
RCP-72 9.5 ± 1.2 25.6 ± 0.2 3 YCP-2 3.7 ± 0.7
YCP-2 13.3 ± 2.2 50.7 ± 1.5 4 YCP-18 9.1 ± 0.8
YCP-18 20.2 ± 0.3 31.7 ± 3.2 2 YCP-39 > 200
YCP-39 13.0 ± 0.5 31.8 ± 2.4 2 YCP-41 > 200
YCP-41 19.9 ± 2.2 52.1 ± 0.4 3 YCP-48 3.2 ± 0.1
YCP-48 15.4 ± 1.1 50.4 ± 0.4 3 YCP-50 > 200
YCP-50 20.7 ± 1.4 91.0 ± 0.6 4 YCP-60 > 200
YCP-60 26.4 ± 0.4 66.0 ± 4.1 3 Metronidazol 0.4  0.04
Pentamidine 1.3 ± 0.1 11.7 ± 1.7 9
†: IC50: Concentration of drug that caused 50 % of inhibition growth.
†: IC50: Concentration of drug that caused 50 % of inhibition growth. ‡: SD:
‡: SD: Standard deviation.
Standard deviation. §: CC50: Concentration of drug that caused 50 % of mortality.ƒ:
SI: Selectivity index = CC50 / IC50
contained also other prenylated benzophenone derivatives
inhibitory activity against parasites mentioned above. such as garcinielliptone I, hyperibone B and propolone B,
These observations correlate well with the in vitro studies C and D [8a]. RCP is composed by isoliquiritigenin,
obtained herein due to these flavonoids are among the liquiritigenin, biocanin A, formononetin, vestitol,
major components of RCP. neovestitol, isosativan, medicarpin, homopterocarpin and
vesticarpan, mainly [8b]; while YCP samples are rich
Previously, Ayres et al. reported the activity of Brazilian in triterpenoids including lanosterol, - and -amyrin,
propolis on L. amazonensis promastigotes and amastigote -amyrin acetate, -amyrone, germanicol and germanicol
form [3a]. In this sense, Machado et al. compared the acetate, lupeol and lupeol acetate, cycloartenol, lanosterol
antileishmanial activity of Brazilian and Bulgarian acetate and 24-methylene-9,19-cyclolanostan-3β-ol [8c].
propolis against four different species of Leishmania (L.
amazonensis, L. braziliensis and L. chagasi from New Although Cuban propolis has been characterized and
World and L. major from Old World). They also observed grouped into three different types according its main
significant differences in the leishmanicidal activities with chemical components, we could not correlate its
IC50 values that ranged from 2.8 to 229.3 µg/mL [3b]. antiparasitic activity against both protozoa with the
Duran et al., demonstrated that propolis samples from chemical composition. Promissory results were found in
Turkey also reduced the proliferation of L. tropica some samples of each type of propolis, but this activity has
promastigotes [3c]. It is very interesting to note that all not been shown by all samples belonging to a same group.
these results were obtained employing propolis samples This apparent contradiction can indicate that the
from different geographical origins containing very antiparasitic activity can be associated to compounds that
dissimilar chemical constituents. are present in a minor percentage in the sample or due to
interaction of different compounds that act as synergistic
On the other hand, T. vaginalis is a flagellated protist that and enhance the effects or interact as antagonist, which
causes trichomoniasis, a common but overlooked sexually provoke the lost of activity in some samples of the same
transmitted human infection, with approximately 170 type. This unresolved situation is very common in products
million cases occurring annually worldwide [7]. Only five that exist in the nature as complex mixture of substances,
propolis extracts showed activity against T. vaginalis; which can interact and change the expected
while thirteen were inactive (Table 2). A high contrast was pharmacological activity. Further experiments with the
observed since only some samples belonging to each compounds obtained from all types of propolis can be
propolis type caused an important inhibition growth of developed in order to elucidate the responsible of the high
trophozoites. In this sense, YCP samples were the most antiparasitic activity showed in some cases.
active, which should be corroborate using in vivo models
of infection. A previous report demonstrated that propolis In conclusion, some Cuban propolis extracts exhibited
possesses in vitro anti-trichomonas activity [4a]. activity against both intracellular and extracellular
protozoa model. Our results corroborated the high
In both protozoa, the reference drug caused better activity, potentiality of propolis as a natural source of new
which is logical since the pentamidine and metronidazol antiprotozoal agents with a wide spectrum of activity. This
are pure compounds and propolis extracts are complex study also confirms that both the chemical composition of
mixture of substances. Chemical studies revealed that propolis and its biological potentiality should be evaluated
BCP has shown nemorosone as the main component and during the quality control process of this natural product.
Activity of propolis on Leishmania and Trichomonas Natural Product Communications Vol. 6 (7) 2011 975

Experimental Activity of propolis samples on L. amazonensis


Propolis samples: Eighteen samples of Cuban propolis amastigotes: Peritoneal macrophages were harvested from
were provided by “La Estación Experimental Apícola”, normal BALB/c mice in RPMI medium (Sigma) and
Havana, Cuba, between October 2003 and December plated at 106/mL in Lab-Tek 16 chamber slides (Costar,
2004. Samples were collected in nine provinces of Cuba Naperville, US) and incubated at 37ºC and 5% CO2 for 2
including Eastern, Central and Western regions (Table 3). hours. Non-adherent cells were removed and stationary-
Propolis samples were extracted by maceration with phase L. amazonensis promastigotes were added at a 4:1
methanol (10 mL, 3 times) for 1 hour at room temperature parasite/macrophage ratio. The cultures were incubated for
employing agitation. Extracts were filtered on paper filters 4 hours and washed to remove free parasites. Propolis was
and the solvent was evaporated at 40ºC under reduced added at a concentration ranging from 100 to 12.5 g/mL
pressure to obtain dry extracts. Each sample was for 48 hours. The cultures were then fixed with absolute
characterized and classified using a combination of NMR, methanol, stained with Giemsa, and examined under light
HPLC-PDA and HPLC-ESI/MS techniques as previously microscopy [9b]. The number of intracellular amastigotes
was described [1b]. Cuban propolis samples used in this was determined by counting the amastigotes residents on
study, their origin and classification are reported in table 3. 100 macrophage per each sample, and the results were
The extracts were dissolved in dimethylsulphoxide expressed as percent of reduction of the infection rate
(DMSO, BDH, Poole, England) at 20 mg/mL and stored at (%IR) in comparison to that of the controls [%IR = 100 –
4ºC. (infection rate of the treated culture/infection rate of the
untreated culture x 100)]. The infection rates were
Table 3: Cuban propolis samples used in this study, classification and origin. obtained by multiplying the percentage of infected
Samples Province (Municipality) Samples Province (Municipality) macrophages by the number of amastigotes per infected
macrophages [9c].
BCP-1 Ciudad Habana (Jardín RCP-39 Pinar del Río
Botánico) (Candelaria)
BCP-4 Granma (Buey Arriba) RCP-41 Pinar del Río (Bahía Cytotoxicity of propolis samples on macrophage:
Honda)
BCP-5 Guantánamo (Imías) RCP-48 Matanzas (Unión de Peritoneal macrophages were collected from normal
Reyes) BALB/c mice in RPMI medium supplemented with
BCP-16 Las Tunas (Puerto Padre) RCP-50 Matanzas (Unión de
Reyes)
antibiotics, and seeded at 30000 cell/well. The cells were
BCP-17 Guantánamo (Salvador) RCP-60 Holguín (Báguanos) incubated for 2 hours at 37ºC in 5% CO2. Non-adherent
RCP-9 Pinar del Río (Cabo de YCP-2 Ciudad Habana (Jardín cells were removed and dilutions of propolis in 1 L
San Antonio) Botánico) DMSO were added to 200 L medium at 10% HFBS and
RCP-29 Villa Clara (Manicaragua) YCP-18 Ciudad Habana (Jardín
Botánico) antibiotics. The macrophages were treated by eight
RCP-35 Pinar del Río (La YCP-39 Pinar del Río concentrations of the product ranging from 200 to 1.7
Coloma) (Candelaria)
RCP-37 Pinar del Río (Güanes) YCP-41 Pinar del Río (Bahía g/mL for 48 hours. The viability was determined using
Honda) the colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-
RCP-45 Matanzas (Jagüey YCP-48 Matanzas (Unión de
Grande) Reyes)
2,5-diphenyltetrazolium bromide (MTT) (SIGMA, St.
RCP-72 Ciego de Ávila YCP-50 Matanzas (Unión de Louis, MO, USA). MTT solutions were prepared at
Reyes) 5 mg/mL, filtered and sterilized at the moment of use,
RCP-2 Ciudad Habana (Jardín YCP-60 Holguín (Báguanos)
Botánico) 15 L was added to each well. After incubation for 4 hours
RCP-18 Ciudad Habana (Jardín the formazan crystals were dissolved by addition of 100
Botánico)
L DMSO. The optical density was determined using an
EMS Reader MF Version 2.4-0, at a test wavelength of
Parasites culture: The strain of L. amazonensis
560 nm and a reference wavelength of 630 nm [9d].
(MHOM/77BR/LTB0016) was kindly provided by the
Department of Immunology, Oswaldo Cruz Foundation,
Activity of propolis samples on T. vaginalis trophozoites:
Brazil. The parasites were routinely isolated from mouse
The assay was performed in 96 well microtiter plates.
lesions and maintained as promastigotes at 26ºC in
First, 99 µL of TYI-S-33 medium were added, followed of
Schneider’s medium (Sigma Chem Co, St. Louis, Mo,
1µL of the dilution of the propolis in DMSO. The assayed
US), containing 10% heat-inactivated foetal bovine
concentrations of the propolis varied from 200 until 1.6
serum (Sigma), 200 U penicillin/mL and 200 g µg/mL. Finally 100 µL of the parasites suspension (25 x
streptomycin/mL (Sigma). The parasites were not used 104 trichomonas/mL) were seeded. Plates were incubated
after the fifth passage. For examining the effect of these
during 46 hours, in a candle jar at 37C. Each product
propolis samples on T. vaginalis, the isolate C173,
concentration and controls were tested in quadruplicate
axenized from a symptomatic woman suffering of
and the experiment was repeated three times. To determine
trichomoniasis, was used [9a]. Parasites were cultured in
the viability of the parasites the medium was aspirated and
TYI-S-33 supplemented with heat-inactivated bovine
replace by one lacking ascorbic acid and L-cysteine,
serum at final concentration of 10%, under anaerobic
followed of the addition of 20 µL of MTT to each well.
conditions, at 37C. MTT was prepared as explained above. After a period of
incubation of 2 hours, the plates were centrifuged (5 min,
976 Natural Product Communications Vol. 6 (7) 2011 Monzote et al.

800 x g), and the supernatant medium was aspirated from Statistical analysis: The 50 % inhibition concentration
the wells, as completely as possible, without disturbing the (IC50) value was determined from the lineal concentration-
formazan crystals or the cells on the plastic surface. response curves in each parasite evaluation. The 50 %
DMSO (100 µL) was added to each well to dissolve the cytotoxic concentration (CC50) was obtained from dose-
formazan crystals, the plates were shaked for 1 min and response curves fit to data by means of the equation for the
immediately the optical density was determined at 560 nm, sigmoidal Emax model [9e]. Selectivity indices (SI) were
using 630 nm as the reference wavelength. then calculated through of division the CC50 for host cells
by the IC50 for L. amazonensis [9f].
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2011
NPC Natural Product Communications Vol. 6
No. 7
Antioxidant Capacity and Phenolic Content of four 977 - 982
Myrtaceae Plants of the South of Brazil
Marcos José Salvador*a, Caroline C. de Lourençoa, Nathalia Luiza Andreazzaa,
Aislan C.R.F. Pascoala and Maria Élida Alves Stefanellob
a
Instituto de Biologia, Departamento de Biologia Vegetal, Curso de Farmácia,
Universidade Estadual de Campinas (UNICAMP), 13083-970, Campinas, SP, Brasil
b
Departamento de Química, Universidade Federal do Paraná (UFPR), 81531-990, Curitiba, PR, Brasil

marcosjs@unicamp.br

Received: December 11th, 2010; Accepted: March 26th, 2011

Antioxidant compounds can be useful to prevent several degenerative diseases or as preservative in food and toiletries. Species of the
Myrtaceae family are able to accumulate phenolic substances and those are closely related to the antioxidant activity due to their capacity to
scavenge free radicals, protect against lipid peroxidation and quench reactive oxygen species. These facts prompted us to investigate the
antioxidant capacity of the ethanolic extracts of the leaves of four Myrtaceae plants collected of the south of Brazil: Eugenia chlorophylla O.
Berg., Eugenia pyriformis Cambess, Myrcia laruotteana Cambess and Myrcia obtecta (Berg) Kiacrsk. The antioxidant potential was
performed using the DPPH (a single electron transfer reaction based assay) and ORAC (Oxygen Radical Absorbance Capacity, a hydrogen
atom transfer reaction based assay) assays. Moreover, the total soluble phenolic content was also measured using the Folin-Ciocalteu reagent.
A preliminary evaluation of the ethanolic extracts of these Myrtaceae plants revealed high levels of phenolic compounds (343.7-429.3 mg
GAE) as well as high antioxidant activity according to both methods (1338 a 3785 µmol of TE/g of extract in ORAC and SC50 in the range of
1.70 and 33.7 µg/mL in the DPPH). The highest antioxidant activity obtained by DPPH assay was exhibited by ethanol extract of the leaves
of E. pyriformis (1.70 g/mL), followed by extracts of M. laruotteana (3.38 g/mL) and M. obtecta (6.66 g/mL). In comparison
with controls, in the DPPH assay, the extract of E. pyriformis was more active than trolox (SC50 = 2.55 g/mL), while the extracts of
M. laruotteana and M. obtecta were more actives than quercetin (SC50 = 7.80 g/mL). In the ORAC assay, all species also show good
antioxidant capacity (1000 µmol of TE/g). Initial HPLC-UV/DAD and ESI-MS confirmed the presence of phenolic acids constituents in the
ethanol extracts. The results indicate the presence of compounds possessing promising antioxidant/free-radical scavenging activity in the
analyzed extracts of Myrcia and Eugenia plants of the south of Brazil.

Keywords: Myrtaceae, Myrcia, Eugenia, radical scavenger, DPPH, HPLC-UV/DAD, ESI-MS.

Antioxidant compounds can be useful to prevent several The selected plants are trees, growing in Southern region
degenerative diseases or as preservative in food and of Brazil [11]. Previous studies have reported the essential
toiletries products. Currently there is an increasing interest oil composition of these plants (12-17], identification of
in searching natural antioxidants to replace synthetic the flavonoids myricitrin, rutin and quercitrin in the leaves
compounds, which can be dangerous for human health. of E. pyriformis [18] and the antimicrobial and antioxidant
Thus many plants have been screened for a possible source activities of the fruits of E. pyriformis [7,19-20]. No
of non-toxic and effective antioxidants [1-3]. Compounds phytochemical reports were found on E. chlorophylla,
with antioxidant activity are generally phenolics and M. laruotteana and M obtecta.
the free radical-scavenging is a suitable method for
preliminary search of them in plants [4]. Myrtaceae is an In this study, the phytochemical screening showed the
important family in Brazil, with more than 1000 species presence of triterpenes/sterols, phenolic compounds,
over the country. Myrcia and Eugenia, with around 400 tannins and saponins in all samples. Theses results are in
and 350 species, respectively, are the major genera [5]. agreement with previous reports of flavonoids, triterpenes
Many species produce edible fruits and, some have an and phenolic compounds in Eugenia and Myrcia [21-26].
essential ecological role in the conservation of Brazilian The ethanol extracts showed a total phenol content in
biodiversity [6]. Screening for antioxidant activity of the range of 343.7-429.3 mg GAE/ g extract (Table 1).
Myrtaceae has been focused on its edible fruits [7-9], with Phenolic compounds (including simple phenols, tannins
only one report on leaves [10]. The aim of this work was and flavonoids) are recognized as one of most important
evaluate the antioxidant capacity of leaves of Eugenia class responsible for antioxidant capacity in plants [27].
chlorophylla O. Berg., Eugenia pyriformis Cambess,
Myrcia laruotteana and Myrcia obtecta (Berg) Kiacrsk.
978 Natural Product Communications Vol. 6 (7) 2011 Salvador et al.

All samples exhibited antioxidant activity concentration- may be suppressed due to protonation on antioxidant
dependent in DPPH assays, with SC50 varying from 1.70 to compounds, whereas in basic conditions, proton
33.72 μg/mL. In DPPH assays the highest antioxidant dissociation of phenolic compounds would enhance a
activity was exhibited by ethanol extract of the leaves of sample’s reducing capacity [28,29].
E. pyriformis (1.70 μg/mL), followed by extracts of
M. laruotteana (3.38 μg/mL), M. obtecta (6.66 μg/mL) and Thus, the results documented in this study demonstrated
E. chlorophylla (33.72 μg/mL). In comparison with that all extracts analyzed showed antioxidant capacity
controls, the extract of E. pyriformis was more active (measured by DPPH and ORAC assays) and this activity
than trolox (SC50 = 2.55 μg/mL), while the extracts of present a positive correlation with the total phenolic
M. laruotteana and M. obtecta were more actives than content (measured by FCR assay). Moreover the caffeic
quercetin (SC50 = 7.80 μg/mL) (Table 1). Moreover, in and chlorogenic acids presented considerable antioxidant
ORAC-FL kinetic assay the extracts showed a good (Table 1) capacity and were detected in some of the
antioxidant capacity with value between 1338.58 and extracts studied (Table 2).
3785.70 µM of Trolox equivalent per gram of extract
(µM of TE/g). In accordance to literature data, samples Phenolic compounds has been presented as important
with values ≥1000.00 µM of TE/g can be considered substances in combating free radical production mainly
samples with good antioxidant capacity in this assay due to its chemical structure and redox capacity, allowing
[28,29]. In ORAC-FL assays, highest antioxidant activity them to act as reducing agents, hydrogen donating,
was exhibited by ethanol extract of the leaves of neutralizing free radicals [30], chelating of transition
M. laruotteana, followed by extracts of E. chlorophylla, metals and inhibiting lipid peroxidation [31]. In biological
E. pyriformis and M. obtecta (Table 1). systems this capacity confers pharmacological properties
to this compounds that act preventively against diseases
Table 1: Total phenol content and antioxidant capacity by the DPPH and related to oxidative stress.
ORAC assays of ethanol extracts of Myrtaceae plants.

Sample Phenol contenta (mg DPPH assay, SC50a ORAC assaya The results for E. pyriformis corroborate previous work
Ethanol extract of GAE/g of extract (µg/mL)c (µmol of that reported the inhibition of xanthine oxidase, an enzyme
or fraction)b TE/g)d responsible by super-oxide anion production [32] and
Eugenia chlorophylla 429.3 (5.2) 33.72 (2.25) 2197.20 (1.12)
Eugenia pyriformis 396.2 (4.5) 1.70 (1.19) 1456.50 (4.92) antioxidant activity of fruits [7,19].
Myrcia laruotteana 401.4 (2.5) 3.38 (3.38) 3785.70 (2.67)
Myrcia obtecta 343.7 (4.9) 6.66 (5.30) 1338.58 (4.85) The ESI-MS fingerprints technique with direct infusion
Quercetin* - 7.80 (2.00) 5.62 (0.89)e
Caffeic acid* - 10.80 (2.60) 2.86 (2.02)e [33-35] was used to characterize the presence of
Chlorogenic acid* - 12.15 (1.80) 2.65 (1.50)e compounds with potent free-radical scavenging activity in
Trolox* - 2.55 (1.40) - this work. The extracts were analyzed by direct insertion
a
Mean value (%RSD, relative standard deviation) of triplicate assays. both in the negative and positive ion modes. However,
b
Total phenolics data expressed as milligrams of gallic acid equivalents
per gram (mg of GAE/g).
ESI(+)-MS fingerprints produce by far the most
c
DPPH assay data expressed as SC50 (concentration that inhibited 50% of characteristic mass spectra; hence only the ESI(-)-MS data
the DPPH radical) in micrograms per milliliters (µg/mL). will be presented and discussed. This method in the
d
ORAC data expressed as micromol of Trolox equivalents per gram negative ion mode provides a sensitive and selective
(µmol of TE/g).
e
ORAC data expressed as relative Trolox equivalent, mean (%RSD,
method for the identification of polar organic compounds
relative standard deviation) of triplicate assays. with acidic sites, such as the phenolic organic acids.
*Experimental positive controls. Deprotonated forms of the compounds of interest were
-: not evaluated. then selected and dissociated and their ESI-MS/MS were
compared to those of standards.
The differences in best antioxidant capacity of the species
studied among the two assays happens due to differences Chlorogenic acids are esters of trans-cinnamic acids
of sensibility and on the basis of the chemical reactions (coumaric, caffeic, ferulic, and 3,4-dimethoxycinnamic)
involved in each test: ORAC is a hydrogen atom transfer with quinic acid. The trans-cinnamic acids can be
reaction based assay (HAT) and DPPH is a single electron esterified at one or more of the hydroxyls at positions 1, 3,
transfer reaction based assay (ET). It is apparent that the 4, and 5 of quinic acid, originating series of positional
hydrogen atom transfer reaction is a key step in the radical isomers. In HPLC-UV/DAD and ESI-MS analysis the
chain reaction. Therefore, the HAT based method is more Myrcia samples presented m/z 353 as base peaks (bp) in
relevant to the radical chain-breaking antioxidant capacity. negative ionization mode mass spectra and UV spectra
Overall, there are a multitude of ET-based assays for characteristics of caffeoylquinic derivatives (UV max:
measuring the reducing capacity of antioxidants. The ≈298 and 325 nm) which, when taken together, suggest
assays are carried out at acidic (FRAP and DPPH), neutral positional isomers of a quinic acid (QA) esterified with a
(TEAC and ORAC), or basic (total phenols assay by Folin- single caffeoyl (CAF) unit. The product ion spectra
Ciocalteau reagent, FCR assay) conditions. The pH values obtained by negative ion MS/MS for precursor ions m/z
have an important effect on the reducing capacity of 353 were different from each other, and comparison with
antioxidants. At acidic conditions, the reducing capacity
Antioxidant capacity of four Myrtaceae plants Natural Product Communications Vol. 6 (7) 2011 979

Figure 1: ESI-MS fingerprints of ethanol extracts from the leaves of four Myrtaceae plants of the South of Brazil (a: Eugenia chlorophylla; b: Eugenia
pyriformis; c: Myrcia obtecta; d: Myrcia laruotteana).

Table 2: Compounds identified in ethanol extracts from the leaves of four Myrtaceae plants of the South of Brazil using ESI(-)-MS/MS.
ESI-MS ions (m/z)
Compound Myrtaceae plants Deprotonated ions [M-H]- m/z MS/MS ions m/z
ECE EPE MOE MLE
Caffeic acid - + + + 179 15 eV: 179→135
Quinic acid + + + + 191 25 eV: 191→173, 127, 111, 93, 85
Ferulic acid - + - - 195 15 eV: 193→178, 149, 134
Chlorogenic acid (5-O-(E)-caffeoylquinic acid) - - + + 353 15 eV: 353→191, 179, 173
+: detected; -: not detected. (ECE: Eugenia chlorophylla; EPE: Eugenia pyriformis; MOE: Myrcia obtecta; MLE: Myrcia laruotteana).

identification of peak as 5-O-(E)-caffeoylquinic acid (5-


CQA). Furthermore, the identity of this compound was
also confirmed through co-elution with a 5-CQA authentic
Caffeic acid Ferrulic acid standard. In the ESI-MS fingerprints of the samples of
Myrtaceae extracts (Figure 1, Table 2) the following
components were identified in their deprotonated forms:
caffeic acid (m/z 179), quinic acid (m/z 191), ferulic acid
(m/z 193), and chlorogenic acid 5-CQA (m/z 353), figure
2. The content of phenolic compounds in the extracts,
Quinic acid Chlorogenic acid
possibly could explain the high antioxidant activity
verified for these extracts of Myrtaceae.
Figure 2: Phenolic organic acids identified in antioxidant ethanol extracts
from the leaves of four Myrtaceae plants of the South of Brazil. The investigation by direct infusion electrospray ionization
mass spectrometry (ESI-MS) provided important
the caffeoylquinic acids (CQA) identification keys [36,37] information about bioactive components present in the
led to the individualization of three CQA positional Myrtaceae extracts, that are widely reported as potent
isomers. The product ion spectrum for m/z 353 showed m/z antioxidants, probably explaining the antioxidant activity
191 (bp) and m/z 179 at 4% ri. The greater relative of the studied extracts [35, 38-40].
intensity of m/z 179 in the product ion spectrum, led to the
980 Natural Product Communications Vol. 6 (7) 2011 Salvador et al.

Experimental 37°C. The procedure was carried out according to the


Plant Material: The leaves of Eugenia chlorophylla O. method established by Ou and co-workers [29] with
Berg. (I), Eugenia pyriformis Cambess (II), Myrcia modifications [45]. The data were expressed as
laruotteana Cambess (III) and Myrcia obtecta (Berg) micromoles of Trolox equivalents (TE) per gram of extract
Kiacrsk. (IV) were collected in Curitiba, Paraná State, on dry basis (μmol of TE/g) and as relative Trolox
Brazil. Voucher specimens were deposited at the equivalent for pure compounds. Quercetin and 6-hydroxy-
herbarium of Universidade Federal do Paraná (UPCB 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)
53304, 16741, 53303 and 60504, respectively). were used as positive controls. The analyses were
performed in triplicate.
Extracts preparation and chemical analysis: Dried and
powdered leaves of each plant (50 g) were extracted HPLC analysis: HPLC analyses were conducted using a
with ethanol (3 X 300 mL) at room temperature. The RP-18 column (Lichrospher, 5 μm, 225\4.6 mm, Merck).
solvent was removed under reduced pressure to give the The mobile phase consisted of a linear gradient combining
crude extracts (E. chlorophylla 5.4%, E. pyriformis 5.7%, solvent A (acetonitrile) and solvent B (water/acetic acid,
M. laruottena 14.1% and M. obtecta 21.4%), which were 99:1, v/v, pH 2.88) as follows: 15% A (15 min), 15-40% A
used in DPPH assays. (5 min), 40-60% A (5 min), 60-100% A (5 min), 100-15%
A (5 min), 15% A (5 min). The analyses were carried out
Phytochemical Analysis: Phytochemical tests for in triplicate at a flow rate of 0.8 mL/ min and an injection
sterols/triterpenes, phenolic compounds, tannins, saponins volume of 20 μL. UV-DAD detector was set to record
and alkaloids were carried on according usual between 200 and 600 nm, and the UV chromatograms
methodology [41]. were measured at 254 and 330 nm. The samples were
analyzed at 1 mg/mL. The standard sample of quinic acid
Quantitative determination of total soluble phenols: and chlorogenic acid (5-O-(E) caffeoylquinic acid) were
The extracts, dissolved in methanol, were analyzed for also analyzed and then used for co-elution with authentic
their total soluble phenolic content according to the Folin- standard.
Ciocalteau colorimetric method [42-43], using gallic acid
as reference. The results were expressed as milligrams of Electrospray ionization mass spectrometry
gallic acid equivalents (GAE) per gram of extract or fingerprinting: Crude extracts of Myrtaceae plants were
fraction (mg of GAE/g). The analyses were performed in diluted in a solution containing 50% (v/v) chromatographic
triplicate. grade methanol and 50% (v/v) deionized water and 0.5%
of ammonium hydroxide (Merck, Darmstadt, Germany). In
the fingerprinting ESI-MS analysis, the general conditions
Radical scavenging activity using the DPPH method: were: source temperature of 100 oC, capillary voltage of
The antiradical activity of extracts was determined using 3.0 kV and cone voltage of 30 V. For measurements in the
the stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)
negative ion mode, ESI(-)-MS, 10.0 L of concentrated
[44]. The test was performed in 96-well microplates. Fifty
NH4OH were added to the sample mixture having a total
microliters of a 250 μM DPPH solution in MeOH was
volume of 1000 L yielding 0.1% as final concentration.
added to a range of solutions of different concentrations
For measurements in the positive ion mode ESI(+)-MS,
(seven serial 3-fold dilutions to give a final range of 100 to
1.6 μg mL-1) of extracts to be tested in MeOH (10μL). 10.0 L of concentrated formic acid were added giving a
Absorbance at 517 nm was determined 30 min after the final concentration of 0.1%. ESI-MS was preformed by
addition of each of the compounds tested, and the direct infusion with a flow rate of 10 L min mL-1 using a
percentage of activity was calculated. Quercetin and 6- syringe pump (Harvard Apparatus). Structural analysis of
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid single ions in the mass spectra from extract was performed
(Trolox) were used as positive controls. All samples were by ESI-MS/MS. The ion with the m/z of interest was
tested in triplicate. The antioxidant activity of each sample selected and submitted to 15–45 eV collisions with argon
was expressed as the SC50 value, which is the in the collision quadrupole. The collision gas pressure was
concentration in μg mL-1of each extract that scavenged optimized to produce extensive fragmentation of the ion
50% of the DPPH radicals. All of the results are expressed under investigation. The compounds were identified by
as mean of three different trials. comparison of their ESI-MS/MS fragmentation spectra
with literature data [33-35].
Evaluation of antioxidant capacity by ORAC assay:
The antioxidant capacity of the ethanolic extract was Statistical analysis: Data are reported as mean (%RSD,
assessed through the oxygen radical absorbance capacity relative standard deviation) of triplicate determinations.
(ORAC) assay. This assay measures antioxidant The statistical analyses were carried out using the
scavenging activity against peroxyl radicals using Microsoft Excel 2002 software package (Microsoft Corp.,
fluorescein as the fluorescent probe. ORAC assays were Redmond, WA)
carried out on a Synergy HT multi-detection microplate
reader system. The temperature of the incubator was set at
Antioxidant capacity of four Myrtaceae plants Natural Product Communications Vol. 6 (7) 2011 981

Acknowledgments - The authors are grateful to Dr. and to FAPESP, CNPq and FAEPEX-UNICAMP for
Armando C. Cervi, from Departamento de Botânica, financial support.
Universidade Federal do Paraná, for plant identification

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major components by electrospray ionization mass spectrometry. Food Chemistry, 104, 1048-1054.
[36] Clifford MN, Johnstton K, Knight S, Kuhnert N. (2003) Hierarchical scheme for LC-MSn identification of chlorogenic acids.
Journal of Agricultural and Food Chemistry, 51, 2900-2911.
[37] Clifford MN, Johnstton K, Knight S, Kuhnert N. (2005) Discriminating between the six isomers of dicaffeoylquinic acid by LC-
MS . Journal of Agricultural and Food Chemistry, 53, 3821-3832.
[38] Kim HJ, Kim EJ, Seo SH, Shin CG, Jin C, Lee YS. (2006) Vanillic acid glycoside and quinic acid derivatives from Gardeniae
fructus. Journal of Natural Products, 69, 600–603.
[39] Roche M, Dufour C, Mora N, Dangles O. (2005) Antioxidant activity of olive phenols mechanistic investigation and
characterization of oxidation products by mass spectrometry. Organic & Biomolecular Chemistry, 3, 423–430.
[40] Kweon M, Hwang H, Sung H. (2001) Identification and antioxidant activity of novel chlorogenic acid derivatives from bamboo
(Phyllostachys edulis). Journal of Agricultural and Food Chemistry, 49, 4646–4655.
[41] Harbone JB. (1998) “Phytochemical Methods. A guide to modern techniques of plant analysis”, Chapman & Hall, London.
[42] Piccinelli AL, De Simone F, Passi S, Rastrelli L. (2004) Phenolic constituents and antioxidant activity of Wendita calysina leaves
(burrito), a folk Paraguayan tea. Journal of Agricultural and Food Chemistry, 52, 5863-5868.
[43] Aquino SD, Angioni M, Schirru S, Agabbio M. (2001) Quality and physiological changes of film packaged ‘Malvasio’ mandarins
during long term storage. Lebensmittel-Wissenschaft und-Technologie 34, 206-214.
[44] Cuendet M, Hostettman K, Potterat O, Dyatmko W. (1997) Iridoid glucosides with free radical scavenging properties from
Fagraea blumei. Helvetica Chimica Acta, 80, 1144-1152.
[45] Salvador MJ, Ferreira EO, Mertens-Talcott SU, Castro WV, Butterweck V, Derendorf H, Dias DA. (2006) Isolation and HPLC
quantitative analysis of antioxidant flavonoids from Alternanthera tenella Colla. Zeitschrift fuer Naturforschung C, 61, 19-25.
2011
NPC Natural Product Communications Vol. 6
No. 7
Cytotoxicity of Active Ingredients Extracted from Plants of 983 - 984
the Brazilian “Cerrado”
Veronica CG Soaresa, Cibele Bonacorsib, Alana LB Andrelad, Lígia V Bortolotie,
Stepheny C de Campose, Fábio HR Fagundesd,e, Márcio Piovanie, Camila A Cotrima,
Wagner Vilegasc and Marcos H Toyamaf
a
Institute of Biology, UNICAMP, Campinas, Sao Paulo, 13083-862. Brazil
b
Department of Microbiology, UNESP, Araraquara, Sao Paulo, 14801-902. Brazil
c
Institute of Chemistry,UNESP, Araraquara, Sao Paulo, 14801-970. Brazil
d
Department of Pharmacy, Unianchieta, Jundiai, Sao Paulo, 13207-270. Brazil
e
Department of Pharmacy, UNIP, Jundiai, Sao Paulo, 13214-525.Brazil
f
Institute of Biology, UNESP, Campus Litoral Paulista, Sao Vicente, Sao Paulo,11330-150. Brazil

vcgsoares@gmail.com

Received: December 10th, 2010; Accepted: March 16th, 2011

Cytotoxicity assays are needed for the screening of natural products with potential anti-inflammatory. The purpose of this study was to
compare the basal cytotoxicity of active ingredients extracted from plants of the Brazilian “cerrado”. The viability was assayed with the
neutral red uptake assay in Mac Coy cells after 24h of exposition. The dose evaluated was 50 µg/µL. The test substances were: cinnamic
acid, p-coumaric acid, chlorogenic acid, syringic acid, vannilic acid, homogentisic acid, scandenin, palustric acid, diosgenin, cabraleone.
Studies of cytotoxicity demonstrated that all active compounds evaluated have low toxicity in vitro. The substances showed cell viability
above 60% for the concentration used. However, the cinnamic acid, sacandenin and palustric acid showed highest toxicity with a 50%
reduction in cell viability for the dose of 50 µg/µL. Cytotoxic screening results are useful to estimate the best concentrations of those
compounds with potential anti-inflammatory without their cause cell death.

Keywords: Natural products, Cytotoxicity, Brazilian Cerrado.

The Cerrado is one of the world's threatened biodiversity Table 1: Viability was performed with the neutral red uptake assay in Mac
Coy cells after 24h of exposure to the test substances.
hotspots [1a]. About 60% of its vegetation has already
been removed [1b] and the remaining areas are isolated in Natural Products (50 µg/mL nm (±SD)* (%)
Control + 0.519 ± 0.010 100%
forest fragments [2a]. Due to the devastation many natural Cinnamic acid 0.155 ± 0,015 30%
compounds with potential biological activities were lost. Scandenin 0.207 ± 0.012 40%
Palustric acid 0.249 ± 0.016 48%
Screenings of natural compounds using cytotoxicity assays Homogentisic acid 0.259 ± 0.013 50%
Diosgenin 0.404 ± 0.014 78%
offer the advantage of evaluate several compounds p-coumaric acid 0.415 ± 0.013 30%
simultaneously. The comparative sensitivity of cells to Cabraleone 0.337 ± 0.012 65%
toxicity tests that evaluate direct-acting and indirect-acting Syringic acid 0.337 ± 0.015 65%
Vannilic acid 0.363 ± 0.016 70%
cytotoxicants may be important to explaining their mode Chlorogenic acid 0.415 ± 0.013 80%
of action [2b]. Natural products with low cytotoxicity * Values presented with standard deviation.
activities constitute an excellent alternative search for
complementary treatments for inflammatory disease. The compounds showed cell viability above 60% for the
concentration used. However, the cinnamic acid, scandenin
The values obtained from the cytotoxicity test performed and palustric acid showed highest toxicity with a 50%
on cells MacCoy can be used as parameters for doses used reduction in cell viability for the dose of 50 µg.
in trials of anti-inflammatory activity. The purpose of this
study was to compare the basal cytotoxicity of active The natural products obtained from the Brazilian cerrado
compounds extracted from plants of the Brazilian show large potential for pharmaceutical product
“cerrado” and find those whose toxicity is low to further development. Some researches have shown the use of
anti-inflammatory assays. Due to this purpose dose of these compounds as an alternative for the treatment of
50 µg was used. The percentage of cell viability was cancer [3]. In addition, the potential of these products
established for each of the compounds evaluated and the mighty be exploited to treat other inflammatory disorders
results are presented in Table 1. such as physiological dysfunctions. Several xanthones,
984 Natural Product Communications Vol. 6 (7) 2011 Soares et al.

coumarins, terpenoids and phenolic compounds were Culture Section of the Adolfo Lutz Institute, São Paulo,
isolated from plants Brazilian cerrado and with remarkable Brazil) was maintained in Eagle medium with 7.5% fetal
activities including antiHIV [4], antibacterial [5], bovine serum at 37ºC.
trypanocidal [6], and anticancer against the cell lines
KM12 (colon adenocarcinoma), U251 (glioma), PC3 Cytotoxicity assay: After trypsinization, 0.2 mL aliquots
(prostate), and K562 and HL60 (leukemia) [7-9a]. Our of Eagle, containing approximately 104-105 cells/mL were
results agree with those previously reported. According to transferred to 96-well microtiter tissue-culture plates and
the results, all the products have shown low cytotoxicity. incubated at 37°C. After 24 hr, the medium was removed
and the cells were covered in unmodified medium
The differences between the chemical structures of the (control) or in medium modified with various
substances evaluated, was the determining factor for concentrations of the test compound. After incubating for
difference of cytotoxicity. Complex structures (diosgenin, another 24 hr, the medium was removed and the plates
cabraleone) showed less toxic, possibly because these were prepared for the neutral red (NR) assay [9b]. After
compounds are not able to go into de cells. Structures brief agitation, the plates were transferred to a microplate
with coumarin ring were the most toxic (cinnamic acid, reader (Spectra (Shell) & Rainbow (Shell) Reader, Tecan
p-coumaric acid) Glycoside radical (acid chlorogenic) Austria GMBH) and the optical density of each well at
usually shows low toxicity due to the presence of 620 nm was measured, using a 540 nm filter. All
hydroxyls that make difficult the transport of the experiments were performed at least four times, using
compounds to into the cells. Natural compounds with low three wells for each concentration of chemical tested. The
toxicity and potential as anti-inflammatory drugs have cytotoxicity data was standardized by determining
been a source of research, once the search for new drugs absorbance and calculating the corresponding chemical
with fewer side effects has increased significantly. concentrations [9c].

Experimental Compounds: The test substances were obtained after


Chemical and culture media: Neutral red was purchased maceration of different plants species from Brazilian
from Sigma (St. Louis, MO, USA). Fetal bovine serum cerrado, resulted in hexane, dichloromethane, ethanol and
(FBS) was obtained from Cultilab (Campinas, SP, Brazil). hydro-ethanol extracts, depending on extraction procedures.
Dulbecco´s Modified Eagle Medium (DMEM) was The substances, cinnamic acid, p-coumaric acid, chloro-
obtained from Instituto Adolfo Lutz (São Paulo, SP genic acid, syringic acid, vannilic acid, homogentisic acid,
Brazil). scandenin, palustric acid, diosgenin, cabraleone were
obtained using methods described by dos Santos [9d].
Cell Line: Mac Coy mouse fibroblast cell line (CCL1;
American Type Culture Collection, USA, from Cell Acknowledgments - Financial Support: FAPESP, CNPq.

References
[1] (a) Myers N, Mittermeier RA, Mittermeier CG, Fonseca GAB, Kent J. (2000) Biodiversity hotspots for conservation priorities.
Nature, 403, 853-858; (b) Machado RB, Ramos Neto MB, Pereira PGP, Caldas EF, Gonçalves DA, Santos NS, Tabor K, Steininger
M. (2004) Estimativas de perda da área do Cerrado brasileiro. Brasília: Conservação Internacional, 1-6.
[2] (a) Durigan G, Siqueira MF, Franco GADC. (2007) Threats to the Cerrado remnants of the State of São Paulo, Brazil. Scientia
Agricola, 64, 355-363; (b) Soares VCG, Varanda EA, Raddi MSG. (2006) In vitro basal and metabolism-mediated cytotoxicity of
flavonoids. Food and Chemical Toxicology, 44, 835-838.
[3] Mesquita ML, de Paula JE, Pessoa C, de Moraes MO, Costa Lotufo LV, Grougne R, Michel S. (2009) Cytotoxic activity of
Brazilian Cerrado plants used in traditional medicine against cancer cell lines. Journal of Ethnopharmacology, 123, 439-445.
[4] Huerta-Reyes M, Basualdo MdelC, Abe F, Jimenez-Estrada M, Soler C, Reyes-Chilpa R. (2004) HIV1 inhibitory compounds from
Calophyllum brasiliense leaves. Biological & Pharmaceutical Bulletin, 27, 1471–1475.
[5] Pretto JB, Cechinel-Filho V, Noldin VF, Sartori MRK, Isaias DEB, Cruz AB. (2004) Antimicrobial activity of fractions and
compounds from Calophyllum brasiliense (Clusiaceae/Guttiferae). A Journal of Biosciences Zeitschrift fur Naturforschung C, 59,
657–662.
[6] Abe F, Nagafuji S, Okawa M, Kinjo J, Akahane H, Ogura T, Martinez-Alfaro MA, Reyes-Chilpa R. (2005) Trypanocidal
constituents in plants 5. Evaluation of some Mexican plants for their trypanocidal activity and active constituents in the seeds of
Persea americana. Biological & Pharmaceutical Bulletin, 28, 1314–1317.
[7] Reyes-Chilpa R, Estrada-Muniz E, Ramirez Apan T, Amekraz B, Aumelas A, Jankowski CK, Vazquez Torres M. (2004) Cytotoxic
effects of mammea type coumarins from Calophyllum brasiliense. Life Sciences, 75, 1635–1647.
[8] Ito C, Murata T, Itoigawa M, Nakao K, Kaneda N, Furukawa H. (2006) Apoptosis inducing activity of 4 substituted coumarins
from Calophyllum brasiliense in human leukaemia HL60 cells. Journal of Pharmacy and Pharmacology, 58, 975–980.
[9] (a) Suffredini IB, Paciência MLB, Varella AD, Younes RN. (2007) In vitro cytotoxic activity of Brazilian plant extracts against
human lung, colon and CNS solid cancers and leukemia. Fitoterapia, 78, 223–226; (b) Borenfreund E, Puerner JA. (1985) Toxicity
determined in vitro by morphological alterations and neutral red absorption. Toxicology Letters, 24, 119-124; (c) Barile FA. (1994)
In vitro cytotoxicology. New York: CRC Press, 96p; (d) dos Santos LC, da Silva MA, Rodrigues CM, Carbone V, Napolitano A,
Bassarello C, Mari A, Piacente S, Pizza C, Vilegas W. (2009) Characterization of flavonoid and naphthopyranone derivatives from
Eriocaulon ligulatum using liquid chromatography tandem mass spectrometry. Natural Product Communications, 4, 1651-1656.
2011
NPC Natural Product Communications Vol. 6
No. 7
Propagation and Conservation of Native Forest Genetic 985 - 988

Resources of Medicinal Use by Means of in vitro and


ex vitro Techniques
Sandra Sharry, Marina Adema, María A. Basiglio Cordal, Blanca Villarreal, Noelia Nikoloff,
Valentina Briones and Walter Abedini

Centro Experimental de Propagación Vegetativa (C.E.ProVe)Facultad de Ciencias Agrarias y


Forestales. Universidad Nacional de La Plata. CICPBA. Diagonal 113 N° 469 (1900) La Plata,
Buenos Aires, Argentina

ssharry@gmail.com

Received: December 13th, 2010; Accepted: March 10th, 2011

In Argentina, there are numerous native species which are an important source of natural products and which are traditionally used in
medicinal applications. Some of these species are going through an intense extraction process in their natural habitat which may affect their
genetic diversity. The aim of this study was to establish vegetative propagation systems for three native forestal species of medicinal interest.
This will allow the rapid obtainment of plants to preserve the germplasm. This study included the following species which are widely used in
folk medicine and its applications: Erythrina crista-galli or “seibo” (astringent, used for its cicatrizant properties and for bronchiolitic
problems); Acacia caven or “espinillo” (antirheumatic, digestive, diuretic and with cicatrizant properties) and Salix humboldtiana or “sauce
criollo” (antipyretic, sedative, antispasmodic, astringent). The methodology included the micropropagation of seibo, macro and
micropropagation of Salix humboldtiana and the somatic embryogenesis of Acacia caven. The protocol for seibo regeneration was adjusted
from nodal sections of seedlings which were obtained from seeds germinated in vitro. The macropropagation through rooted cuttings of
“sauce criollo” was achieved and complete plants of this same species were obtained through both direct and indirect organogenesis using in
vitro cultures. The somatic embryogenesis for Acacia caven was optimized and this led to obtain a high percentage of embryos in different
stages of development. We are able to support the conservation of native forest resources of medicinal use by means of vegetative
propagation techniques.

Keywords: Micropropagation, native forest species, macropropagation, somatic embryogenesis.

Medicinal plants have been used since ancient times as In Buenos Aires province (Argentina), there are different
new therapeutic agents and their uses have been native forest species of medicinal interest and some of
transmitted from generation to generation, either in oral or them are Salix humboldtiana or “sauce criollo”, Erythrina
written forms, up to the present, and this is known as the crista-galli or “seibo” and Acacia caven or “espinillo”.
“traditional therapeutic practice”, the use of extracts or
active principles of plants, which has been essential to take Salix humboldtiana is distributed in river banks or islands,
care of people’s health in the first level of attention. sometimes in sandy places, too. The different parts of the
plant contain salicin. The bark is used in folk medicine as a
The developed countries as well as the developing ones quinine substitute (it contains glucosides). The decoction
have increased the use of medicinal plants or their products of this bark is used “against intermittent fever” (rubber).
[1]. Medicinal plants have played a vital role in societies The bark is bitter and has febrifugal, tonic, sedative and
including Argentina for centuries. Most of these plants are spasmodic properties. It is also astringent [2]. Erythrina
wild plants which were available in some forest crista-galli has several important pharmacological uses. It
ecosystems. With the intensity of development and is an astringent and a sedative to heal wounds (3% of bark
clearing of land many of these wild plants used for decoction). It is antihemorrhoidal and used for vaginal
medicinal purposes are no longer available in their natural lavage in candidiasis cases (bark). It is disinfectant and
habitat. Now we can address this environmental situation deodorant, it has cicatrizant properties and it is
by ex situ conservation and propagation of medicinal ahemostatic, emollient for colds, coughs, catarrh,
plants for its sustainable utilization using new and bronchitis and asthmatic pains (the leaves are smoked in a
traditional techniques. pipe or rolled up like a cigar). It has narcotic, sedative and
hypnotic properties: this is attributed to the most inner part
986 Natural Product Communications Vol. 6 (7) 2011 Sharry et al.

of the bark when it is used in an infusion (it contains


several alkaloids). This plant is also used for muscular and
rheumatic pains (a balm prepared with its bark and flowers
in 70% of alcohol). The leaves are used as
antihemorrhoidal for external use and they are antiseptic
and astringent [2]. Acacia caven or “espinillo” is a
medicinal plant that lives in South America and it is not
tropical. It grows in the arid highlands in the centre and
north of the country as well as on the islands in the Paraná Figure 1 a-b: Macropropagation of Salix humboldtiana. Plants
River, in very humid areas. The parts of the plant which obtained by rooting of cuttings.
are used for medicinal purposes are its leaves, stems and
seeds. The leaves of this medicinal plant have cicatrizant
properties, and the seeds are used as a digestive. The
leaves and stems have antiphlogistic and sedative
properties (http//www.tusplantasmedicinales.com/).

Vegetative reproduction keeps the parental genotype and


its characteristics are preserved in its off springs. Thus, the
genotypes of selected trees which are propagated
vegetatively reproduce identically and form clones. Grafts,
cuttings and layering are the traditional methods of
vegetative propagation [3].

In the last few years the use of in vitro culture techniques


in trees has facilitated the clonation of select phenotypes,
the preservation and the manipulation of vegetal material.
These techniques allow the multiplication of clones in a Figure 2a-f: In vitro culture of Salix humboldtiana. a. In vitro seedlings
short period of time, in any season of the year and in a of Salix humboldtiana obtained from immature embryos. b. Calli obtained
from the seedling leaves. c. Shoots formed from calli. d. Shoots from
limited space [4]. By using these techniques, the long time
nodal sections. e. Induction of roots from microcuttings. f. Whole plants
it takes a plant to reach maturity, the low viability of seeds, under greenhouse conditions (acclimatization and hardening).
and the difficulties some species have to propagate by
traditional methods can be reduced [5]. Besides, hundreds The percentage of contamination of the nodal sections
of clones from the same species can be reproduced in vitro. which grew from fully grown plants was of 80-90% when
Later, they are taken to a plant nursery and then, they are they were treated with 50% of sodium hypochlorite during
cultivated in fields where they will develop and finally 25 minutes and of 60% when they were treated with 50%
become a product with a specific economic interest of sodium hypochlorite during 45 minutes. The WPM
(http://www.biologia.org/). culture medium without growth regulators was the best
one to obtain preformed shoots and whole plantlets. The
The aim of this study was to establish vegetative first shoots were observed approximately 20 days later,
propagation systems for three native trees of medicinal and the roots, 25-30 days after they were cultured in vitro.
interest: Salix humboldtiana (sauce criollo), Erythrina
crista-galli (seibo) and Acacia caven (espinillo). This will The culture media with BAP promote the formation of
help to obtain plants to conserve germplasm in a rapid way. callus on the base of the microcuttings. This callus turned
out to be organogenic and the production of shoots was
Macropropagation of Salix humboldtiana: The substrate induced by using cytokinins (Figure 2).
with the mixture of soil, perlite and vermiculite (6:3, 5:0, 5)
was adequate to place the Salix humboldtiana cuttings. The Micropropagation of Erythrina crista-galli : Growth of
cuttings rooted 15 days after they were placed in water. the preformed shoots was induced in MS with 1 mg.L-1
90% of the cuttings which were treated with IBA rooted, of BAP and 0.5 mg.L-1 of NAA. These shoots were
and 75% of them originated complete plants (Figure 1). subcultured in a WPM medium with 0.1 mg.L-1 of IBA
where they elongated. Whole plants were obtained in a
In vitro culture of Salix humboldtiana: The culture media WPM rooting medium with 0.1 mg.L-1 of NAA. These
used for callus, shoot and root induction were adequate. plants were acclimatized under controlled light and
Callus with de novo shoots (indirect organogenesis) were temperature conditions (16 hours of light and 8 hours of
obtained approximately 35 days after the leaves were darkness, T 21°C +/-2°C) (Figure 3).
cultured. The shoots formed roots 25 days later. In this
way, vitroplants were obtained and they became Somatic embryogenesis of Acacia caven: Direct
acclimatized successfully. and indirect somatic embryogenesis were obtained on the
Vegetative propagation systems for three medicinal plants Natural Product Communications Vol. 6 (7) 2011 987

Figure 3a-d: Micropropagation of Erythrina crista-galli. a. In vitro seedlings. b. Shoot elongation from stem section. c. Induction of roots. d.
Acclimatization of whole plants.

Figure 4a-e: Somatic embryogenesis of Acacia caven.a. Mature fruits of Acacia caven. b y c Seeds in chemical scarification. d. Somatic embryos in a
globular and heart stage formed fron cotyledons. e. Embryos in torpedo and cotiledonar stage.

culture medium and the PGR concentration (1 mg.L-1 of For Salix humboldtiana or “sauce criollo”, we used micro
2,4 D and 0.1 mg/L-1 of BAP) used. The somatic embryos and macropropagation systems. Macropropagation was
occur directly over the cotyledons, on their adaxial side, or done through the rooting of cuttings with the exogenous
indirectly from the formation of the callus after 6 months application of growth regulators and the optimization of
of culture. Somatic embryos were observed in different their nutritional requirements. The micropropagation was
stages of development, and this showed there is no done through the use of in vitro plant tissue culture
synchronicity in their maturation (Figure 4). The somatic techniques, following the direct and indirect organogenic
embryos in a globular stage were subcultured on MS pathways.
medium free from PGR, and they germinated there. 30%
of them were converted into complete plants. For Erythrina crista-galli or “seibo” and Acacia caven or
“espinillo” we used micropropagation following the
Conclusion: When the vegetative propagation techniques pathway of organogenesis and somatic embryogenesis.
of the species mentioned before are adjusted, they Mother plants which were in good sanitary conditions,
contribute to the preservation of the forest genetic optimal growth, and adaptability to the particularities of
resources. Excellent opportunities for scientific research the local site were chosen as a donating source for the
are generated by installing germplasm banks with a wide different explants. The methodology included:
range of genetic material. This becomes very important if
we consider that these species have a great importance in Macropropagation of Salix humboldtiana: Between 100
the medicinal use and among others. and 150 plant stem cuttings with single node each were
cut of approximately 30 cm long and from 0.8 to 1.5 cm
These studies pretend to be preliminary stages to achieve in diameter. They were treated superficially with 1000 mg.
the production of native plants of medicinal interest, L-1 of Bennomyl fungicide for 3 hours in order to avoid
without the degradation of the genetic base of this the presence of fungi. After that, the bases of the cuttings
resource, which is vitally important for the preservation of were dippedin 50 mg. L-1 of indole-3-butyric acid (IBA)
the forest resources. for 24 hours to induce rooting. The control was not treated
with IBA. Then, they were placed in running water for 15
Experimental days and they were planted in plastic flowerpots N° 14
This study has been developed at the Centro Experimental (1500 cm3) with a mixture of soil, perlite and vermiculite
de Propagación Vegetativa (C.E.Pro.Ve.) in the Facultad (6:3.5:0.5) as substrate [6]. The pots were arranged in
de Ciencias Agrarias y Forestales at Universidad Nacional randomized block design and replicated three times.
de La Plata, Buenos Aires, Argentina. In order to reach the
objectives, we used vegetative propagation strategies for In vitro culture of Salix humboldtiana: Explants were
each species. nodal sections with internodes (microcuttings) from adult
plants and leaves from in vitro seedlings obtained from
988 Natural Product Communications Vol. 6 (7) 2011 Sharry et al.

immature embryos. The embryos were surface sterilized Micropropagation of Erythrina crista-galli: In order to
with 10% of commercial sodium hypochlorite (55% active induce the shoot proliferation nodal sections from in vitro
chlorine) for 5 minutes in order to place them in vitro, and germinated seedlings were used as source of explants.
later obtain the seedlings. The leaves from the seedlings Different culture media with different growth regulators
were placed on Murashige & Skoog (MS) [7] basal were tested. The explants were cultured in MS medium in
medium at full strength supplemented with 1 mg.L-1 of a complete, half, and a quarter concentrations, with the
benzyl amino purine (BAP) and 0.5 mg.L-1 of naftalen addition of different growth regulators: 1 and 2 mg.L-1 of
acetic acid (NAA), 2% sucrose and 7.5 g. L-1 agar. Calli BAP, 0.5 mg.L-1 of NAA, 1mg.L-1 of IBA, 2% sucrose and
were subcultivated on MS medium at full strength with 7.5 g.L-1 of agar, alone or combined. Cultures were
1mg/L-1 of BAP and 1 mg.L-1 of NAA. The nodal sections incubated at 21°C +/-2°C with a 16 hours photoperiod.
or microcuttings of adult “sauce” plant were washed with
running water for 5 minutes, and they were surface Somatic embryogenesis of Acacia caven: In this case,
disinfected with 2000 mg/ L-1 of Bennomyl fungicide for 3 cotyledons from mature seeds were used as explants.
hours and 50% of commercial sodium hypochlorite (55% These seeds were treated with 98% of sulfuric acid in
active chlorine) for 25 and 45 minutes. For shoot order to scarification for two hours and they were washed
induction, the following culture media were tested: Woody with running water for 10 minutes. Then, they were
Plant Medium (WPM) without growth regulators and disinfected with 70% of ethanol during 5 minutes and 20%
WPM supplemented with 0.1; 0.5; 1; 1.5; and 2 mg.L-1 of of sodium hypochlorite (55% active chlorine) for 30
the 6- benzilaminopurine (BAP). minutes. After that, they were washed 3 times with
distilled water under a laminar flow hood and they were
The cultures were maintained in the culture room under a put in sterile water during 7 days in order to soften the
regime of 16 h photoperiod (intensity - 40 µEcm- seed coat and obtain the cotyledons. The latter were sowed
2/min/sec) at 21°C +/-2°C. All experiments were in a Murashige & Skoog (MS) basal medium, at half
conducted at least three times with 15 replicates each. concentration of macro and micronutrients, supplemented
with 1 or 2 mg.L-1 of 2,4-Dichlorophenoxyacetic acid
Shoots from both explants were placed in a rooting (2.4-D) and 0.1 mg.L-1 of BAP, 3% of sucrose and 7.5 g.
medium with the macro and micronutrients of WPM [8] L-1 agar (Table 2). The cotyledons were placed with their
supplemented with 0.1 mg.L-1 of Indolebutyric acid (IBA) abaxial side in contact with the culture medium and were
(Table 1). maintained in the culture room under complete darkness at
21°C +/-2°C.
Table 1: Culture media used to the differents explants (leaves and nodal
sections) of Salix humboldtiana. Table 2: Culture media used in the somatic embryogenesis of Acacia
Basal media Growth mg.L -1
Type of explant caven.
regulator PGR
Explants Media 2.4D BAP Condition
MS complete BAP/ANA 1:0.5 Leaves
(mg.L-1) (mg.L-1)
MS complete BAP/ANA 1:1 Leaves
Cotyledon (Control) MS/2 0 0 25 +/- 2°C
WPM IBA 0,1 Shoots
Cotyledon MS/2 1 0.1 in the
WPM BAP 0.1; 0.5; 1; 1.5 y 2 Nodal sections
Cotyledon MS/2 2 0.1 darkness.
WPM - - Nodal sections

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[1] García González M. (2000) Plantas medicinales científicamente validadas. II Congreso de Ciencias “Exploraciones dentro y fuera
del aula”. Fundación CIENTEC. INBioparque, Santo Domingo, Heredia, Costa Rica.
[2] Lahitte HB, Hurrell JA. (1994) Flora arbórea de la Isla Martín García nativa y naturalizada. Reserva Natural y Cultural. Provincia
de Buenos Aires. República Argentina. ISSN 0325-1225. Serie informe Nº 47. 27-229
[3] Hartmann HT, Kester DE. (1998) Propagación de plantas. Ed. CECSA. México, D.F. Parte II. 220, 221-760
[4] Mroginski L, Sansberro P. y Flaschland E. (2010) Herramientas básicas. En: Biotecnología y Mejoramiento Vegetal II. Ediciones
INTA-Eds: Gabriela Levitus, Viviana Echenique, Clara Rubinstein, Esteban Hopp, Luis Mroginski.
[5] Neumann K. (2009) Plant Cell and Tissue Culture - A Tool in Biotechnology: Basics and Application (Principles and Practice),
Springer.
[6] Abedini W, Adema M, Herrera J, Sharry S, Villarreal B, Nikoloff N. (2008) Recursos Forestales nativos de la provincia de Buenos
Aires: la Biotecnología como una estrategia de conservación. III Congreso Nacional de Conservación de la Biodiversidad. Facultad
de Ciencias Exactas y Naturales, UBA. Ciudad Autónoma de Buenos Aires. CD ROM
[7] Murashige T, Skoog F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia
Plantarum, 15, 473-497.
[8] Lloyd G, McCown B. (1980) Commercially feasible micropropagation of mountain laurel Kalmia latifolia, by use of shoot tip
culture. Combined Proceedings International Plant Propagation Society, 30, 421–427.
2011
NPC Natural Product Communications Vol. 6
No. 7
Genotoxic Evaluation of a Methanolic Extract of Verbascum 989 - 991

thapsus using Micronucleus Test in Mouse Bone Marrow


Franco Matías Escobara, María Carola Sabinia, Silvia Matilde Zanona, Laura Noelia Cariddia,
Carlos Eugenio Tonnb and Liliana Inés Sabinia
a
Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico-Químicas y
Naturales.Universidad Nacional de Río Cuarto, Río Cuarto, Córdoba. Argentina
b
INTEQUI-CONICET, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San
Luis, San Luis. Argentina

fescobar@exa.unrc.edu.ar

Received: November 15th, 2010; Accepted: March 25th, 2011

Verbascum thapsus L. is a medicinal plant and has been used to treat numerous pulmonary diseases, asthma, inflammatory disease,
spasmodic coughs and migraine headaches. Several studies have demonstrated that different extracts of V. thapsus present antimicrobial
activity. Thus, the goal of this study was to evaluate the genotoxic and cytotoxic activities of a methanolic extract of Verbascum thapsus,
using micronucleus test in mouse bone marrow. No toxicity in bone marrow was detected in the extract-treated groups. The methanolic
extract of V. thapsus at doses of 100, 300 and 500 mg / kg, did not produce a significant increase in the frequency of MNPCE in bone
marrow and neither altered the relationship PCE / NCE respect to negative control. These cytogenotoxic findings contribute the preclinical
knowledge of methanolic extract of V. thapsus and provide security in its use as herbal medicine.

Keywords: Verbascum thapsus L., methanolic extract, micronucleus, bone marrow, genotoxicity.

The use of medicinal plants in therapy or as dietary mullein, great mullein [3b]. V. thapsus is distributed
supplements remounts centuries ago, but it has increased worldwide. In Argentina this species is abundant however;
substantially in the last decades [1a,1b]. The popularity of it is considered an exotic plant. Many studies carried
herbal medicines is related to their easy access, therapeutic out with plants collected in other countries, have shown
efficacy, relatively low cost, and assumed absence of toxic that different extracts have antimicrobial, antitumor
effects. and cytotoxic activities [4a-4c]. Therefore, given the
abundance of the species in Córdoba province, Argentina,
Widespread public opinion is that being a natural product, cytotoxic and antiviral properties of methanolic extract of
herbal medicines are harmless and free from adverse V. thapsus were investigated. The results of these previous
effects. However, the safety of their use has recently been studies have indicated that the extract markedly inhibits
questioned due to the reports of illness and fatalities Herpes suis virus type 1 at non cytotoxic concentrations
[2a-2c]. Considering the complexity of herbals in general [5a,5b]. Since this information is hopeful, it is necessary to
and their inherent biological variation, it is now necessary define the cytogenotoxic potential to ensure the use of the
to evaluate their safety, efficacy and quality [1b]. Thus, an extract at safe levels.
assessment of their mutagenic and cytotoxic potential is
necessary to ensure the relatively safe use of plant-derived The aim of this study was to determine the genotoxic and
medicines. cytotoxic activities of a methanolic extract of Verbascum
thapsus, using micronucleus test in mouse bone marrow.
Scrophulariaceae is an important family of plants Evaluation of micronucleus induction is the primary in
comprising over 200 genera and about 2500 species. It vivo test in a battery of genotoxicity tests and is
includes Mimulus, Penstemon, Digitalis, Veronica and recommended by the regulatory worldwide agencies to be
Verbascum [3a]. Different members have been valued for conducted as part of product safety assessment.
their curative properties and are widely employed in
domestic and regular medicine. At least 250 species of The results of micronucleus (MN) test in BALB/c mice
Verbascum are known. Among the species traditionally treated with different doses of the extract are summarized
used in medicine, the most important is Verbascum in Table 1. In all cases these results are expressed as mean
thapsus L., commonly known as mullein, common (± standard deviation).
990 Natural Product Communications Vol. 6 (7) 2011 Escobar et al.

Table 1: Mean of polychromatic erythrocytes with micronuclei (MNPCE) observed in bone marrow cell of female (F) and male (M) BALB/c mice treated with
a Verbascum thapsus methanolic extract, and respective controls.
Dose MNPCE PCE/NCE
Treatments Number of MNPCE per animal
mg/kg (mean ± SD) (mean ± SD)
F1 F2 F3 M1 M2 M3
Negative control (saline) 0 2 3 3 2 2 2 2.50 ± 0.55 1.69 ± 0.14
V. thapsus methanolic extract 100 1 2 5 1 3 1 2.17 ± 1.60 1.77 ± 0.12
V. thapsus methanolic extract 300 3 1 3 1 2 2 2.00 ± 0.89 1.79 ± 0.23
V. thapsus methanolic extract 500 5 1 1 2 1 2 2.00 ± 1.55 1.74 ± 0.15
Positive control (cyclophophamide) 20 11 14 11 13 9 12 11.7 ± 1.7* 1.69 ± 0.10
Thousand cells were analyzed per animal, for a total of 6000 cells per group. SD = Standard deviation. * p< 0.001, statistically significant difference from
saline group (ANOVA. Tukey`s test).

25 Male
Female
20

MNPCE / 1000 PCE


MNPCE 15

10

NCE
0

kg

kg

kg
(-)

)
(+
NCE PCE

g/

g/
g/
ol

l
ro
m

m
tr

t
on

on
10

30

50
C

C
Figure 2: Frequency of Micronucleated Polychromatic Erythrocytes
(MNPCE) induced in bone-marrow cells of female (F) and male (M)
Figure 1: A photomicrograph of mice whole bone-marrow smear BALB/c mice treated with a V. thapsus methanolic extract: negative
showing nucleated as well as enucleated cells (PCEs and NCEs). One the control (saline solution), positive control (cyclophosphamide 20 mg/kg
polychromatic erythrocyte also contains micronuclei. body weight). Values are shown as mean ± SD.

Examples of polychromatic and normochromatic Therefore, the results obtained in the present study allow
erythrocytes unaltered, normal, and the presence of concluding that the methanolic extract of Verbascum
micronucleated polychromatic erythrocytes are shown in thapsus does not contain genotoxic and cytotoxic
Figure 1. compounds since its administration in mice at doses of
100, 300 and 500 mg/kg, showed no evidence of
The percentage frequency of MN in the groups treated genotoxicity or cytotoxicity in vivo. The extract did not
with 100, 300 and 500 mg / kg of methanolic extract, produce a significant increase in the frequency of MNPCE
which were 2.17 (±1.60), 2.00 (±0.89), 2.00 (±1.55) in bone marrow and neither altered the relationship PCE /
respectively, showed no significant differences from the NCE respect to negative control.
saline-treated group: 2.5 (±0.55). However, there was a
significant increase in the frequency of micronucleus in These cytogenotoxic findings contribute the preclinical
PCE from the positive control group treated with knowledge of methanolic extract of V. thapsus and provide
cyclophosphamide (Figure 2). security in its use as herbal medicine.

No citotoxicity in bone marrow was detected in the Experimental


extract-treated groups. Statistical analysis of the proportion Plant material and extraction: Aerial parts of Verbascum
PCE / NCE revealed no differences in any study group. thapsus L. were collected in San Luis province, Argentina.
There were no sex-dependent changes in any treatment. The plant material was identified by Ing. Luis A. del Vitto.
A voucher specimen (N° #514) was preserved and
V. thapsus methanolic extract contains iridoid glycosides deposited in herbal library of the “Herbario de la
(laterioside, harpagoside, ajugol, picroside IV), three Universidad Nacional de San Luis, Argentina”. The leaves
iridoid ((+)-genipin, α-gardiol and β-gardiol), one were dried and chopped finely using a blender. Eight
phenylethyl glycoside (verbacoside), two sesquiterpenes hundred grams of dried material were successively
(buddlindeterpene A and buddlindeterpene B), one extracted with 3.5 L of the following solvents: n-hexane,
diterpene (buddlindeterpene C), and one biflavonoid chloroform and methanol at room temperature for 48 h.
(amentoflavone) [6]. The evaporation of the extracts in vacuum at 40ºC yielded
Genotoxic evaluation of a methanolic extract of Verbascum thapsus Natural Product Communications Vol. 6 (7) 2011 991

the hexane, chloroform and methanol extracts. The Slides were stained with May-Grünwald and Giemsa
methanolic extract was dissolved in saline solution and solutions [7b] which maximized the differentiation
subsequently diluted to appropriate working between the polychromatic (PCE) and normochromatic
concentrations. (NCE) erythrocytes. To determine index of genotoxicity
the number of micronucleated polychromatic erythrocytes
Animal’s treatments: Two months old male/female (MNPCE) was obtained at an average of 1000 PCE,
BALBc mice weighing ca. 20 g were intraperitoneally counted per animal per dose. In order to evaluate any
injected with a single dose of V. thapsus methanolic cytotoxic effect of extract, the ratio of PCE/NCE was
extract (volume 0.2 ml). Three doses were selected (100, determined in the same sample. Statistical significance was
300 and 500 mg/kg) considering previous citotoxicity data determined by analysis of variance (ANOVA), applying
obtained with Vero cells. Cyclophosphamide (Sigma) at 20 software GraphPad Prism 5.0.
mg/kg and saline solution were used as positive and
negative controls respectively. Acknowledgments - The authors are grateful to
CONICET, MinCyT of Córdoba, Universidad Nacional de
Mouse bone marrow micronuclei assay: Six mice per Río Cuarto and PICTOR program, BID 1728 /OC-AR for
dose were sacrificed at 24 h post-injection and femurs financial support. We also would like to thank Ing. Luis A.
were removed. Femurs were prepared for the boned- del Vitto for taxonomic determination of the plant
marrow micronucleus test as previously described [7a]. specimen.

References
[1] (a) Woods PW. (1999) Herbal healing. Essence, 30, 42-46; (b) WHO (World Health Organization). (2002) Drug Information Herbal
Medicines. Vol. 16. World Health Organization, Geneva.
[2] (a) Stewart MJ, Moar JJ, Steenkamp P, Kokot M. (1999) Findings in fatal cases of poisoning attributed to traditional remedies in
South Africa. Forensic Science International, 101, 177-183; (b) Ernst E. (2002) Toxic heavy metals and undeclared drugs in Asian
herbal medicines. Trends in Pharmacological Sciences, 23, 136-139; (c) Veiga-Junior VF, Pinto AC, Maciel MAM. (2005)
Medicinal plants: safe cure? Química Nova, 28, 519-528.
[3] (a) Grieve M. (1981) A Modern Herbal Vol 2. Dover Publications: New York, 562-566; (b) Turker AU, Gurel E. (2005) Common
Mullein (Verbascum thapsus L.): Recent Advances in Research. Phytotherapy Research, 19, 733-739.
[4] (a) McCutcheon AR, Ellis SM, Hancock REW, Towers GHN. (1992) Antibiotic screening of medicinal plants of the British
Columbian native peoples. Journal of Ethnopharmacology, 37, 213-223; (b) McCutcheon AR, Roberts TE, Gibbons E, Ellis SM,
Babiuk LA, Hancock REW, Towers GHN. (1995) Antiviral screening of British Columbian medicinal plants. Journal of
Ethnopharmacology, 49, 101-110; (c) Turker AU, Camper ND. (2002) Biological activity of common mullein, a medicinal plant
Journal of Ethnopharmacology, 82, 117-125.
[5] (a) Escobar F, Gallotti V, Salas M, Sabini C, Giordano O, Contigiani M, Sabini L. (2008) Comparative assays of cytotoxicity
induced by extracts of Verbascum thapsus. Biocell, 32, 108; (b) Escobar F., Konigheim BV, Aguilar J, Sabini C, Giordano O,
Contigiani M, Sabini L. (2008) Preliminary studies of antiherpetic activity exerted by extracts of Verbascum thapsus. Biocell, 32,
109.
[6] Hussain H, Aziz S, Miana GA, Ahmad VU, Anwar S, Ahmed I. (2009) Minor chemical constituents of Verbascum thapsus.
Biochemical Systematic and Ecology, 37, 124-126.
[7] (a) Schmid W. (1975) The micronucleus test. Mutation Research, 31, 9-15; (b) Cole RJ, Taylor NA, Cole J, Arlette CF. (1979)
Transplacental effect of chemical mutagens detected by micronucleus test. Nature, 277, 317-318.
2011
NPC Natural Product Communications Vol. 6
No. 7
Study of Antiviral and Virucidal Activities of Aqueous 993 - 994
Extract of Baccharis articulata against Herpes suis virus
Cristina Vanesa Torres, María Julia Domínguez, José Luis Carbonari, María Carola Sabini,
Liliana Inés Sabini and Silvia Matilde Zanon

Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Fisico-Químicas y


Naturales. Universidad Nacional de Río Cuarto. Río Cuarto, Córdoba. Argentina

ctorres@exa.unrc.edu.ar

Received: December 10th, 2010; Accepted: March 16th, 2011

Baccharis articulata is native of América and traditionally used for the treatment of digestive disorders and urinary infections. Cytotoxicity
of aqueous extracts of B. articulata was investigated in Vero cells. As the maximal non cytotoxic concentration has been established, this
concentration has been used to evaluate antiviral and virucidal activities against Herpes suis virus type 1, member of the same subfamily of
Herpes simplex virus. Aqueous extracts of B. articulata exhibited more than 95% of virucidal activity. These findings support their potential
application as a disinfectant or antiseptic with low toxicity and provide a valuable knowledge to ethnopharmacology properties of Baccharis
articulata.

Keywords: cytotoxicity, virucidal, antiviral activity, aqueous extract, Baccharis articulata, Herpes suis virus.

Baccharis articulata, commonly known as carqueja, Table 1: Cytotoxic effect and inhibition of viral replication of aqueous
extracts of Baccharis articulata.
frequently found in the hills region of Cordoba province of
Cytotoxicity Antiviral activity (%) Virucidal (%)
Argentina, exhibit antioxidant, antibacterial, anti-HIV and MNCC* A B C MNCC MNCC 2X
antifungal abilities, [1a-1c]. In treatment of herpetic CAE 1000 g/mL 25.6 33 0 31.5 97.8
infections one or more drugs currently available were used, HAE 600 g/mL 54 6.8 11 80 96
but both continuous use and self-medication promote the * Maximal non-cytotoxic concentration, A: viral adsorption, B: post-viral adsorption,
development of resistance and tolerance of these viruses C: pre-treatment cell.

[2]. This background encouraged the study of cytotoxic,


antiviral and virucidal activities of aqueous extracts of B. (54%) during the stage of viral adsorption and penetration.
articulata against Herpes suis type 1, virus closely related This value showed that Herpes suis virus were more
to Herpes simplex types 1 and 2. sensitive at HAE than CAE of B. articulata.

The aim of the present study was to determine Only the pre-treatment of Vero cells with HAE slightly
concentration of aqueous extracts that do not affect interfered Herpes suis type 1 adsorption to cellular
monolayer cell and to be used in later assays. Therefore receptor. Therefore, both extracts would not induce
cytotoxic effect on Vero cells of aqueous extracts was antiviral state in the cells and neither would interfere to the
evaluated by daily microscopic observation of treated cells mechanism of endocytosis used in the entry of virus into
to determine the MNCC (Maximal Non Cytotoxic the cell. The CAE and HAE demonstrated to exert strong
Concentration). The MNCC values were 1000 µg/mL and extracellular virus inactivation mostly when the assays
600 µg/mL for cold aqueous extract (CAE) and hot were carried out with extracts at double concentration of
aqueous extract (HAE), respectively. The cultures exposed MNCC (97.8 and 96% respectively). Results obtained in
to extract concentrations lower than MNCC exhibited this work allow concluding that the aqueous extracts of
morphology similar to control cultures. The cytotoxic B. articulata exert slight antiviral activities against Herpes
effect was characterized by retraction cell and suis virus type 1.
disruption of cell monolayer. Virucidal and antiviral
It is known that plant aqueous extracts, among other
assays performed at different stages of virus replication
components, contain anthocyanins, saponins, polypeptides
revealed percentages of inhibition shown in Table 1. The
and terpenes [3a]. Studies on the chemical composition of
CAE inhibited 25 and 33% viral replication when the
B. articulata have reported presence of several terpenes
extract was added at 1000 µg/mL (MNCC) during the viral
such as articulina, germacrene and ɑ-pinene [3b]. These
adsorption and later that step, respectively. The HAE,
compounds in other plant species (Glyptopetalum
at 600 µg/mL, demonstrated to exert the inhibitory activity
sclerocarpum, Thymus vulgaris) have exhibited antiviral
994 Natural Product Communications Vol. 6 (7) 2011 Torres et al.

activity [3a,3c]. As a consequence they could be virus was discarded. The cells were overlaid with an
responsible of the antiviral activity demonstrate in this overlay medium containing 1% of methylcellulose. The
work. Furthermore, additional studies are needed in order plates were further incubated at 37ºC for 72 h. Later cell
to identify which compounds could be responsible for this monolayer was fixed with 10% formalin. The virus
effect and how they exert antiviral action. plaques formed on Vero cells were stained with 1% crystal
violet. Percentage of viral inhibition was determined.
Experimental
Plant material: Baccharis articulata was collected in the Post-adsorption and penetration of virus: Confluent
hills of Cordoba, Argentina. Taxonomic identification was monolayer of Vero cells grown in 24-well culture plates
performed by Prof. Margarita Grosso of the Universidad were infected with Herpes suis type 1 (105 PFU/mL) and
Nacional de Río Cuarto. A specimen of the plant was incubated at 37ºC for 90 min. After residual virus was
deposited (Nº RCV 1810) in the Herbarium. Dried aerial removed, the cells were covered with the overlay medium
parts (branch and leaf) (15 g) were submitted to extraction containing 1% of methylcellulose and extract at MNCC,
with 700 mL of cold water at 4°C for 2 days (cold aqueous and incubated at 37ºC for 72 h. Percentage of viral
extract, CAE) and at 70°C for 2 days (hot aqueous extract, inhibition was determined.
HAE). The extracts were filtered and lyophilized. Pretreatment: Monolayer cells grown in 24-well culture
Viruses and cells: Vero cells (African green monkey plates were incubated for 2 h with extract at MNCC. After
kidney) were grown in Eagle´s minimum essential medium the extract was discarded, culture cells were inoculated
(MEM) supplemented with 8% FCS, 1% gentamicin and with Herpes suis type 1 (105 PFU/mL) and incubated at
1% L-glutamine and maintained at 37ºC in 5% CO2 37ºC for 90 min. The remainder virus was discarded and
atmosphere. Herpes suis virus type 1 strain RC/79 was the cells were incubated with the overlay medium
isolated in Río Cuarto in 1979 [4a]. containing 1% of methylcellulose at 37°C for 72 h.
Percentage of viral inhibition was determined.
Cytotoxicity assays: Confluent cell monolayers cultivated
in 96-well culture plates were treated with different Virucidal activity assay: Viral suspensions (105 PFU/mL)
concentrations of extracts and incubated at 37ºC for 72 h. were incubated with extract at MNCC and at double
At this time, maximal non cytotoxic concentration concentration. After incubation of virus at 37°C for 2 h,
(MNCC) was determined by microscopic observation. monolayer cells grown in 24-well culture plates were
infected with treated virus. The infected cells were
Antiviral assays: The antiviral activity of tested extracts incubated at 37°C for 90 min. The remainder virus was
was evaluated at different stages of viral replication by discarded and the cells were incubated with overlay
plaque reduction method. Virus titres were calculated by medium containing 1% of methylcellulose at 37°C for 72
plaque forming units per mL (PFU/mL) [4b]. The h. Percentage of viral inactivation was determined.
percentage of inhibition was calculated as the ratio
between virus titres in treated cells and in untreated cells. Acknowledgments - The authors thank Universidad
Nacional de Río Cuarto and PICTOR program, BID 1728
During adsorption and viral penetration: Monolayer /OC-AR for financial support. We are grateful to Prof.
cells grown in 24-well culture plates were incubated for 90 Margarita Grosso for help in identification of the plant
min with Herpes suis virus type 1 (105 PFU/mL) in specimen.
combination or not with extract at MNCC. Then, residual

References
[1] (a) Palacios PS, Wilson EG, Debenedetti SL. (1999) HPLC analysis cafeilquínicos acid present in three species of Baccharis.
Dominguezia, 15, 39; (b) De Oliveira SQ, Dal-Pizzol F, Gosmann G, Guillaume D, Moreira JC, Schenkel E. (2003) Antioxidant
activity of Baccharis articulata extracts: isolation of new compound with antioxidant activity. Free Radical Research, 37,
555-559; (c) Vivot Lupi EP, Sanchez Brisuela CI, Casik Jeifetz F, Sequin Acosta CJ. (2009) Screening of antifungal activity of
extracts of plant species in Entre Ríos. Revista Cubana de Farmacia, 43, 74-84.
[2] Kimberlin DW, Whitley RJ. (1996) Antiviral resistance: Mechanisms, Clinical Significance and Future Implications. Journal of
Antimicrobial Chemotherapy, 37, 403-421
[3] Ambrogi A, Giraudo J, Busso J, Bianco O, Bagnat E, Segura de Aramburu M, Ramos B, Ceriatti F. (1981) Primer diagnóstico de la
enfermedad de Aujeszky en cerdos en la República Argentina. Gaceta Veterinaria. Buenos Aires, Tomo XLIII, 357, 58-64
[4] Dulbecco, R. (1962) Production of plaques in monolayer tissue culture by single particles of an animal virus. Proceedings of the
National Academy of Sciences, USA, 38, 747-752
2011
NPC Natural Product Communications Vol. 6
No. 7
Evaluation of Cytogenotoxic Effects of Cold Aqueous 995 - 998

Extract from Achyrocline satureioides by Allium cepa L test


María C. Sabinia*, Laura N. Cariddia, Franco M. Escobara, Romina A. Bachettia, Sonia B. Sutila,
Marta S. Contigianib, Silvia M. Zanona and Liliana I. Sabinia
a
Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico-Químicas y
Naturales. Universidad Nacional de Río Cuarto. Río Cuarto, Córdoba, Argentina
b
Instituto de Virología “José María Vanella”. Facultad de Ciencias Médicas. Universidad Nacional
de Córdoba. Córdoba, Argentina

csabini@exa.unrc.edu.ar

Received: December 14th, 2010; Accepted: March 16th, 2011

Achyrocline satureioides (“marcela del campo”) is native to America. Numerous investigations have reported several bioactive properties
such as anti-inflammatory, hepatoprotective, immunomodulatory, antimicrobial and antiviral. Nowadays, few medicinal plants have been
scientifically evaluated to test its safety, efficacy and potential benefits, despite the great public interest in these herbs. The aim of this work
was to evaluate the cytotoxic and genotoxic activities of cold aqueous extract obtained from A. satureioides using Allium cepa L test. The
results demonstrated the absence of genotoxicity of the extract. Only higher concentrations induced cytotoxicity but interestingly this effect
was reversible and was not associated with mutagenicity. The contribution of this research provides assurance of safety in the application of
Achyrocline satureioides in treatment of microbial diseases and other pathologies helping to define selective toxicity.

Keywords: Achyrocline satureioides (Lam.) DC, Allium cepa L test, cytogenotoxicity, cold aqueous extract.

Asteraceae (Compositae) family includes some of the For all previously described, it is of paramount importance
oldest and most valued plants for medicinal purposes [1a]. to know the cytotoxicity and genotoxicity of extracts from
It is known that certain genus of this family contain toxic A. satureioides. In order to evaluate the cytotoxic and
compounds such as tannic, cyanide, formic and malic acid genotoxic activities of cold aqueous extract (CAE)
[1b]. Achyrocline satureioides (Lam.) DC. is a relevant obtained from Achyrocline satureioides experiments were
species that belongs to the family Asteraceae. This plant, performed using Allium cepa L test with modifications.
commonly known as “marcela del campo”, is native to
America and extends throughout the continent, as well as Table 1 summarizes the results of the effects of CAE of
Europe and Africa. In our country it is often found A. satureioides on root of Allium cepa. Taking into account
in the hills of Córdoba, San Luis and Buenos Aires the number of bulbs roots, the different concentrations of
[2]. Numerous investigations have reported several CAE induced normal development, similar to control.
bioactive properties, such as anti-inflammatory [3a],
sedative [3b], hepatoprotective [3c], antioxidant [3d,3e], The analysis of length of roots treated with CAE for 5 days
immunomodulatory and antimicrobial [3f], antitumoral showed statistically significant differences (p<0.05)
[3g], antiviral [3h,3i,3j] and photoprotective [3k]. among all treatments vs. negative control and absence of
dose-response relationship. The inhibition of roots length
Nowadays, few medicinal plants have been scientifically was ≥ 50% (p<0.05) for all tested concentrations.
evaluated to test its safety, efficacy and potential benefits, Considering the bulbs treated for 2 days (with reversion),
despite the great public interest in these herbs [4]. there were no significant differences between treatments of
Regulatory worldwide authorities require information on 0.5 and 2 mg/mL vs. negative control. By the contrary,
the genotoxic potential of new drugs as part of the safety there were significant differences for the remaining
evaluation process. Allium cepa L test allows assessment concentrations of CAE compared to negative control
of toxicity of substances in terms of macroscopic (Figure 1). The average root length for treatment of 2 days
parameters such as growth and form of roots and, with CAE (with reversion) was always higher than of 5
evaluation of genotoxicity from microscopic parameters days, although there was no statistically significant
such as types and frequencies of chromosomal aberrations difference within the same concentration considered. The
and abnormal cell divisions. concentration of 0.5 mg/mL was the exception because it
996 Natural Product Communications Vol. 6 (7) 2011 Sabini et al.

Table 1: Macroscopic parameters analyzed in roots of Allium cepa L after treatment with different concentrations of CAE of A. satureioides for 5 and 2 days.
TREATMENTS
(n = 4 bulbs)
PARAMETER
CAE (mg/ml) for 5 days CAE (mg/ml) for 2 days
C (-) C (+) C (-) C (+)
0.5 1 2 3 4 0.5 1 2 3 4
Mean root number 54 37 67 41 46.5 60.5 56 48.5 34.5 43.5 62.5 39 38 39.5
Mean root length 20.41 13.97 10.03 7.78 8.42 7.51 6.61 25.91 17.55 18.98 11.51 13.07 7.91 10.95
(mm) ± SEM ±2.00 ±0.65 ±0.62 ±0.56 ±0.72 ±0.60 ±0.45 ±2.63 ±0.94 ±1.48 ±0.64 ±1.03 ±0.66 ±0.86
Ha 8 32 11 0 2 10 3 10 20 7 5 10 6 4
Geb 0 0 0 10 14 16 25 0 0 0 15 6 15 37
Abnormalities
Nec 0 0 16 17 6 4 0 0 0 12 17 1 0 18
Tud 10 5 2 0 0 0 1 9 5 0 1 3 0 0

Ha: hook; Geb: gelling; Nec: necrosis; Tud: tumor.

2 days (with reversion) 10


30
MI 1 MI 3
5 days 8 MI 2 C+

Mitotic Index (%)


Roots length

20
6
(mm)

4
10

0
0
C- C+ 0.5 1 2 3 4 C- 0.5 1 2 3 4 C- 0.5 1 2 3 4 C- 0.5 1 2 3 4 C+
Concentration (mg/mL)
Treatment (mg/mL)
Figure 1: Roots length of bulbs of Allium cepa L treated with different Figure 2: Comparison of mitotic indices 1, 2 and 3 of the roots of the
concentration of CAE of A. satureioides for 5 days vs. 2 days (with Allium cepa bulbs treated with different concentrations of CAE of
reversion). Achyrocline satureioides.

showed a statistical difference (p<0.001), indicating the inhibition of cell division. For the MI 2, the trend of the
recovery of the roots by mineral water action, after curve revealed a drastic reduction for MI% at the
treatment with the extract. This result suggests that bulbs concentrations 2, 3 and 4 mg/mL. This behaviour could be
would have the ability to recover from damage induced by due to these concentrations are extremely toxic when they
this concentration of extract. are used for 5 days. The values of MI 3 (treatment with
reversion) did not show statistically significant differences
Respect to macroscopic abnormality, the study revealed between treatments, neither between these treatments and
the presence of hooks and tumours in negative control with negative control, indicating the ability of roots to recovery
low frequency. These spontaneous changes are normal and of the toxic action of CAE. The comparative analysis of
concordant with other authors [5a,5b]. Positive control, MI 1 vs. MI 3, for each concentration, showed significant
paracetamol (acetaminophen 0.3 mg/mL), showed a high difference (p<0.5), indicating that the damage was
incidence of root hooks and also induced the appearance of reversed (Figure 2).
tumours. None of the roots treated with CAE induced the
development of hooks. The results of all treatments with Analysis of phases index for bulbs treated for 2 and 5 days
the extract were markedly different to the effect caused by showed that cell division in roots treated with 0.5 and
paracetamol. 1 mg/mL was similar to negative control. By the contrary,
cell cycle stages were modified in the bulbs treated with 2,
Gelling and necrosis were present with varying frequency, 3 and 4 mg/mL. Prophase was the most observed phase,
having the first one the highest incidence. Roots treated demonstrating an arrest of cell division at this stage.
with extract did not show significant values in the number Application of CAE at these concentrations would be
of tumours. Pigmentation was observed in most of roots affecting the formation of chromosomes and avoiding their
treated due to intense colour of extract. placement in equatorial plane of the cell.

Statistical analysis of mitotic index of bulbs treated with Statistical analysis of the results obtained in treatment with
CAE did not show significant difference between negative reversion did not show significant differences in phase
control and treatments with 0.5 and 1 mg/mL, for 2 days index of treated roots vs. negative control, indicating the
(MI 1) and 5 days (MI 2). So, these concentrations did reversion of the changes induced by CAE of A.
not exert toxicity. In contrast, there were significant satureioides. These results point out that mitosis was
differences between negative control and treatments of normally developed as it confirms by the value of MI 3,
2, 3 and 4 mg/mL. These concentrations would exert an (Figure 3).
Cytogenotoxic evaluation of the extract of Achyrocline satureioides Natural Product Communications Vol. 6 (7) 2011 997

A
A A A A
Phase index (%)

100

50

B C D E
0
C- 0,5 1 2 3 4

Treatment
F F
B

100
Phase index (%)

Figure 4: Microphotographs of meristematic cells of root tips of Allium


50 cepa. Squash preparations, stained in acetocarmine and observed in light
microscope. (A) A typical view of different size, shape and basophility of
nuclei and interphase and mitotic phases; (B) cells with sticky
chromosomes; (C) cells with delayed chromosomes; (D) cells with
0
chromosomal bridges; (E) physiological aberrations: c-mitosis; (F)
C- C+ 0,5 1 2 3 figures similar to apoptotic bodies in interphase cells.

Treatment
Experimental
C Plant material: Healthy plants of A. satureioides species
were collected manually from Villa Jorcoricó, southern
Phase index (%)

100
Córdoba hills in 2009. The plant material was identified by
Dr. Luis Del Vitto, Facultad de Farmacia y Bioquímica,
50 Universidad de San Luis, San Luis, Argentina. A voucher
specimen was deposited in the Herbarium of the
University of San Luis (Nº 6362).
0
C- 0,5 1 2 3 4 Obtention of cold aqueous extract: Dried aerial vegetal
Treatment parts (15 g) were submitted to extraction with 700 mL of
Prophase index Anaphase index
cold water at 4°C for 2 days. The mixture was filtered and
Metaphase index
lyophilized. This extract was identified as cold aqueous
Telophase index
extract (CAE).
Figure 3: Phase index of cell division in roots treated with CAE of
Achyrocline satureioides (A) MI 1, (B) MI 2, and (C) MI 3. Determination of genotoxic activity of cold aqueous
extract from A. satureioides by the Allium cepa test:
Microscopic evaluation of cells showed physiological and Allium test as described [7a,7b,7c] was developed with
clastogenic aberrations as previously described [6]. The some modifications. Qualitative and quantitative changes,
physiological aberrations observed were c-mitosis and, macro and microscopic, induced by treatment with CAE in
sticky and delayed chromosomes, while the other ones plant cells were assessed.
presented chromosomal bridges. These alterations were
found more often in roots with normal cell division. Onion root tips of Allium cepa L grown in mineral water,
Similar figures to apoptotic bodies were observed in inter- in darkness, with aeration and constant temperature of 25 ±
phase cells treated for 2 and 5 days with CAE, (Figure 4). 0.5 °C were employed. CAE was assayed at 0.5, 1, 2, 3
and 4 mg/mL of mineral water. Positive (paracetamol 0.3
This study demonstrated the absence of genotoxicity of mg/mL) and negative (mineral water) controls were
CAE from A. satureioides. High concentrations of the included in the system. Extract concentrations were
extract induced citotoxicity but this effect was reversible applied for different times: 2 and 5 days, and 2 days
and was not associated with mutagenicity. The followed by 3 days with water (reversion). At the end of
contribution of this research provides assurance of safety each treatment, 2-3 root tips from these bulbs were cut and
in the application of Achyrocline satureioides in treatment fixed in a mixture of absolute alcohol:glacial acetic acid
of microbial diseases and other pathologies helping to (3:1, v/v). These roots were hydrolyzed in 1N HCL for 5
define selective toxicity. Nowadays, studies referred to minutes after which they were washed in distilled water.
chemical characterization of CAE from A. satureioides are Two root tips were then squashed on each slide, stained
in progress. with acetocarmine for 10 min and cover slips carefully
998 Natural Product Communications Vol. 6 (7) 2011 Sabini et al.

lowered on to exclude air bubble. The cover slips were Previously, macroscopic changes of the roots in terms of
sealed on the slides with clear fingernail polish as root number, length and presence of abnormalities, were
suggested by [8]. These roots were reserved for evaluating evaluated.
the cytogenetic abnormalities (microscopic). Six slides
were prepared for each concentration and controls (at 1000 Statistical significance was determined by analysis of
cells per slide) were analyzed at ×1000 magnification for variance (ANOVA) using GraphPad Prism 5.0 software.
induction of chromosomal aberration. The mitotic index
was calculated as the ratio between the number of cells in Acknowledgments - The authors thank to CONICET,
division and 1000 observed cells. In addition, phases MinCyT of Córdoba, Universidad Nacional de Río Cuarto
indices were also calculated [7a,7c]. The percentage and the PICTOR programme, BID 1728 /OC-AR, for
frequency of aberrant cells was calculated based on the providing financial support. The authors are also grateful
number of aberrant cells per total cells scored at each to Dr Luis Del Vitto for taxonomic classification of the
concentration of the extract, [5b,7c,9]. plant specimen.

References
[1] (a) Paulsen E. (2002) Contact sensitization from Compositae containing herbal remedies and cosmetics. Contact Dermatitis, 47,
189–198; (b) Duke JA. (2000) Toxins: their toxicity and distribution in plant genera. In: Handbook of medicinal herbs, pp.
525-568.
[2] Instituto Nacional de Investigación Agropecuaria (INIA). (2004) Estudios en domesticación y cultivos de especies medicinales y
aromáticas nativas. Estación experimental Las Brujas. Ruta 48 km 10, Rincón del Colorado, Canelones, Uruguay.
[3] (a) De Souza K, Bassani VL, Schapoval E. (2007) Influence of excipients and technological process on anti-inflammatory activity
of quercetin and Achyrocline satureioides (Lam.) D.C. extracts by oral route. Phytomedicine, 14, 102-108; (b) Hnatyszyn O,
Moscatelli V, Rondina R, Costa M, Arranz C, Balaszczuk A, Coussio J, Ferraro G. (2004) Flavonoids from Achyrocline
satureioides with relaxant effects on the smooth muscle of Guinea pig corpus cavernosum. Phytomedicine, 11, 366-369; (c)
Kadarian C, Broussalis AM, Miño J, Lopez P, Gorzalczany S, Ferraro G, Acevedo C. (2002) Hepatoprotective activity of
Achyrocline satureioides (LAM). Pharmacology Research, 45, 57-61; (d) Arredondo M, Blasina F, Echeverry C, Morquio A,
Ferreira M, Abin-Carriquiry J, Lafon L, Dajas F. (2004) Cytoprotection by Achyrocline satureioides (Lam.) D. C. and some of its
main flavonoids against oxidative stress. Journal of Ethnopharmacology, 91, 13-20; (e) Polydoro M, Souza KC, Andrades ME, Da
Silva EG., Bonatto F, Heydrich J, Dal-Pizzol F, Shapoval EE, Bassani VL, Moreir JC. (2004) Antioxidant, a pro-oxidant and
cytotoxic effects of Achyrocline satureioides extracts. Life Sciences, 74, 2815–2826; (f) Calvo D, Cariddi N, Grosso M, Demo M,
aldonado A. (2006) Achyrocline satureioides (LAM.) DC (Marcela): Actividad antimicrobiana sobre Staphylococcus spp. y efectos
inmunomoduladores sobre linfocitos humanos. Revista Latinoamericana de Microbiología, 48, 247-255; (g) Ruffa MJ, Ferraro G.,
Wagner ML, Calcagno ML, Campos RH, Cavallaro L. (2002) Cytotoxic effect of argentine medicinal plant extracts on human
hepatocellular carcinoma cell line. Journal of Ethnopharmacology, 79, 335-339; (h) Zanon SM, Ceriatti FS, Rovera M, Sabini LI,
Ramos BA. (1999) Search for antiviral activity of certain medicinal plants from Córdoba, Argentina. Revista Latinoamericana de
Microbiología, 41, 59-62; (i) Bettega JMR, Teixeira H, Bassani VL, Barardi CRM, Simões CM. (2004) Evaluation of the
antiherpetic activity of standardized extracts of Achyrocline satureioides. Phytotherapy Research, 18, 819-23; (j) Sabini MC,
Escobar FM, Tonn CE, Zanon SM, Contigiani MS, Sabini LI. (2010) Evaluation of antiviral activity of aqueous extracts from
Achyrocline satureioides against Western equine encephalitis virus. Natural Product Research, DOI:
10.1080/14786419.2010.490216; (k) Morquio A, Rivera-Megret F, Dajas F. (2005) Photoprotection by topical application of
Achyrocline satureioides (‘Marcela’). Phytotherapy Research, 19, 486-90.
[4] Calixto JB. (2000) Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic
agents). Brazilian Journal of Medical and Biological Research, 33, 179-189.
[5] (a) Fiskesjö G. (1994) Allium test II: Assessment of a chemical genotoxic potential by recording aberrations in chromosomes and
cells divisions in root tips of Allium cepa. Environmental Toxicology and Water Quality, 9, 235-241; (b) Bidau CJ, Amat AG., Yajia
M, Martí D, Riglos GA, Silvestroni A. (2004) Evaluation of the genotoxicity of aqueous extracts of Ilex paraguariensis St. Hil.
(Aquifoliaceae) using Allium Test. Cytologia, 69, 109-117.
[6] Rani G, Kaur K, Wadhwa R, Kaul SC, Nagpal A. (2005) Evaluation of the anti-genotoxicity of leaf extract of Ashwagandha. Food
and Chemical Toxicology, 43, 95-98.
[7] (a) Fiskesjö G. (1985) The Allium test as a standard in environmental monitoring. Hereditas, 102, 99-112; (b) Fiskesjö G, Levan A.
(1993) Evaluation of the first ten MEIC chemicals in the Allium test. ATLA, 21, 139-149; (c) Fiskesjö G. (1997) Allium test for
screening chemicals; evaluation of cytological parameters. Plants for Environmental Studies, CRC, Lewis Publishers, New York,
pp. 307-333.
[8] Grant WF, (1982) Chromosome aberrations assays in allium report of the USEPA gene tox program. Mutation Research, 99,
273-291.
[9] Bakare AA, Mosuro AA, Osibanjo O. (2000) Effect of simulated leachate on chromosomes and mitosis in roots of Allium cepa (L).
Journal of Environmental Biology, 21, 263-271.
2011
NPC Natural Product Communications Vol. 6
No. 7
Toxic Plants Used in Ethnoveterinary Medicine in Italy 999 - 1000

Lucia Viegi* and Roberta Vangelisti

Dipartimento di Biologia, Unità di Botanica generale e sistematica, Pisa University,Via L. Ghini 5,


56126 Pisa, Italy

lviegi@biologia.unipi.it

Received: November 10th, 2010; Accepted: March 16th, 2011

This study was conducted to document the use of toxic or potentially toxic plants for the treatment of ailments in livestock and pets in
ethnoveterinary practice in Italy. More than 250 of the entities used (81% for curative purposes) can be toxic unless dosed appropriately.
Many (55%) are dietary supplements. The list included 186 species (45%) for internal and 175 (55%) for external use, many used in places
where animals are kept. The species belong to 71 families, among which the Fabaceae predominate. The purpose of the study was to provide
information that can be validated and associated with correct determination, permitting even potentially dangerous plants to be used in
veterinary practice.

Keywords: Toxic plants, ethnoveterinary medicine, Italy.

The number of plants used in Italy to treat domestic Toxic plants belong to 71 families, 62 of which are
animals was previously reported to be 260 [1] and is now Angiosperms, one Gymnosperm, seven Pteridophytes,
more than 500 [2a-c]. These plants include fungi, ferns, one fungus (Amanitaceae). The most common families
gymnosperms and angiosperms. Most are dietary were Fabaceae, followed by Asteraceae, Ranunculaceae,
supplements, chosen for their positive effect on growth Labiatae, Euphorbiaceae, Apiaceae and Liliaceae. This
and ease of administration. Many are used for prevention, differs slightly from the general statistics for
but more than 60% of all uses are curative. Some plants ethnoveterinary medicine, which indicate species of the
are valued against parasites and as repellents, others for family Asteraceae to be the most numerous [3a,3b], as
their toxic effects on fish. Other plants are considered to found in the Mediterranean area in general [4].
have magic properties. In this study we analyze use of
toxic and potentially toxic plants in ethnoveterinary The toxic or potentially toxic species identified had largely
medicine in Italy. curative uses. The main methods of administration were as
such, decoction, crushed and macerated. The main active
Bibliographic and unpublished data in our database were ingredients were glycosides and alkaloids, with saponins
examined and screened for toxic and potentially toxic and triterpenoids accounting for more than 13%, followed
plants. Shepherds and farmers generally avoid by tannins, volatile oils, terpenoids and resins [5a-5d]. In
administering such plants, though many were used many species, the toxic substances are not distributed
traditionally and considered relatively safe. More than throughout the plant: many are concentrated in certain
250 toxic or potentially toxic plants were identified, about organs while the rest of the plant is innocuous; sometimes
50% of all species used in ethnoveterinary medicine. substances are influenced by the vegetative period or age
Most (81%) are used for curative purposes and can be of the plant. Toxic substances are often more abundant in
toxic if not appropriately dosed. A good number (55%) certain phases of the life cycle, usually in seeds and
are dietary supplements. 186 species (45%) are used juvenile plants, and in certain phases of the vegetative
internally and 175 externally (55%) (Table 1). The active cycle, usually spring. Sometimes plants can be toxic if not
ingredients, largely glycosides and alkaloids, are listed in appropriately dosed, if infected by fungi, if they
Table 2. The types of animals treated were cattle accumulate harmful substances or if combined with
(23.93%), sheep (10.73%), poultry (9.5%), horses conventional remedies [6a-6c].
(7.83%), pigs (6.38 %), goats (5%), dogs and cats
(3.19%), rabbits (2.18%) and animals in general (27%). Toxic plants were certainly used with caution in
Many plants were used in places where animals are kept, ethnoveterinary traditions, because loss of an animal was a
such as stables, chicken houses, drinking troughs (3.92%) serious event. Their use cannot be encouraged. The aim of
(e.g. Alnus glutinosa, Artemisia absinthium, Datura the present study was to provide information that can be
stramonium, Nerium oleander, Sambucus ebulus, S. validated and associated with correct determination,
nigra). permitting even potentially dangerous plants to be used in
1000 Natural Product Communications Vol. 6 (7) 2011 Viegi & Vangelisti

veterinary practice. The study is part of a series concerned pharmacological research and for the conservation of
with the enormous heritage of empirical experience and native flora. Benefits may range from local to European
knowledge still traceable in Italy. Such documentation is community level.
useful to save animal lives, for phytochemical-
Table 1: Uses and examples of toxic or potentially toxic plants used in ethnoveterinary medicine in Italy.
Internal use Vermicides Ichthyotoxic Flea, mouse and mole repellents Pesticides and repellents
Agrostemma githago Allium sativum Achillea ligustica Aconitum napellus Colchicum autumnale,
Amanita muscaria Artemisia absinthium, Anthirrinum majus Alnus glutinosa Anemone hortensis
Anemone hortensis A. vulgaris Conium maculatum Artemisia absinthium Colchicum autumnale
Crocus neapolitanus Calamintha nepeta Cyclamen repandum Calamintha nepeta Cestrum parqui
Daphne mezereum Cucurbita pepo Daphne gnidium Conium maculatum Daphne laureola
Dryopteris filix-mas Dryopteris filix-mas Euonymus europaeus Delphinium consolida, Delphinium consolida
Euphorbia dendroides, Fraxinus ornus Euphorbia characias, D. staphysagria Euphorbia helioscopia
E. lathyris Glechoma hederacea E. dendroides, E. helioscopia, Ficus carica Helichrysum italicum
Eucalyptus resinifer Juglans regia E. lathyris, E. paralias, Laburnum alpinum Veratrum album
Helleborus sp.pl. Mercurialis annua E. pinea, E. pithyusa L. anagyroides
Ilex aquifolium Polypodium australe Juglans regia Lupinus albus
Ligustrum vulgare Ruta angustifolia, Marrubium vulgare Ruscus aculeatus
Glechoma hederacea R. chalepensis Oenanthe crocata Ruta graveolens
Mercurialis annua R. graveolens Pistacia lentiscus Tanacetum vulgare
Papaver rhoeas, Santolina insularis Plumbago europaea Veratrum album
P. somniferum Sempervivum tectorum Sambucus sp.
Polypodium australe, Verbascum thapsus Solanum nigrum
P. vulgare Teucrium chamaedrys
Polystichum setiferum Thapsia garganica
Rhamnus sp.pl. Urginea maritima
Ricinus communis Verbascum pulverulentum,
Veratrum album V. sinuatum, V. thapsus

Table 2: Active principles of toxic or potentially toxic plants used in ethnoveterinary medicine in Italy.
% no. of species Notes (active compounds and the no. of species that contain them)
Glycosides 25.82 110 coumarin and furocoumarin (11); saponinic glycosides (15)
Alkaloids 19.48 83
Saponins and triterpenoids 13.38 57 saponins (non saponinic glycosides) (51)
Tannins 8.92 38
Volatile oils, terpenoids, resins 8.22 35 resins (5)
Organic acids 6.10 26 oxalates (13)
Pigments (flavonoids) 4.23 18
Diterpenoids 3.29 14
Phytoestrogens 3.05 13 phytoestrogens, phytosterols
Nitrates and nitrites 2.11 9
Lignans and lignins 2.11 9
Toxic if infected with fungus or in cases of accumulation 1.88 8
Enzymes 1.41 6 thiaminase, urease, ficin

References
[1] Viegi L, Pieroni A, Guarrera PM, Vangelisti R. (2003) A review of plants used in folk veterinary medicine in Italy as basis for a
databank. Journal of Ethnopharmacology, 89, 221-244.
[2] (a) Viegi L, Camarda I, Piras G. (2005) Some aspects of ethnoveterinary medicine in Sardinia (Italy). Proceed. IV International
Congress of Ethnobotany (ICEB 2005), 21-26 August, Istanbul, Turkey, 135-136; (b) Bullitta S, Piluzza G, Viegi L. (2007) Plant
resources used for traditional ethnoveterinary phytotherapy in Sardinia (Italy). Genetic Resources and Crop Evolution, 54,
1447-1464; (c) Felicioli A, Giusti M, Vangelisti R, Viegi L. (2008) Plants used as antiparasitic in italian ethnoveterinary medicine.
Parassitologia, 50, Suppl. 1, 210.
[3] (a) Fossati F, Bianchi A, Favali MA. (1999) Farmacopea popolare del parmense: passato e presente. Informatore Botanico Italiano,
31, 171-176; (b) Barbini S, Tarascio M, Sacchetti G, Bruni A. (1999) Studio preliminare sull'etnofarmacologia delle comunità
ladino dolomitiche. Atti Colloquio S.B.I. "Botanica Farmaceutica ed etnobotanica alle soglie del duemila: passato e futuro a
confronto", Genova, 9-11 aprile 1999. Informatore Botanico Italiano, 31, 181-182.
[4] Pieroni A, Giusti ME, de Pasquale C, Lenzarini C, Censorii E, Gonzáles-Tejero MR, Sánchez-Rojas CP, Ramiro-Gutiérrez J,
Skoula M, Johnson C, Sarpaki A, Della A, Paraskeva-Hadijchambi D, Hadjichambis A, Hmamouchi M, El-Jorhi S, El-Demerdash
M, El-Zayat M, Al-Shahaby O, Houmani Z, Scherazed M. (2006) Circum-Mediterranean cultural heritage and medicinal plant uses
in traditional animal healthcare: a field survey in eight selected areas within the RUBIA project. Journal of Ethnobiology and
Ethnomedicine, 2, 16-28.
[5] (a) Debelmas AM, Delaveau P. (1978) Guide des Plantes Dangereuses. Ed. Maloine, Paris; (b) Maugini E. (1994) Manuale di
Botanica Farmaceutica, VII edizione, Piccin, Padova; (c) Lorgue G, Lechenet J, Rivière A. (1999) Tossicologia clinica
veterinaria. C. Giraldi Ed.; (d) Frohne D, Pfänder HJ. (2004) Poisonous plants. 2nd Edition. Manson Publishing Ltd, London
[6] (a) Bruneton J. (1999) Toxic Plants Dangerous to Humans and Animals. Lavoisier Publishing, Paris; (b) Frohne D, Pfänder HJ.
(2004) Poisonous plants. 2nd Edition. Manson Publishing Ltd, London; (c) Wynn SG, Fougère BJ. (2007) Veterinary Herbal
Medicine. Mosby, Elsevier.
2011
NPC Natural Product Communications Vol. 6
No. 7
Diagnosis of Public Programs focused on Herbal Medicines 1001 - 1002
in Brazil
Ely Eduardo Saranz Camargoa, Mary Anne Medeiros Bandeirab and
Anselmo Gomes de Oliveirac
a
Programa de Pós-graduação em Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas,
Universidade Estadual Paulista Julio de Mesquita Filho-Unesp, Rodovia Araraquara-Jaú, km 01,
14801-902, Araraquara, SP. Brazil
b
Universidade Federal do Ceará, Faculdade de Farmácia, Odontologia e Enfermagem,
Rua Alexandre Baraúna, 949, Fortaleza, CE

elycamargo@ipcs.com.br

Received: November 12th, 2010; Accepted: March 25th, 2011

The present study is aimed to diagnose the current public programs focused on herbal medicines in Brazil by means of in loco visits to 10
programs selected by means of questionnaires sent to 124 municipalities that count on herbal medicine services. The main purpose of the
implementation of program programs is related to the development of medicinal herbs. 70% of them are intended for the production of herbal
medicines and 50% are aimed to ensure the access of the population to medicinal plants and or herbal medicines. The initiative of the
implementation of these programs was related to the managers (60%). The difficulties in this implementation were due to the lack of funding
(100%) of the programs. In 60% of the programs, the physicians did not adhere to herbal medicine services due to the lack of knowledge of
the subject. Training courses were proposed (80%) to increase the adhesion of prescribers to the system. Some municipalities use information
obtained from patients to assess the therapeutic efficiency of medicinal plants and herbal medicines. Of the programs underway, cultivation
of medicinal plants was observed in 90% and 78% of them adopt quality control. In most programs, this control is not performed in
accordance with the legal requirements. The programs focused on medicinal plants and herbal medicines implemented in Brazil face some
chronic problems of infrastructure, management, operational capacity and self-sustainability, which can be directly related to the absence of a
national policy on medicinal plants and herbal medicines.

Keywords: Medicinal plants, herbal medicines, public health.

The use of medicinal plants dates back thousands of years. being developed. These historical aspects of the use of
However, the traditional allopathic medicine faces medicinal plants clarify the importance of the interaction
increased competition from alternative treatments due to between local communities and their natural environment
the production of herbal medicines in accordance to the to the entire society in the present and in the future [1c]. At
standards recommended by the legislation these days [1a]. the same time, there is the urgent need to investigate the
The development of quality assurance within the abundant biodiversity and, particularly, the medicinal
pharmaceutical industry involves the concern with the flora, its proper and rational use, in order to find out how
production of seeds, planting, harvesting, drying, medicinal plants can be used by the population for
extraction, production practices and storage of drugs, and medicinal purposes in an efficient and safe way [2a].
all these processes must be carried out according to a strict
quality control, pre-clinical and clinical trials and data Data generated from the questionnaires completed has
record. Quality assurance has enabled the health demonstrated the existence of well-structured programs,
professionals to prescribe safely herbal medicines that the and the activities carried out within these programs
population has been taking for quite a long time [1b]. involved pharmaceutical care, corroborating the expansion
and effectiveness of the National Policy of Integrative and
The use of medicinal plants for therapeutic purposes (both Complementary Practices at the SUS (Brazilian Public
the traditional and popular usage of these plants and their Health System) [2b]. Thus, visits to 10 programs focused
use based on scientific evidence) is a common practice on herbal medicines were scheduled and had excellent
nowadays. The use of plants to mitigate symptoms or cure results, as demonstrated in the previously described
diseases has been a very common practice for long, research. [2c]. The visits were conducted from December
especially because the resources used in the production of 2008 to December 2009 and were aimed to collect
drugs are not available according to the needs of the information on the activities developed within the scope
population, or else, many of these drugs are currently of the programs, as well as information on their
1002 Natural Product Communications Vol. 6 (7) 2011 Camargo et al.

implementation, difficulties faced and solutions involved herbal medicine handling a pharmacist was the
encountered to ensure the maintenance of the referred technical responsible person, according to the country’s
programs. The programs selected for visit were carried out legislation. 7 out of these professionals (80%) are
in all the Brazilian regions as follows: 1 in the Southern registered with their respective regional board of pharmacy
region, 5 in the Southeastern region, 2 in the Center-West and only 2 (20%) were not registered with their regulatory
region, 1 in the Northeastern region and 1 in the Northern bodies. The pharmaceutical forms handled in the programs
region. The selection was based on the data generated from are distributed according to the number of quotations in the
the questionnaires sent to 124 Brazilian municipalities. It herbal medicine workshops: 67% of the programs deliver
was found that in all programs visited (10) the main solutions, 22% suspensions, 55% capsules, 78% dyes, 11%
purpose of their implementation was the development of elixirs, 89% syrups, 89% creams, 55% ointments and 44%
medicinal plants. In 70% of them the purpose was the liquid soaps. It has also been found that 78% of the programs
production of herbal medicines, 50% were aimed to ensure that handle herbal medicines performed quality control.
the access of the population to these medicinal drugs and
only one program (10%) was aimed to decrease the The distribution of medicinal plants and herbal medicines
healthcare costs incurred by the population. 60% of the is performed by the pharmacist in all the cases, and in
programs stemmed from the initiative of municipal some of them, it is also performed by physicians (50%),
governments, 50% were suggested by experts, 20% by nurses (50%), technicians (40%), other professionals, such
users and 30% had different origins. The same results were as dentists, biologists and nutritionists (20%). Therefore, in
obtained when the initiatives were carried out by more 80% of the total programs visited there was some kind of
than one group. Regarding the inclusion of herbal follow-up of patients taking medicinal plants or herbal
medicine in pharmaceutical care programs in the medicines, 20% did not have any kind of follow-up
municipalities, such inclusion was found to occur in 70% service. However, in some of the programs that reported
of them and in 30% of them it did not occur. These follow-up services, the evaluation was not recorded.
findings may have a direct influence on the maintenance of Regarding efficiency assessment, 80% of the programs
the programs, since funding issues can compromise the performed this assessment and 20% of them did not
survival of those programs not included in the perform any efficiency assessment.
pharmaceutical care programs of the municipalities.
80% of the programs provide training courses to the
The difficulties in the implementation of these programs professionals involved in herbal medicines and 20% of
included: lack of funding (100%), poor adhesion by them do not count on any kind of training courses or
prescribers (60%), lack of space (70%) lack of qualified activities. However, it was found that all the programs
professionals (40%) and 1 (10%) mentioned the lack of visited had educational activities targeted at the local
interest of the population. Still regarding the difficulties community. One issue that has been greatly discussed
encountered, it was found that 90% of the programs were with the coordinators of the programs visited was the
funded by the municipality and 40% were funded by the partnership with universities or other institutions, both
state, but only 1 (10%) was exclusively funded by the public and private, Only 3 (30%) of them have established
state. Regarding the cultivation of medicinal plants, only 1 partnerships, mostly with universities, and 70% have no
(10%) did not count on a garden and raw material for kind of partnership. Nevertheless, most programs had tried
herbal medicines was obtained from suppliers. The unsuccessfully to establish partnerships, and the main
selection of the medicinal species cultivated was based on reason for this was the lack of funding.
literature research (100%), an in 80% of them it was also
based on popular knowledge and in 40% the local species Finally, the results obtained in this study conclude that
were used. Only 1 (10%) program did not count on a herbal medicine is getting considerable attention and has
herbal medicine workshop, being focused on the become a valuable asset that ensures the access of the
distribution of medicinal plants. In all the programs that population to basic health care.

References

[1] (a) Brasil. (2010) Ministério da Saúde. Agencia Nacional de Vigilância Sanitária. Resolução de Diretoria Colegiada (RDC) no. 10,
de 09 de março de 2010. Dispõe sobre a Notificação de Drogas Vegetais Junto a Agencia Nacional de Vigilância Sanitária. Diário
Oficial de União, Brasília; (b) Goodman LS, Gilman AG, Hardman JG. (2003) As Bases Farmacológicas Da Terapêutica. 10a ed.
Rio de Janeiro: Mc GrawHill, 2003, 1436 p; (c) Brasil. (2006) Ministério da Saúde. Secretaria de Ciência, Tecnologia e Insumos
Estratégicos. Departamento de Assistência Farmacêutica. A fitoterapia no SUS e o programa de pesquisa de plantas medicinais da
central de medicamentos/ Ministério da Saúde, Secretaria de Ciência, Tecnologia e Insumos Estratégicos, Departamento de
Assistência Farmacêutica. – Brasília: Ministério da Saúde. 148p. – “serie B. textos básicos de saúde”.
[2] (a) Alzugaray D. (1988) Enciclopédia da plantas medicinais, Editora três Ltda São Paulo; (b) Brasil. (2006) Ministério da Saúde.
Secretaria de Atenção a Saúde. Departamento de Atenção Básica. Política nacional de práticas integrativas e complementares no
SUS – PNPIC-SUS/Ministério da Saúde, Secretaria de Atenção a Saúde, Departamento de Atenção Básica. – Brasília: Ministério
da Saúde, 92 p. – “serie B. texto básico de saúde”; (c) Camargo EES. (2010) Avaliação dos programas de plantas medicinais e
medicamentos fitoterápicos, visando subsidiar sua reorientação no sistema único de saúde. Tese (Doutorado em Ciências
Farmacêuticas) Universidade Estadual Paulista. Araraquara – SP, 230p
2011
NPC Natural Product Communications Vol. 6
No. 7
Identification of Thiosildenafil in a Health Supplement 1003 - 1004

Marcello Nicoletti

Dep. Enviromental Ecology, University Sapienza, Rome, Italy

marcello.nicoletti@uniroma1.it

Received: December 11th, 2010; Accepted: March 16th, 2011

The presence of a sildenafil derivative, the thiosildenafil, in an herbal product has been evidenced first by HPTLC and later determined by
isolation and analysis of spectroscopic data. The analyzed product is nowadays marketed as dietary supplement containing herbal extracts
and claimed for male and female sexual improvement. This report is noteworthy since it is clear that adulterated materials can cause serious
health problems if they are consumed as herbal “natural” products, generally considered deprived of toxicity by the consumers. The use of a
simple and reliable method, based on HPTLC, to determine synthetic adulterations is reported in this paper.

Keywords: thiosildenafil, sildenafil, dietary supplement, HPTLC, NMR.

The presence of synthetic drugs in the formulation of


R
10

herbal products in order to improve the efficacy has been HN 6 5


4
N

N
reported in several cases. The case seems to be very 24
O2
S
7
8 9
1
N
16 15 N
important in adulteration of herbal products marked as N
25
28 17
18
14
19 20
21
11 12

27
“natural” in cases of erectile dysfunction, since an abuse 29
O 13

can be very dangerous for patients who unwittingly R = O sildenafil


R = S thiosildenafil
consume a synthetic potent drug instead of a botanical.
prescription drugs and must be used under medical
In the recent years, in particular, several cases have been supervision, herbal products are self administered and
reported about such herbal products produced in China generally regarded as being harmless because of their
[1-3]. The reported monitoring aspects were essentially natural origin. Confusion between the two categories is
based on the analytical analyses concerning the detection therefore very dangerous.
of the adulterants that in these cases are synthetic selective
inhibitors of cyclic guanosine monophosphodiesterase-5 Recently, a survey of the analysis of the presence of
(PDE-5). Such active principles are the well known synthetic PDE-5 inhibitors in dietary supplements has been
registered drugs, but also a plethora of analogues obtained reported [3]. Among the seventeen considered commercial
by minor modifications to the basic structure of PDE-5 formulations of herbal drugs or dietary supplements
inhibitors was found. The reason of this proliferation is marketed for sexual dysfunction, eight resulted
mainly due to the intention to escape analytical controls. adulterated, containing sildenafil, tadalafil, vardenafil,
Therefore, several efforts were focused on the best hydroxyhomosildenafil and/or thiomethisosildenafil.
analytical tool to catch the adulterant, since first HPLC We were able to examine the content of an herbal
determination [4], followed by NMR [5], LC/MS and supplement heavily marketed in internet sites as Sensual
LC/MS/MS [6], until the recent 2D and 3D DOSY 1H Tea or Jinshenkang, commercialized as able to rapidly
NMR [7]. In this paper we report a further case of solve any sexual problem of females and males. The
adulteration and propose the use of HPTLC as simple, product, also marketed in Italy, came from Spain, where
direct and low cost method to detect such and other now the product has been removed from the market. First,
adulterants, also in case of their presence in complex a HPTLC in dichloromethane: methanol (9:1, v/v) in a
herbal mixtures. horizontal chamber (Camag 20X10) after saturation with
the same mobile phase was performed. The plate showed a
Whereas sildenafil citrate (Viagra®, manufactured by great spot very strong at UV lamp at 250 nm; its position
Pfizer), vardenafil hydrochloride (manufactured by and its intensity, in comparison with the other spots due to
Levitra) and tadalafil (Cialis®, manufactured by Lilly) are components of the herbal extracts, were an evident clue of
well known compounds approved by the U.S. Food and the adulteration. Direct HPTLC comparison with
Drug Administration for the treatment of erectile sildenafil, verdenafil and tadalafil excluded the identity
dysfunction, the analogues usually are not subjected to with these substances. Also densitometry analysis
any control. In any case whereas registered products are confirmed the differences.
1004 Natural Product Communications Vol. 6 (7) 2011 Nicoletti

Easily, extraction with ethyl acetate afforded a complete in CDCl3 using CHCl3 signal (7.23 and 77.0 ppm) as
remove of the substance from the product and NMR internal reference. MS by hyphenated LC/MS LXQ
spectrum of the extract resulted in a highly pure compound Thermo Electron.
identified as thiosildenafil, by comparison with reported
data and analysis of 2D spectra. In particular, making a Samples and extraction: The analyzed samples were
1
H-NMR comparison with sildenafil [3], it is diagnostic imported from Spain and sold as a dietary supplement in
the upfield shift (δH = + 0.12) of the N-Me, due the package containing white granules. The reported content
influence of the thiocarbonyl. The LC-MS analysis of the granules was mainly sugar and a series of herbal
confirmed the presence of thiosildenafil, as well as a little extracts. Sildenafil, tadalafil and vardenafil were obtained
quantity of sildenafil, probably as remaining product from corresponding marketed products.
of the conversion into the thioderivative. Quantitative
determination was obtained by the NMR method proposed HPTLC analysis: The granules of a package (40 g) were
by Balyssac [1] and gave for each package a quantity equal grounded and extracted with ethyl acetate (50 mL) for 4 h.
to that of a tablet of Viagra. Independently, HPLC analysis After filtration over 0.45 m filter, the filtrate was
was performed confirming the non identity with previous evaporated and dissolved in 50 mL of ethyl acetate/H2O
compounds and affording similar quantitative results. 1:1 (v/v). The content of the organic phase was directly
used for analysis, including HPTLC using sildenafil and
Herbal products spiked with synthetic drugs are dangerous vardenafil as reference standards. At UV lamp at 350 nm
to consumers and noxious for future correct developing of the Rf value of the unknown constituent (0.81) resulted
use of natural products. Controls must be based on simple, significantly higher than those of the two reference
viable and low cost analyses. Therefore, HPTLC is a compounds (0.79 and 0.74 for sildenafil and vardenafil,
strong candidate to be used in the detection of anomalous respectively). The layers, treated with H2SO4 2N spray
constituents in botanicals to obtain easy clues of the reagent and subsequent worming at 110°C, showed other
adulteration. minor spots at lower RF, probably due to the natural
products. For complete identification and improve the
Experimental quality of spectroscopic analyses, pure thiosildenafil was
HPTLC in silica gel 60 in dichloromethane: methanol (9:1, obtained after silica gel CC in CHCl3:MeOH 40:1 of the
v/v) developed in a horizontal chamber (Camag 20X10). above organic phase.
Deposition with CAMAG Linomat IV, TLC scanner 3
WINCATS software. For the densitometric analysis a Thiosildenafil: The ethyl acetate extract was evaporated
Camag TLC scanner 3 linked to winCATS software was and the residue directly examined by NMR. 1H NMR (400
used after multi-wavelength scanning between 250 and MHz, CDCl3) and ITMS data were in accordance with
400 nm. NMR by BRUKER AM400 at 400 MHz for 1H reported ones [7].
NMR and 100 MHz for 13C NMR. Spectra were recorded

References

[1] 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. Journal of
Pharmaceutical and Biomedical Analysis, 50, 602-612.
[2] Blok-Tip L, Zomer B, Bakker F, Hartog KD, Hamzink M, ten Hove J, Vredenbregt M, de Kaste D. (2004) Structure elucidation of
sildenafil analogues in herbal products. Food Additives and Contamination, 21, 737-748.
[3] Singh S, Prasad B, Savaliya AA, Shah RP, Gohil VM, Kaur A (2009) Strategies for characterizing sildenafil, vardenafil, tadalafil
and their analogues in herbal dietary supplements, and detecting counterfeit products containing these drugs. Trends in Analytical
Chemistry, 28, 13-26.
[4] Daraghmeh N, Al-Omari M, Badwan AA, Jaber AMY. (2001) Determination of sildenafil citrate and related substances in the
commercial products and tablet dosage form using HPLC. Journal of Pharmaceutical and Biomedical Analysis, 25, 483-491.
[5] Wawer I, Pisklak M, Chilmonczyk Z. (2005) 1H, 13C, 15N NMR analysis of sildenafil base and citrate (Viagra) in solution, solid
state and pharmaceutical dosage forms. Journal of Pharmaceutical and Biomedical Analysis, 38, 865-870.
[6] Park HJ, Jeong HK, Chang MI, IM MH, Jeong JY, Choi DM, Park K, Hong MK, Youm J, Han SB, Kim DJ, Park JH, Kwon SW.
(2007) Structure determination of new analogues of verdenafil and sildenafil in dietary supplements. Food Additives and
Contaminations, 24, 122-129.
[7] Trefi S, Gilard V, Balayssac S, Malet-Martino M, Martino R. (2009) The usefulness of 2D DOSY and 3D DOSY-COSY 1H NMR
for mixture analysis: application to genuine and fake formulations of sildenafil (Viagra). Magnetic Resonance in Chemistry, 47,
S163-173.
2011
NPC Natural Product Communications Vol. 6
No. 7
Hypolipidemic Effect of Seed Oil of Noni (Morinda citrifolia) 1005 - 1008

Diana C. Pazosa, Fabiola E. Jiménezb, Leticia Garduñoa, V. Eric Lópezb and M. Carmen Cruzb,*
a
Departamento de Farmacia, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional,
CP 11340, México DF, México
b
Centro de Investigación en Biotecnología Aplicada, Instituto Politécnico Nacional, CP 90700,
Tepetitla, Tlaxcala, México

dianadelcpg@yahoo.com, ccruz25@hotmail.com

Received: November 14th, 2010; Accepted: March 16th, 2011

Morinda citrifolia, has been reported to posses different biological activities and almost all parts of this have been studied phytochemically.
However there are few studies on the seeds of fruit. The objective of present study was investigated the effect to Noni Seed Oil (NSO) on
serum lipid levels in normolipidemic and hyperlipidemic induced mice. We find that administration of noni oil causes a reduction in total
cholesterol and triglycerides levels in both models. However hypolipidemic effect is higher when hyperlipidemia is presented.

Keywords: Morinda citrifolia seed oil, Atherogenic Index, Linolenic acid, hypolipidemic effect, tyloxapol.

Morinda citrifolia L. (Rubiaceae) commonly known as Hyperlipidemic is defined as elevated lipid levels in
noni, is an evergreen tree may reach heights of 3 to 8 m plasma, and represent one of the factors associated with
tall. Its leaves range from 10 to 45 cm long and it bears cardiovascular diseases, which are a worldwide death
tubular white flowers and a green fruit. This fruit turns cause [11-13]. Treatment of dyslipidemia reduces
yellow and then white as it ripens, has a pungent odor and cardiovascular events. The modern pharmacological
contains seeds of about 3 mm in length. This plant is therapy for abnormal lipids is effective but is expensive
native to Asia, Australia and Polynesia [1]. The plant is and it is associated with side-effects leading to patient
used in the treatment of arthritis, headaches, digestive incompliance. For this reason, our work evaluates the
problems, diabetes mellitus, high blood pressure, and effect of NSO on lipid levels (total cholesterol (chol),
angina pectoris among others [2]. The Leaves, steam, root, triglyceride (Tg), high density lipoprotein (chol-HDL), in
fruit and seeds of noni are used in various forms such as normolipidemic and hyperlipidemic mice. Castelli’s
capsules, teas, juice and oil [2,3]. Atherogenic Index (AI) was calculated for determined risk
factor of cardiovascular disease with noni seed oil
Due to the great popularity of this plant many consumed.
phytochemical studies have been carried out in which have
reported compounds as iridoids, anthraquinones phenolics, Table 1: Gas chromatographic retention times (Rt) and molecular weights
of fatty methyl esters from NSO.
glycosides of fatty acids and alcohols, cumarins,
flavonoids, alkaloids and terpenes [4]. Regarding the Compound Rt (min) M+ (amu) %
biological activity of this specie, the antimicrobial effect Methyl palmitate 38.5 270 9.4
Methyl palmitoleate 39.2 268 0.7
was the first observed property [5], however other effects Methyl stearate 42.5 298 4.2
like antitubercular [6], hypoglycemic [7], anti- Methyl oleate 43.1 296 15.9
inflammatory [8], antitumor [9] and analgesic [10] have Methyl linoleate 44.2 294 67.8
been reported.
Analysis of NSO
Among these one evaluated the toxicity and nutritional The total yield of oil for two extractions of dried seeds was
value, as well as the determination of the fatty acid 12%. GC-MS analysis of the FAME (fatty acid methyl
composition to assess if it is usable as edible vegetable oil. esters) prepared by transesterification procedure indicated
Seeds constitute 2.5% of the whole fruit and are the presence of five fatty acids with the relative
considered a waste in the industrial process for making composition shown in Table 1. The FAME showed mass
juice [3]. Although anthraquinones and fatty acids such as spectra with molecular ions at m/z 270, 268, 298, 296 and
arachidonic and palmitoleic have been isolated from these 294, corresponding to the retention times of 38.5, 39.2,
seeds, it has not been established whether they show any 42.5, 43.1, 44.2 min. These times indicated the presence of
biological activity [1]. palmitic (C16), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1)
1006 Natural Product Communications Vol. 6 (7) 2011 Pazos et al.

Table 2: Hypolipidemic effect produced by NSO in normolipidemic mice.


Doses
Total-Chol (%) Tg (%) chol-HDL (%)
(mg/kg/day)
CONTROL 100±2.1 100±0.18 100±1.58
NSO 150 4.16±1.07 11.26±0.17 -13.91±1.49
NSO 300 -11.01±1.73 -12.2±0.22 -10.36±2.11
NSO 600 -19.09±2.69 -33.7±0.19 -22.42±3.2

Table 3: Hypolipidemic effect produced by NSO in hyperlipidemic mice


male.
Doses
Total-chol (%) Tg (%) chol-HDL (%)
(mg/kg/day)
CONTROL 100±6.6 100±1.08 100±0.95
NSO 150 -22.13±1.72 -74.2±0.09* 33.54±1.06
NSO 300 5.73±7.16 -64.97±0.31 39.54±1.49
NSO 600 -15.57±1.8 -31.79±0.24 42.85±1.29
* Represents significant difference when compared with the control when Figure 1: Castelli’s Atherogenic index for normolipidemic and
analyzed by ANOVA ± standard error. hyperlipidemic mice treated with different doses of NSO.

and linoleic (C18:2) fatty acids respectively, being the inhibition of cholesterol biosynthesis by inhibition of
principal component linoleic acid. However further studies HMG Co-A. However, the failure of NSO to cause
are necessary to determine the exact chemical complete inhibition indicates the involvement of additional
composition. mechanisms.

Biological assay: Data on the effect on lipid-lowering Various indices have been used for the diagnosis and
activity of the three parameters evaluated are summarized prognosis of cardiovascular disease, one of them was
in Tables 2 and 3. All animals showed good health during reported by Castelli [11]. Castelli’s Atherogenic index (AI)
the period of administration. At necropsy no changes were is total cholesterol/HDL ratio and it is considered that a
found in the organs of the treated animals. value below than four units represents a low risk of
cardiovascular disease [11-13]. AIs were calculated for
Animals administered with 600 mg/Kg (Table 2) registered the different groups, being that it is smaller for
the most important effect with a reduction of -19.09% and normolipidemic group when administered NSO (Figure 1).
-33.7% for total cholesterol and triglycerides respectively. While for the hyperlipidemic group a slight decrease
These results suggest a hypolipidemic activity of seed oil regarding the control is observed only at dose of 300
and a relationship between dose and effect. mg/Kg. However the values for this group stay below the
one it limits.
Treatment of ICR mice with Triton WR 1339 (tyloxapol)
resulted in a significant elevation in total serum We find that the administration of noni oil causes a
cholesterol, HDL-cholesterol and triglycerides respect to hypolipidemic effect, mainly evident when hyperlipidemia
control. occurs. The results from this study rationalize the
medicinal use of noni seed oil in dislipidemia. However
Tyloxapol is a non-ionic surfactant being widely used to further studies are required to prove efficacy of noni seed
explore possible mechanism of lipid lowering drugs, it oil in dyslipidemia and to prove that it can be used as a
causes drastic increase in serum triglycerides and potential medicine for cardiovascular diseases.
cholesterol levels due to increase in hepatic cholesterol
synthesis particularly by the increase in HMG Co-A The main constituent of noni seed oil is linoleic acid,
(3-hydroxy-3-methyl-glutaryl Co-A) activity and by the however hypolipidemic activity may be due to the
inhibition of lipoprotein lipase responsible for hydrolysis presence of other compounds in oil.
of plasma lipids [14].
Experimental
The Table 3 shows data about the use of NSO in Extraction seed oil (NSO): The noni fruit was obtained
hyperlipidemic mice. The lowest reduction values for from Córdoba Veracruz, México. The seeds were obtained
cholesterol (-22.15%) and triglycerides (-74.2%) were from fresh fruits. The batch of seeds were washed and
obtained with dose of 150 mg/kg, exhibiting the best dried at room temperature. Dried seeds were ground and
hypolipidemic effect. Not relationship dose-effect in extracted by maceration with food-grade hexane at 1:5
cholesterol was observed. Nevertheless for triglycerides is ratio at room temperature for 24 h. The extract was filtered
notorious inverse relationship dose-effect. and the solvent removed by distillation under reduced
pressure. The crude oil was used in the bioassay.
Significant inhibition of lipid levels increase by noni seed
oil of Morinda citrifolia in this model is indicative of the
Hypolipidemic effect of Morinda citrifolia seed oil Natural Product Communications Vol. 6 (7) 2011 1007

Oil analysis: Fatty acid composition was determined for administration of Triton WR 1339 (Tyloxapol) was
gas chromatography (GC) of methyl ester obtained after dissolved in water at 400 mg/Kg. The seed oil was
transesterification of the crude oil [15] on a Hewlett- administered 1h before and 22 and 48 h after the tyloxapol
Packard 5890 Series II Gas Chromatograph equipped with injection [16].
a 5971A Mass Selective detector and using an HP-
Innowax capillary column (30 m x 0.2 mm x 0.25 μm thick Mice were treated with the oil seed suspended in a
coating). Helium was used as the carrier gas at a flow rate 1:4.5:4.5 tween 80: mineral oil: saline solution and
of 1 mL/min with the injector set to split mode at 250ºC. administered orally by an incubation needle at doses of
The oven was programmed from 100 to 140°C at 150, 300 or 600 mg/Kg/day for 28 days. Animals receiving
1.5°C/min and then from 140 to 250°C at 5°C/min and the vehicle were used as the non-cholesterol control group.
held at 250ºC for 10 min. The detector was operated at
70 eV in scanning mode over the range of 50–550 amu. For tyloxapol-treated mice blood samples were taken 48 h
Mass spectra were compared with data in NIS library and after injection. On the other hand, animals receiving
% of fatty acid was calculated for integration value for treatment for 28 days were fasted for 12 h before sacrifice.
chromatogram. Blood samples were collected by periorbital plexus
bleeding and centrifuged at 3000 rpm for 15 min. Total
Hypolipidemic Evaluation: Hypolipidemic activity was cholesterol (Col), high-density lipoprotein cholesterol (col-
studied in (ICR) male mice weighing 25-30g (Birmex, S. HDL), and triglycerides (Tg) levels were determined in the
A., Mexico City). All animals were housed in hanging serum, using a Wiener lab, Selectra 2000 automatic
metal cages and maintained at 24±2 ºC and 50±10% analyzer.
relative humidity, with 12 h light/dark cycle. They were
fed on standard pellet diets (Rodent Diet 5001, PMI All data are expressed as the percentage of the cholesterol
Nutrition International, Inc. Brenwood, MO) and drinking group control (the mean± standard error) by using Student
water was freely available. All animals appeared healthy 9t test. P values less than 0.05 considered statistically
throughout the dosing period, maintaining normal food significant.
intake and weight gain. At sacrifice, no gross
abnormalities were observed in any treated mice. Castelli Atherogenic index (AI) were calculated using the
equation IA= Total cholesterol/HDL-chol [11].
All animals were treated in accordance with ethical
principles and regulations specified by the Animal Care Acknowledgments – We thank SIP/IPN by financial
and Use Committee of our institution and the standards of support (20090806). D.C-P is grateful to PIFI-IPN for the
the National Institutes of Health of Mexico. scholarships awarded. M.C-C, L-C., and V.E.-L are
fellows of the EDI/IPN and COFAA/IPN programs.
The mice were randomly divided into groups of six
animals. Hyperlipidemia was induced in the mice by

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[15] Halket J. (1993) Derivatives for Gas Chromatography–Mass Spectrometry. In Handbook of Derivatives for Chromatography, Blau
K. and Halket J. (Eds.). John Wiley, Chichester, 317.
[16] Silva RM, Santos FA, Maciel MA, Pinto AC, Rao VSN. (2001) Effect of trans-dehydrocrotonin, a 19-nor-clerodane diterpene from
Croton cajucara on experimental hypertrigliceridemia and hypercholesterolaemia induced by Triton WR 1339 (Tyloxapol) in
Mice. Planta Medica, 67, 763-765.
2011
NPC Natural Product Communications Vol. 6
No. 7
Composition of Egyptian Nerolì Oil 1009 - 1014

Ivana Bonaccorsia*, Danilo Sciarronea, Luisa Schipillitia, Alessandra Trozzib,


Hussein A. Fakhryc and Giovanni Dugoa
a
Dipartimento Farmaco-chimico, Università di Messina, V.le Annunziata, 98168 Messina, Italy
b
Dipartimento Farmaco-biologico, Università di Messina, V.le Annunziata, 98168 Messina, Italy
c
A. Fakhry & Co. 1081 Cornich El-Nil Cairo 11451, Egypt

bonaccor@pharma.unime.it

Received: November 10th, 2010; Accepted: March 3rd, 2011

The bitter orange flower oil (or nerolì) is an essential product, largely used in perfumery. Nerolì is obtained by hydrodistillation or steam
distillation, from the flowers of bitter orange (Citrus aurantium L.). Since a long time nerolì production is limited and its cost on the market
is considerably high. The annual production in Tunisia and Morocco is ca. 1500 Kg, representing more than 90% of the worldwide
production. A small amount of nerolì is also produced in Egypt, Spain and Comorros (not exceeding 150 kg totally). Due to the high cost, the
producers and the users have tried to obtain less expensive products, with odor characters close to that of nerolì oil to be used as substitute
and sometimes as adulterants of the genuine oil. In this study are investigated five samples of Egyptian nerolì oils produced in 2008 and
2009, in the same industrial plant, declared genuine by the producer. For all the samples the composition was determined by GC/FID and by
GC/MS-LRI; the samples were also analyzed by esGC to determine the enantiomeric distribution of twelve volatiles and by GC-C-IRMS for
the determination of the 13CVPDB values of some mono and sesquiterpene hydrocarbons, alcohols and esters. The analytical procedures
allowed to quantitatively determining 86 components. In particular the variation of the composition seems to be dependent on the period of
production. In fact, the amount of linalool decreases from March to April while linalyl acetate presents an opposite trend, increasing in the
same period. The RSD determined for the 13CVPDB are very small (max. 3.89%), ensuring the authenticity of all samples. The results are
also discussed in function of the limits provided by the European Pharmacopoeia (EP) (2004), AFNOR (1995) and ISO (2002) regulations for
genuine nerolì oils.

Keywords: Nerolì oil, Citrus aurantium L., GC, GC/MS-LRI, GC-C-IRMS, es-GC.

The oil of nerolì is obtained by hydrodistillation or The annual world production of nerolì is today less than
by steam distillation of the flowers of bitter orange 2000 Kg; most of it is concentrated in Morocco and in
(C. aurantium L.). Tunisia. Small amounts of nerolì, about 150 Kg/year, are
produced in Egypt, Spain, and Comorros. The market
Nerolì, rose and jasmine are often cited as “the three pearls price of nerolì is considerably high and the organic product
of perfumery”. Nerolì is the classic ingredient of the most can be sold at more than 4,500 USD/Kg. It is therefore
famous and prestigious perfumes and eau de cologne. It is predictable that this oil can be subject to adulteration by
also used as flavor ingredient in food and beverages. In the addition of less valuable natural products, such as the
traditional Chinese medicine the extracts from bitter oils obtained from flowers of citrus different from C.
orange flowers are used to treat digestive problems and aurantium, or by addition of leaf oils or of synthetic
insomnia. compounds. The adulteration of nerolì oil is not easily
identifiable, mainly because the reference data available in
Nerolì is the product of a laborious work: the flowers, literature, relative to oils produced industrially and
which bloom between the end of April and the beginning extracted in laboratory [1-3], ranges widely and is also
of June are collected manually during the first hours of the probably affected by the geographic origin of the trees.
day; one worker can collect about 20 Kg per day for a Based on the rules AFNOR (Association Francaise de
period of 20 days; the flowers are hydro- or steam-distilled Normalization) [4a], ISO (International Organization for
with a yield ranging from 0.08% at the beginning of the Standardization) [4b] and EP (European Pharmacopeia)
season to a maximum of 0.13% under the most favorable [5], some of the results available in literature should not
conditions. The production in the European Mediterranean indicate genuine samples. Very few results are available in
Countries (mainly France) is subject to a strong decrease, literature on the enantiomeric distribution of volatiles
mainly for the specialized working cost necessary to determined in nerolì oils [6-10]. In the authors knowledge
collect the flowers, and for the contraction of the cultivated the IRMS analysis was never performed before on nerolì.
fields of bitter orange.
1010 Natural Product Communications Vol. 6 (7) 2011 Bonaccorsi et al.

Usually essential oils quality assessment is obtained by (5-6%) and by geraniol (3-4%). The sesquiterpene alcohols
traditional chromatographic techniques (GC-FID, GC-MS, (E)-nerolidol and (E,E)-2,6-farnesol are also well
Es-GC, HPLC) as recently reported by our research group represented ranging together between 2-5%. The main
[11], recognized for their validity in the quality control ester is linalyl acetate (2-15%) followed by geranyl
field. Advanced chromatographic techniques have been acetate (about 3%) and neryl acetate (about 1.5%).
also exploited for different essential oils as fast GC/MS The most abundant monoterpene hydrocarbon is limonene
[12] and multidimensional GC-GC for quantitative [13] (8-12%) followed by (E)--ocimene (3-5%), and by
and enantiomeric ratio assessment [14,15]. Recently has -pinene (2-4%); the main sesquiterpene hydrocarbon is
gained importance the Gas Chromatography-Combustion- -caryophyllene (0.6-0.9%).
Isotope Ratio Mass Spectrometry (GC-C-IRMS) that,
determining small differences in the isotopic carbon The sample produced in 2008 by hydrodistillation,
composition of the matrices, can be exploited to compared to all the other oils obtained by steam
discriminate between products of different origin [10,16- distillation, has the highest amount of linalool and of total
18a]. In this regards GC-C-IRMS can be an useful tool in alcohols, and the lowest amount of linalyl acetate and of
the flavour and fragrance authenticity control, unveiling total esters.
illicit essential oils production methods, such as the oils
adulteration by the addition of synthetic or natural In the samples produced in 2009 the composition varies
compounds, different from the genuine ones [18b,19,20]. gradually but significantly during the productive season.
The total monoterpene hydrocarbons and the single
To our knowledge nerolì oil was never investigated by components of this class of compounds, the total
IRMS. It is therefore particularly interesting to provide monoterpene alcohols and the single components of this
information useful for the genuineness assessment of class of compounds as well as the ratio linalool/linalyl
nerolì, also in function of the geographic origin. acetate decrease during the season. Total esters and linalyl
acetate present an opposite behavior, as well as the
The present article reports the results relative to the sesquiterpene hydrocarbons and aldehydes. Neryl and
composition of five samples of Egyptian nerolì produced geranyl acetate remain constant during the whole season.
in 2008 and 2009, to the enantiomeric distribution and the
isotopic ratio of selected components. In Figure 1 are graphically described the seasonal variation
of class of substances and some single components. The
The samples analyzed are described below: results confirm, as reported in literature, that the main
Sample Description components of nerolì oil are linalool, linalyl acetate and
1 hydrodistilled from Egypt (2008) limonene. The amount of these components determined in
2 steam-distilled March 23th 2009 this study fall in the ranges hitherto determined for nerolì
3 steam-distilled March 28th 2009
4 steam-distilled April 7th 2009 oil. It should be however mentioned that in one Egyptian
5 steam-distilled April 9-11th 2009 oil [1a] it was determined the 30% of linalool content and
1% of linalyl acetate; the highest value (74%) of linalool
Table 1 reports the composition determined by GC-FID, of was reported for a Chinese oil [1a] which presented a very
the samples analyzed. To facilitate comparison with unusual low amount of limonene (1%); in some Spanish
information already available in literature the results are oils [3] were reported very low values of linalyl acetate
here reported as raw peak area %. The correction factors (0.6%).
(C.F.) for each class of substances determined by GC-FID
are however reported in Table 1 to provide complete The results determined in the present study also confirm
information to the reader. In the case of distilled oils, as the presence of some Key compounds such as methyl
nerolì, the volatile fraction should represent the whole oil. anthranilate, methyl N-methyl anthranilate, phenyl ethyl
The quantitative results obtained from triplicates show alcohol, (E)-nerolidol, and some newly identified
CV% values always below 5%. The 86 components components such as the (E,Z)- and (E,E)-2,6-farnesals,
identified by GC/MS with the use of LRI as filters useful for the characterization of this product.
interactively applied during the mass spectral identification
process [21] represent about 99% of the whole oils. In Table 2 reports the enantiomeric distribution of some
comparison with literature information this study led to the components determined by es-GC. The values are
identification of numerous components (indicated by * in determined from triplicates with CV% never exceeding
Table 1), while the presence of numerous minor 5.5% with the exception of that relative to the (-)--
components previously reported were not here confirmed. thujene isomer which is 8.9% due to the chromatographic
behavior of this component. Figure 2 shows the chiral
Hydrocarbons range between 20-25%, oxygenated chromatogram of one of the samples analyzed.
compounds vary between 73-78%; among these alcohols
range from 58 to 70% and esters from 7 to 19%, while The enantiomeric ratios of camphene, sabinene, - and
aldehydes are present at small amounts (0.16-0.26%). The -phellandrene and citronellal were determined in
main alcohol is linalool (44-53%), followed by -terpineol nerolì oils for the first time. The values of the enantiomeric
Composition of Nerolì oil Natural Product Communications Vol. 6 (7) 2011 1011

Table 1: Composition of the five samples analyzed (% of peak areas) 2,3-Dihydrofarnesol* 1688 1688 0.02 0.01 0.01 0.01 0.04
determined by GC-MS-LRI and GC-FID. β-Sinensal* 1695 1699 0.09 0.09 0.09 0.08 0.08
(E,Z)-2,6-Farnesal* 1711 1714 0.02 0.01 0.01 0.02 0.03
LRIa LRIb 1 2 3 4 5 (E,E)-2,6-Farnesol 1718 1716 2.03 1.17 1.29 1.59 1.66
-Thujene 925 927 0.01 0.02 0.02 0.02 0.02 (E,E)-2,6-Farnesal* 1739 1737 0.03 0.02 0.02 0.03 0.04
-Pinene 933 933 0.15 0.26 0.22 0.22 0.23 -Sinensal* 1752 1749 0.02 0.02 0.02 0.02 0.01
Camphene 950 953 0.01 0.01 0.01 0.01 0.01 Farnesyl acetate° 1833 1832 0.04 0.02 0.02 0.03 0.03
Sabinene 973 972 0.85 1.56 1.15 1.44 1.33 (E,E)-Geranyl linalol* 2024 2026 0.02 0.02 0.02 0.03 0.03
β-Pinene 979 978 1.89 3.70 3.44 3.29 3.04 C.F. Sum of uncorrected % of peak areas
6-Methyl-5-hepten-2-one 984 978 0.02 0.02 0.02 0.02 0.02 HYDROCARBONS 1.0 21.79 24.95 23.97 22.57 20.10
Myrcene 989 991 1.43 1.74 1.61 1.44 1.33 Monoterpene 20.67 24.16 22.76 20.99 18.30
cis-Dehydrolinalol oxide* 1006 1006 0.02 0.02 0.02 0.02 tr Sesquiterpene 1.09 0.74 1.17 1.52 1.77
-Phellandrene 1007 1002 0.01 0.02 0.01 0.01 0.02 Aliphatic 0.03 0.05 0.04 0.06 0.03
δ-3-Carene 1010 1009 0.09 0.06 0.05 0.06 0.03 ALDEHYDES 1.3 0.26 0.16 0.22 0.25 0.26
(E)-2-Hexenyl acetate 1014 1017 - 0.01 0.01 0.01 tr Monoterpene 0.10 0.05 0.08 0.10 0.10
-Terpinene 1018 1018 0.04 0.13 0.09 0.07 0.04 Sesquiterpene 0.16 0.14 0.14 0.15 0.16
o-Cymene* 1020 1022 tr tr tr tr tr KETONES 1.3 0.09 0.08 0.08 0.11 0.09
p-Cymene 1025 1025 0.13 0.09 0.06 0.13 0.13 Monoterpene 0.05 0.05 0.05 0.07 0.05
Limonene 1031 1030 11.89 10.14 10.10 9.18 7.87 Aliphatic 0.04 0.03 0.03 0.04 0.04
(Z)-β-Ocimene 1035 1026 0.48 0.61 0.58 0.50 0.47 ALCOHOLS 1.3 69.72 59.58 58.68 57.81 58.19
(E)-β-Ocimene 1046 1046 3.31 5.11 4.81 4.09 3.40 Monoterpene 65.21 57.17 56.14 54.07 53.17
γ-Terpinene 1058 1049 0.12 0.28 0.19 0.17 0.11 Sesquiterpene 4.49 2.39 2.52 3.71 4.99
cis-Linalool oxide 1071 1069 0.13 0.17 0.17 0.18 0.25 Aliphatic 0.02 0.02 0.02 0.03 0.03
Terpinolene 1087 1086 0.26 0.43 0.42 0.36 0.27 ESTERS 1.6 6.88 13.39 15.55 17.55 19.35
Linalool 1107 1101 53.33 45.58 45.31 43.80 43.69 Monoterpene 6.83 13.36 15.52 17.50 19.30
4,8-Dimethyl-1,3(E),7- Sesquiterpene 0.04 0.02 0.02 0.03 0.03
nonatriene* 1114 1113 0.03 0.04 0.04 0.05 0.03 Aliphatic 0.01 0.01 0.01 0.02 0.02
+ Phenylethyl alcohol* OXIDES + ETHERS 1.5 0.22 0.30 0.32 0.37 0.34
Fenchol° 1122 1123 tr - - - tr OTHERS 0.10 0.14 0.16 0.14 0.10
trans-p-Menth-2,8-dienol 1124 1122 tr - - - tr ALL 99.06 98.60 98.98 98.80 98.43
cis-p-Menth-2-en-1-ol 1127 1124 0.03 0.03 tr 0.02 0.02 Notes a: LRI measured on SLB-5MS column; b: Reference LRI reported in
trans-Limonene oxide* 1135 1142 0.01 0.09 0.09 0.10 0.02 literature (FFNSC 1.3GC-MS library, Shimadzu, Japan; or Adams RP.
trans-p-Menth-2-enol* 1144 1141 0.02 0.02 0.01 0.02 0.03 Identification of essential oil components by gas chromatography/mass
Terpinen-4-ol 1182 1177 0.44 0.79 0.52 0.57 0.59 spectrometry, 4th Edn. Carol Stream, IL, USA: Allured Publishing Corp;
p-Cymen-8-ol* 1189 1189 0.02 0.01 0.01 0.01 0.03 2007; or Hochmuth, D.H., Joulain, D., König, W.A., 2002. MassFinder
(Z)-3-Hexenyl butanoate* 1190 1184 0.01 tr tr 0.01 0.02 Software and Data Bank, University of Hamburg); tr: ≤ 0.005; C.F. Correction
-Terpineol 1199 1195 6.22 6.17 5.90 5.50 4.89 Factor (FID response) for class of compounds; * identified, in the authors
trans-Piperitol 1211 1209 0.01 0.02 0.01 0.01 0.02 knowledge, for the first time in nerolì oils; ° correct isomer not identified.
Nerol 1226 1229 1.28 1.12 1.06 1.01 0.95
cis-Carveol* 1234 1232 tr - - - -
Neral 1239 1238 0.03 tr 0.03 0.03 0.03
Carvone 1246 1246 0.01 0.02 0.02 0.03 0.01
Linalyl acetate 1250 1243 2.19 8.77 10.97 12.97 14.57 Table 2: Enantiomeric distribution of some volatile components in the
Geraniol 1253 1255 3.83 3.42 3.30 3.06 2.94 samples analyzed.
trans-Myrtanol* 1257 1261 tr 0.01 0.02 0.02 0.01
Geranial 1269 1264 0.07 0.05 0.05 0.07 0.07 1 2 3 4 5
Bornyl acetate* 1286 1287 tr 0.01 0.01 0.01 0.01 S-(+)--Thujene 59.02 48.80 33.53 56.53 30.98
Geranyl formate* 1298 1298 0.03 0.01 0.02 tr 0.04 R-(-)--Thujene 40.98 51.20 66.47 43.47 69.02
Methyl geranoate* 1321 1320 0.01 tr tr tr 0.02 R-(+)--Pinene 41.37 36.77 32.96 34.70 n.d.
Linalyl propanoate* 1331 1333 tr - - - 0.02 S-(-)--Pinene 58.63 63.23 67.04 65.30 n.d.
Bicycloelemene* 1334 1338 tr 0.01 0.01 0.01 tr 1S,4R-(-)-Camphene n.d. n.d. n.d. n.d. 90.85
δ-Elemene 1337 1335 0.03 0.05 0.06 0.06 0.06 1R,4S-(+)-Camphene n.d. n.d. n.d. n.d. 9.15
Methyl anthranilate 1342 1337 0.04 0.10 0.12 0.09 0.06 R-(+)-β-Pinene 2.05 2.04 1.72 1.76 2.08
-Terpinyl acetate 1348 1349 0.05 0.06 0.07 0.06 0.07 S-(-)-β-Pinene 97.95 97.96 98.28 98.24 97.92
Citronellyl acetate 1349 1353 0.02 - - - 0.02 R-(+)-Sabinene 80.89 81.40 76.72 81.93 81.14
Neryl acetate 1359 1361 1.45 1.45 1.43 1.44 1.45 S-(-)-Sabinene 19.11 18.60 23.28 18.07 18.86
Geranyl acetate 1379 1380 3.08 3.06 3.02 3.01 3.08
R-(-)--Phellandrene 44.55 27.42 n.d. 14.37 32.01
β-Elemene 1392 1391 0.09 0.11 0.09 0.12 0.18
(E)-Jasmone* 1395 1390 0.02 0.01 0.01 0.02 0.02 S-(+)--Phellandrene 55.45 72.58 n.d. 85.63 67.99
n-Tetradecane* 1400 1400 tr 0.01 tr 0.01 R-(-)-β-Phellandrene 39.17 27.75 53.74 36.50 49.31
0.01 S-(+)-β-Phellandrene 60.83 72.25 46.26 63.50 50.69
cis--Bergamotene* 1404 1411 0.01 tr tr 0.01
Methyl N-methyl anthranilate 1409 1405 0.04 0.02 0.02 0.02 0.01 S-(-)-Limonene 1.65 2.54 2.42 2.58 2.60
β-Caryophyllene 1424 1424 0.56 0.60 0.68 0.82 0.94 R-(+)-Limonene 98.35 97.46 97.58 97.42 97.40
Perillyl acetate* 1435 1435 - tr tr tr 0.01 S-(-)-Citronellal n.d. 47.14 45.09 n.d. n.d.
Aromadendrene 1443 1439 0.01 tr tr tr 0.01 R-(+)-Citronellal n.d. 52.86 54.91 n.d. n.d.
Geranyl acetone* 1448 1453 0.04 0.03 0.03 0.04 0.04 R-(-)-Linalyl acetate 98.95 99.39 99.47 99.41 99.28
(E)-β-Farnesene 1453 1452 0.09 0.13 0.13 0.18 0.22 S-(+)-Linalyl acetate 1.05 0.61 0.53 0.59 0.72
-Humulene 1460 1452 0.06 0.06 0.06 0.08 0.11 R-(-)-Linalol 78.25 78.51 78.54 78.48 78.55
9-epi-β-Caryophyllene* 1464 1464 0.01 tr tr 0.01 0.01 S-(+)-Linalol 21.75 21.49 21.46 21.52 21.45
Geranyl propanoate* 1469 1471 tr tr tr 0.01 0.01 S-(+)-Terpinen-4-ol 62.19 62.74 60.00 61.68 62.13
Germacrene D 1485 1479 0.03 0.07 0.07 0.08 0.08 R-(-)-Terpinen-4-ol 37.81 37.26 40.00 38.32 37.87
Bicyclogermacrene* 1500 1497 0.10 0.14 0.16 0.14 0.05 S-(-)--Terpineol 28.77 28.98 28.88 29.21 28.99
-Muurolene* 1502 1497 0.01 0.02 0.01 0.02 0.01
R-(+)--Terpineol 71.23 71.02 71.12 70.84 70.79
(E,E)--Farnesene 1505 1504 0.05 0.02 0.01 0.02 0.03
γ-Cadinene 1517 1513 tr tr tr tr 0.01 n.d.: not determined.
δ-Cadinene 1522 1518 0.03 0.02 0.03 0.03 0.04
β-Sesquiphellandrene* 1526 1523 0.01 0.01 0.01 0.01 0.01 distribution of limonene, linalol, terpinen-4-ol and
(E)-Nerolidol 1563 1561 2.35 1.15 1.16 2.04 3.21
Spathulenol 1582 1576 0.05 0.03 0.03 0.05 0.05 -terpineol here determined are in good agreement
Caryophyllene oxide° 1587 1587 0.04 0.02 0.02 0.04 0.04 with literature results relative to genuine nerolì oils
Globulol 1591 1587 0.02 0.01 0.01 0.01 0.02
Cadin-4-en-10-ol* 1660 1659 0.02 0.02 0.02 0.01 0.01 [6,7a]; however, if compared to literature, the enantiomeric
n-Tetradecanol* 1677 1680 0.02 0.02 0.02 0.03 0.03 excess of (-)--pinene is slightly lower, that of (-)--pinene
1012 Natural Product Communications Vol. 6 (7) 2011 Bonaccorsi et al.

Table 3: (-)13CVPDB values calculated for the samples analyzed, average uV(x10,000)
Chromatogram

and relative standard deviation% (RSD). 3.0


6
14 15 16 19 24

1 2 3 4 5 Ave RSD
-Pinene 25.24 25.75 25.16 25.24 25.48 25.37 0.95 2.5

Myrcene 24.77 26.26 25.05 24.25 24.30 24.92 3.28


Limonene 27.29 27.78 27.20 27.84 27.32 27.48 1.09 2.0
Linalol 25.64 25.85 25.80 26.08 26.06 25.89 0.72 7
Terpinen-4-ol 28.10 27.74 27.01 27.68 27.81 27.67 1.46 23
1.5
-Terpineol 26.84 27.14 26.57 26.93 27.22 26.94 0.96
Nerol 27.05 27.39 26.27 26.89 28.19 27.16 2.59
Neryl acetate 28.14 27.86 27.48 28.42 28.52 28.09 1.51 1.0
Geranyl acetate 27.42 28.06 27.75 28.54 28.41 28.03 1.65
Caryophyllene* 25.74 25.15 23.18 25.04 24.61 24.74 3.89 0.5 21
8
(E)-Nerolidol 30.60 30.43 30.12 30.79 30.13 30.41 0.96 4
12 22

Farnesol** 30.18 29.3 29.64 29.58 29.79 29.71 1.03 3


1 2
5
910
11
13
17 18
20

Correct isomer identification: *-; **(2E,6E)-


0.0
15.0 20.0 25.0 30.0 min

Table 4: Comparison between the limits reported in the regulations [4,5] and Figure 2: Chiral chromatogram of one sample of neroli oil. Peak identification: 1.
the results experimentally obtained for the five samples of Egyptian Nerolì (+)-α-thujene; 2. (-)-α-thujene; 3. (+)-α-pinene; 4. (-)-α-pinene; 5. (+)-β-pinene; 6. (-
(peak area %). )-β-pinene; 7. (+)-sabinene; 8. (-)-sabinene; 9. (-)-α-phellandrene; 10. (+)-α-
phellandrene; 11. (-)-β-phellandrene; 12. (-)-limonene; 13. (+)-β-phellandrene; 14.
AFNOR ISO EP 2006 Range
(+)-limonene; 15. (-)-linalol; 16. (+)-linalol; 17. (-)-citronellal; 18. (+)-citronellal;
1995 3517:2002
19. (-)-linalyl acetate; 20. (+)-linalyl acetate; 21. (+)-terpinen-4-ol; 22. (-)-terpinen-
Limonene 9-18 9-18 9.0-18.0% 7.87-11.89
4-ol; 23. (-)-α-terpineol; 24. (+)-α-terpineol.
Myrcene 1-4 1-4 - 1.33-1.74
(E)-β-Ocimene 3-8 3-8 - 3.31-5.11
α-Pinene max. 2 tr-2 - - -23

β-Pinene 7-17 7-17 7.0-17.0 1.89-3.70 -24


1
Sabinene - tr-3% - 0.85-1.44 -25
Linalool 28-44 28-44 28.0-44.0 43.31-53.33
-26
α-Terpineol 2-5.5 2-5.5 2.0-5.5 4.89-6.22
(E,E)-Farnesol 1-4 1-4 0.8-4.0 1.29-2.03 -27 2

(E)-Nerolidol 1-5 1-5 1.0-5.0 1.15-3.21 -28


13CVPDB
Linalyl acetate 3-15 3-15 2.0-15.0 2.19-14.57 -29
Geranyl acetate 1-5 1-5 1.0-5.0 3.0-3.08 3
-30
Neryl acetate max. 2.5 tr-2.5 max 2.5 1.43-1.45
Methyl anthranilate - - 0.1-1.0 0.04-0.12 -31
4
-32
Chiral purity

e
Te -ol

l
e

eo

ar l
yo tate
in ol
Li e

(+)-Linalol - - max 30 21.45-21.75

so
(E nyl ate
l

)-F do
m e

ry ero

en
en

en
n

in

ne
i
ce

Te nal

yll
t

ol
ph en-
in

e
ce
on

rp

)-C ac

er
-P

ph
yr

(+)-Linalyl acetate - - max 5 0.53-1.05

la

(2 )-N
M
ta

rp
Li

a-

5
be

6E
(E
ar
Ne

a
er

E,
al

G
70,00
Seasonal variation

60,00
Figure 3: Diagram obtained for 13CVPDB values for each components in
function of the period of production. For sample description see table in text.
50,00

by plotting the 13CVPDB values for each component


40,00 Linalyl acetate analysed in function of the period of production. The
relative standard deviation of the 13CVPDB values range
Linalol
%

Linalol + Linalyl acetate

for the samples analyzed between 3.52 ((E)--


30,00 Monoterp HYDROCARB
Alcohols

caryophyllene) and 0.96 (-terpineol). These low values


Esters
20,00

indicate very narrow ranges of variation, therefore it is


10,00
posible to assume that the 13CVPDB can be considered
0,00
characteristic of authenticity and of the geographic origin
1 2
sample #
3 4
of the samples.

Table 4 provides a comparison of the ranges determined


Figure 1: Seasonal variation of class of components, of linalool and linalyl
acetate in the four samples of nerolì produced during 2009.
from the present results with the limits provided by the
AFNOR, ISO and EP regulations [4,5]. Some of the results
extremely lower and that of (-)-linalyl acetate is higher. fall within these limits; others fall only slightly outside
With the exception of -pinene, the results here obtained them; in three of the samples analyzed linalol is present at
for the components analyzed are overall in good agreement levels sensibly higher than the limits provided by the
with the enantiomeric purity previously determined by aformentioned regulations; -pinene is always below the
Mosandl [6] considered characteristic of genuine nerolì oils. minima reported for nerolì oils. These behavior could be
due to the geographic origin of the oils analyzed.
Table 3 reports the (-)13CVPDB values calculated for the
nerolì oils samples. This study reports for the first time the Considering the high commercial value of nerolì, its
GC-C-IRMS analysis of selected volatile components in limited production in different geographic areas and the
nerolì oil. It is impossible to compare these values with high possibility that this product can be subject to
literature information. Figure 3 shows the graph obtained adulteration, it is necessary to fix quality parameters in
Composition of Nerolì oil Natural Product Communications Vol. 6 (7) 2011 1013

consideration of its variability among different geographic GC-C-IRMS device and analyses: Trace GC Ultra
areas. To accomplish this, for a correct evaluation, not equipped with a TriPlus autosampler, retrofitted to the
only the indices given by these regulations should be taken combustion interface GC/CIII and hyphenated to the
into account, but also the 13CVPDB of selected compounds isotope ratio mass spectrometer Delta V Advantage (all
and the chiral purity of more compounds than the two purchased from Thermo Fisher Scientific, Milan, Italy).
already indicated, thus providing adequate tools for quality GC: column: SLB-5ms (silphenylene polymer) 30 m x
and genuineness assessment. 0.25 mm i.d., 0.25 m df (Supelco, Milan, Italy.);
temperature program: 50°C to 230°C at 3°C/min;
Experimental split/splitless injector (250°C). Inlet pressure: 167 kPa;
Analysis of the essential oils: On the five samples column flow: 2.0 ml/min (constant flow mode); carrier
described in text the following analytical investigations gas: He.
have been carried out: GC/FID, GC/MS of the volatile GC/C III: ox. reactor (Cu/Ni/Pt): 980°C; red. reactor:
fraction; direct enantio-GC for the determination of the 640°C; He: 1 bar; O2: 0.8 bar; CO2: 0.5 bar.
enantiomeric distribution of some volatiles. Each analysis IRMS: EI; electron voltage: 123.99 eV; electron current:
was performed in triplicates. Results are expressed as 1.5 mA; 3 Faraday cup collectors at m/z 44, 45, and 46;
average peak area %. peak center pre-delay and post-delay: 15 s, cup 3;
reference: 60-80 s, 100-120 s, 140-160 s, 180-200 s; split:
GC/FID: The volatile fraction was analyzed by open; evaluation type: CO2_SSH, ref. time: 155.90 s,
HRGC/FID as described. Gas chromatograph: Shimadzu 13C/12C -60.300‰; integration time 0.2 s.
GC2010 equipped with a Flame Ionization Detector, a
split/splitless injector and an AOC-20i series auto-injector. GC-C-IRMS instrument achieves highly precise
Capillary column: 30 m x 0.25 mm I.D. 0.25 m df coated measurement of carbon isotopic abundance, converting the
with SLB-5MS [silphenylene polymer, virtually equivalent eluted volatile components, in CO2 and water into an
in polarity to poly (5% diphenyl/95% methyl)siloxane)] oxidation chamber. After removing water, just behind the
(Supelco, Milan, Italy); column temperature, 50-250°C (10 furnace, by a capillary-shaped phase separator, CO2
min) at 3°C/min; injector temperature: 250°C; detector reaches an ionization chamber where it will be transformed
temperature: 280°C; carrier gas, He at 99.5 kPa (30.0 into three ion traces for the different isotopomers: 12C16O2,
cm/s); injection mode: split; split ratio, 1:100; injected 13 16
C O2 and 12C18O16O, with their corresponding masses at
volume, 1.0 L of diluted oil. Data handling was made by (m/z) 44, 45, 46.The three ion beams are registered
means of GCsolution software. simultaneously by an Universal Faraday collector that
detects the different contributions of ionic fragments
GC/MS Analysis: Samples were analyzed by GC/MS (EI) obtained. Isotopic ratios, 45/44 and 46/44, are expressed in
on a GCMS-QP2010 system equipped with commercially ‰ and are related to a certified standard (VPDB-standard)
available libraries (see notes to Table 1) including the of known value [20]. Exploiting the GC-Combustion
commercial version of the FFNSC ver. 1.3 (Shimadzu, backflush, the most concentrated components were not
Japan) database (created in the authors’ laboratory) introduced into the combustion chamber.
consisting of about 2000 reference standards and their
relative linear retention indices determined on apolar The samples dilutions and the GC-Combustion conditions
column, interactively used as filters for the spectral
were as follows: concentration 1:10 (v/v), 1 L split
interpretation. GC conditions: capillary column and
injection, 1:100 split ratio, backflush: off, for the
temperature program as in GC/FID; carrier gas, He
determination of 13CVPDB of limonene and linalool.
delivered at a constant pressure of 30.6 kPa (30.1 cm/s);
1.0 L of solution (1/10, v/v, essential oil/hexane) injected
Concentration 1:10 (v/v), 1 L split injection, 1:50 split
on a split/splitless injector; injector temperature, 250°C;
ratio, backflush open: 780-830 s and 970-1060 s, for the
injection mode, split; split ratio, 1:50. MS scan conditions:
source temperature, 200°C; interface temperature, 250°C; determination of 13CVPDB of -pinene, myrcene, terpinen-
E energy 70eV; mass scan range, 40-400 amu. Data was 4-ol, -terpineol, nerol, neryl acetate, geranyl acetate, (E)-
handled through the use of GCMSsolution software. caryophyllene, nerolidol, (2E,6E)-farnesol. Data are
collected in triplicate, by using the Isodat 2.5 software
Enantio-GC: Shimadzu GC2010 gas chromatograph (Thermo Fisher Scientific).
equipped with a Flame Ionization Detector, a split/splitless
injector and an AOC-20i series autoinjector. Capillary CO2 reference gas cylinder calibration: The attained
chiral column was a Megadex DETTBS-(diethyl-tert- carbon isotope ratio of the unknown sample is compared to
butil-silyl -cyclodextrin) 25 m x 0.25 mm I.D. x 0.25m that of a calibrated CO2 reference. The CO2 reference gas
df (Mega, Legnano, Italy). Temperature program: 50°- was calibrated by injecting 1 L of a carbon stable isotope
200°C at 2°C/min. Inlet pressure 96.6 kPa (220°C), split ratio reference alkanes mixture comprising C16 to C30
mode 1:20 (gas carrier He); injected volume, 1.0 l; linear (Indiana University, Bloomington, U.S.A.), calibrated
velocity, 30 cm/sec (constant). Data handling was made by against VPDB standard with a defined 13C content. Isotope
means of GCsolution software ratios were expressed as  values (‰), versus a standard.
1014 Natural Product Communications Vol. 6 (7) 2011 Bonaccorsi et al.

(13C/12C)sample - (13C/12C)standard x 1000 Acknowledgements - Authors whish the acknowledge


13CVPDB = Shimadzu for the continuous support.
(13C/12C)sample

Tricosane (C23) was arbitrarily chosen as reference alkane.

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2011
NPC Natural Product Communications Vol. 6
No. 7
Essential oil of Nepeta x faassenii Bergmans ex Stearn 1015 - 1022

(N. mussinii Spreng. x N. nepetella L.): A Comparison Study


Niko Radulovića, Polina D. Blagojevića, Kevin Rabbittb and Fabio de Sousa Menezesb,*
a
Department of Chemistry, Faculty of Science and Mathematics, University of Nis, Serbia
b
School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences,
Trinity College Dublin, Ireland

DESOUZAF@tcd.ie

Received: December 12th, 2010; Accepted: March 16th, 2011

Analysis (GC and GC/MS) of an essential oil sample obtained from dry leaves of Nepeta × faassenii Bergmans ex Stearn, a hybrid species
produced by crossbreeding N. mussinii Spreng. with N. nepetella L., led to the identification of 109 constituents that represented 95.9% of
the oil. The major constituents were 4aα,7α,7aα-nepetalactone (67.8%), 1,8-cineole (6.6%), germacrene D (4.8%), β-pinene (2.7%),
(E)-β-ocimene (2.6%), 4aα,7β,7aα-nepetalactone (2.3%) and (E)-β-farnesene (1.0%). Chemical composition of the oil was compared, using
multivariate statistical analyses (MVA) with those of the oils of other Nepeta taxa, in particular N. mussinii and N. nepetella. This was done
in order to explore the mode of inheritance of the monoterpene biosynthetic apparatus of N. faassenii. Chemical composition of the volatiles
of a Nepeta taxon (different populations) can be subject to variation due to environmental and geographical factors. To accommodate this fact
in the MVAs, along side with N. faassenii essential oil, additional 6 oils (3 different populations of N. nuda L. and N. cataria L. from Serbia)
were included in this study (isolated and analyzed (chemically and statistically)). The MVA analyses recognized N. faassenii as being closely
related to both N. mussinii and N. nepetella. If the relative content of oil constituents per plant and not per chromatogram were used as
variables in the MVA (this was done by simple multiplication of the yields and relative percentages of components) a higher degree of
mutual similarity (in respect to the monoterpene biosynthesis) of N. faassenii to N. mussinii, than to the other parent species, was observed.

Keywords: Nepeta × faassenii Bergmans ex Stearn, Lamiaceae, Nepeta nuda L., Nepeta cataria L., Nepeta mussinii Spreng, Nepeta
nepetella L., essential oil, nepetalactone, chemometrics.

The genus Nepeta L., Lamiaceae, includes over 200 Nepeta × faassenii is cultivated in Europe, C Asia and
species of perennial plants and some annuals [1]. The Persia, and is used as a spice and an ornamental plant [4].
members of this genus are known as catnip or catmint There is only limited data on the composition of the
because of the purported effect on cats, pleasantly volatile oil of the mentioned hybrid taxon [3a]. For this
stimulating cats’ pheromonic receptors, typically resulting reason, the aim of this study was set to analyze in detail
in temporary euphoria [1]. The active substances of catnip (GC and GC/MS) the essential oil of N. faassenii and to
plants, some of which have a sedative effect on humans, compare its chemical composition, using multivariate
have been widely used in medicine, food industry and statistical analyses (MVA: agglomerative hierarchical
perfumery. They are also used in folk medicine as cluster analysis (AHC) and principal component analysis
remedies for bronchitis, flu, cold and other illnesses. (PCA)) with those of the oils of other Nepeta taxa, in
Number of catnips have sudorific, diuretic and particular N. mussinii and N. nepetella. Having in mind
bacteriostatic properties, reduce fever and help treat that the monoterpene diversity and morphology could serve
stomach-aches [1,2]. Generally speaking, Nepeta species as a mirror of the gene flow [5], multivariate analyses were
are essential oil-rich taxa, and some of them contain more primarily done in order to provide an insight into the
than 1% of the essential oil [2,3]. Moreover, many of them inheritance of the chemical characters for this taxon.
are cultivated as garden plants. In order to produce new,
interesting garden/ornamental plants, many Nepeta species The chemical composition of the essential oils of a Nepeta
have been crossbred repeatedly. The aim of the breeding taxon (different populations) can be subject to variation
processes is not only creating plants with an appalling due to environmental and geographical factors. To
look, but also producing larger amounts of biologically accommodate this fact in the MVAs, along side with N.
active compounds [2]. One of the outcomes of these faassenii essential oil, additional 6 oils (3 different
experiments is the sterile hybrid Nepeta × faassenii populations of N. nuda L. and N. cataria L. from Serbia)
Bergmans ex Stearn, which has been produced by were included in this study (isolated and analyzed
crossbreeding N. mussinii Spreng. with N. nepetella L. [4]. (chemically and statistically)).
1016 Natural Product Communications Vol. 6 (7) 2011 Radulović et al.

Table 1: Chemical composition of the essential oils extracted from Table 1 (contd.)
Nepeta faassenii (NF), N. cataria (NC) and N. nuda (NN) 1163 Lavandulol tr M
1165 Sabina ketone tr M
RIa Compound NF NCb NNb Class
1166 Pinocarvone tr 0.20.1 M
765 (Z)-2-Penten-1-ol trc,d tr O
1169 Lavandulol 0.10.0 tr M
778 3-Methyl-2-butenal tr tr O
Methyl 3- 1170 Rosefuran epoxide tr M
778
methylbutanoatee
tr tr tr O 1173 δ-Terpineole tr 1.60.5 MM
801 Hexanale tr tr O 1184 Terpinen-4-ole 0.1 tr 0.30.1 MM
837 Furfurale tr tr O 1193 Cryptone tr MM
845 (Z)-2-Hexenal tr 0.10.0 O 1198 α-Terpineole 0.1 tr 3.91.0 MM
851 (E)-2-Hexenal 0.1 O 1199 Methyl salicylatee tr 0.10.1 tr O
Ethyl 3- 1201 Myrtenale tr 0.30.2 M
852 tr O 1207 Decanale tr O
methylbutanoatee
855 (Z)-3-Hexen-1-ol tr O (E,E)-2,6-Dimethyl-
1208 0.1 M
886 2-Butylfuran tr O 3,5,7-octatrien-2-ol
897 2,6-Dimethypyridine tr O 1218 Verbenone tr M
900 Nonanee 0.10.1 O 1221 β-Cyclocitral tr tr tr O
900 Styrenee,f 0.8 tr tr O 1226 trans-Carveole tr MM
927 Diethyldisulfidee tr O (Z)-3-Hexenyl 2-
1238 tr O
932 α-Thujenee 0.1 tr tr M methyl butanoate
937 α-Pinenee 0.4 tr 1.10.3 M 1239 Nerale tr tr M
951 Allylbenzenee tr O (Z)-3-Hexenyl 3-
1242 0.30.3 O
955 Camphenee tr tr tr M methylbutanoate
963 Hexanoic acide tr O Hexyl 3-methyl
1248 tr O
970 Benzaldehydee tr tr tr O butanoatee
975 Sabinenee 0.8 0.20.1 2.00.5 M 1251 Cumin aldehyde tr MM
978 1-Octen-3-ol tr O 1251 Geraniole tr tr tr M
984 β-Pinenee 2.7 0.40.2 4.00.7 M 1256 Carvonee tr MM
987 3-Octanone tr 0.1 O 1269 Geraniale 0.1 tr M
Methyl 3-
989 Myrcenee 0.3 tr 1.10.1 M 1275 0.3 O
phenylpropanoatee
993 2-Pentyl furan tr tr O
1282 Lavandulyl acetate tr M
995 Dehydro-1,8-cineole tr 0.10.0 MM
1293 Dihydroedulan IA tr 0.10.0 O
997 3-Octanol 0.1 O
1324 (E)-3-Hexenyl tiglate tr O
999 (Z,E)-2,4-Heptadienal tr tr O
Ethyl 3-
1004 Octanale tr O 1350 tr O
phenylpropanoatee
1009 p-Mentha-1(7),8-diene tr tr MM
4aα,7α,7aα-
1014 (E,E)-2,4-Heptadienal tr O 1378 67.8 91.77.8 0.10.0 MI
Nepetalactonee
1021 α-Terpinene tr tr 0.10.1 MM
1381 α-Copaene 0.2 0.20.1 S
1028 p-Cymenee tr tr tr MM
1387 Geranyl acetatee tr M
Methyl 1-cyclohexenyl
1029 tr tr M (Z)-3-Hexenyl (Z)-3-
ketone 1389 0.40.4 tr O
hexenoate
1032 Limonenec 0.5 tr MM
1391 β-Bourbonene 0.2 0.90.6 S
1034 β-Phellandrene 0.1 0.10.0 MM
1392 β-Cubebene tr tr S
1037 1,8-Cineolee 6.6 tr 37.54.9 MM
1393 β-Elemene 0.2 0.40.0 S
1040 (Z)-β-Ocimene tr 0.10.0 M
1393 (E)-β-Damascenone tr O
1045 (E)-β-Ocimene 2.6 0.20.1 0.70.3 M 4aα,7α,7aβ-
1050 2-Phenylacetaldehydee 0.20.2 O 1396 0.3 0.40.3 MI
Nepetalactone
1060 γ-Terpinene tr tr 0.10.0 MM 1397 (E)-Jasmone tr O
1061 (E)-2-Octenal tr O 4aα,7β,7aα-
1062 Bergamal tr M 1404 2.3 1.00.7 12.92.3 MI
Nepetalactone
1070 1-Octen-3-ol tr O 1414 Isocaryophyllene tr S
(E)-3-Hexenyloxy- 1415 α-cis-Bergamotene 0.1 S
1070 tr O
acetaldehyde 1416 α-Gurjunene 0.10.1 S
1071 1-Octanole tr O 1423 β-Ylangene tr S
1073 cis-Sabinene hydrate 0.1 0.40.3 M 1424 Dihydronepetalactone tr MI
cis- Linalool oxide 1425 β-Caryophyllenee 0.9 2.30.8 4.62.5 S
1077 tr tr M
(furanoid)e 1434 β-Copaene 0.1 0.20.0 S
1089 Terpinolenee tr 0.10.0 MM 1441 (Z)-β-Farnesene tr S
1092 Rose furan tr M 1451 Isogermacrene D 0.10.0 S
1101 Linaloole tr tr tr M 1453 (E)-β-Farnesene 1.0 S
0.20.2 4.63.1
1102 Perillene tr MM
1460 α-Humulenee 0.1 0.20.2 tr S
1105 trans-Sabinene hydrate tr tr M
1463 2-Methyltetradecane tr tr O
1106 Nonanale tr tr tr O
1468 allo-Aromadendrene tr S
1107 α-Thujonee tr M
cis-Cadina-1(6),4-
(E)-6-Methyl-3,5- 1470 tr S
1109 tr tr O diene
heptadien-2-one
cis-Muurola-4(14),5-
1128 Dehydrosabina ketone tr M 1473 tr 0.10.0 S
diene
1128 allo-Ocimene tr M
1483 ar-Curcumene tr S
1132 α-Campholenal tr M
1487 γ-Muurolene 0.10.1 S
1139 (E)-Epoxy-ocimene tr M
1145 trans-Pinocarveol tr tr M 1486 Germacrene De 4.8 tr 12.64.4 S
0.20.2
1146 Nopinone tr tr M 1490 (E)-β-Iononee tr O
1147 trans-Sabinole tr M trans-β-Ionon-5,6-
1491 tr O
1151 Citronellal tr M epoxide
1152 trans-Verbenol M 1496 α-Zingiberene 0.1 0.10.1 S
0.10.1
1159 (E)-2-Nonenal tr O trans-Muurola-4(14),5-
1499 tr S
diene
Nepeta x faassenii essential oil Natural Product Communications Vol. 6 (7) 2011 1017

Table 1 (contd.) p-Menthane (MM) 7.4 0.1±0.0 47.3±1.7


1499 Bicyclogermacrene 0.1 0.7±0.6 S Sesquiterpenoids (S) 9.5 3.4±0.5 30.6±5.2
1500 α-Muurolene tr tr S Hydrocarbons 8.6 2.7±0.4 26.4±4.2
1504 (E,E)-α-Farnesene 0.1 tr tr S Oxygenated 0.9 0.7±0.4 26.5±3.1
1509 β-Bisabolene 0.3 0.9±0.2 S Others (O) 1.4 0.9±0.5 0.6±0.1
1512 Germacrene A 0.1 S a
Compounds listed in order of elution from a DB-5MS column with
1520 γ-Cadinene 0.1 tr S
retention indices (RI) determined experimentally by co-injection of a
1523 endo-1-Bourbonanol tr S
homologous series of C7-C28 n-alkanes; bPercentages given as average
1526 β-Sesquiphellandrene 0.2 0.8±0.2 S
1536 trans-γ-Bisabolene tr S values ± absolute deviations of the components’ relative abundances
1538 trans-Cadina-1,4-diene tr S (three oil samples); ctr-trace (<0.05%); dAll compounds present below
1543 α-Cadinene tr S 0.05% in at least one of the analyzed N. nuda and N. cataria oil samples
1550 α-Calacorene tr tr S are given as tr; eThe identity of the constituent was determined by
1561 (E)-Nerolidol tr S comparison of MS and RI matching and confirmed by co-injection of an
1568 1-nor-Bourbonanone 0.1±0.1 S authentic sample; fProbably a contaminant of the isolation procedure;
1569 Dendrolasin tr tr S syn.-synonym.
1572 Mint oxide tr S
1575 Palustrol 0.1±0.0 S
(Z)-3-Hexenyl
1575 tr O
benzoate
1581 Germacrene D-4-ol 0.1 S
1584 Spathulenole 0.2±0.0 S
1587 Caryophyllene oxidee 0.2 0.7±0.1 1.9±0.5 S
1593 β-Copaen-4α-ol tr S
1597 Salvial-4(14)-en-1-one tr tr S
1610 Ledol 0.1±0.0 S
1611 β-Oplopenone tr S
1617 Humulene epoxide II tr 0.4±0.3 S
1626 Junenol tr S
1633 Zingiberenol tr S
1635 1-epi-Cubenol tr S
allo-Aromadendrene
1643 0.1 S
epoxide
Caryophylla-
1644 tr tr S
4(12),8(13)-dien-5-ol
epi-α-Cadinol
1645 tr tr S
(τ-cadinol)
epi-α-Murrolol (syn.g
1648 0.3±0.2 S
τ-muurolol)
1650 3-iso-Thujopsanone tr S
α-Muurolol (syn.
1652 0.1±0.1 S
torreyol)
1659 α-Cadinol 0.1 0.6±0.6 S
14-Hydroxy-9-epi-
1678 0.1±0.0 S
caryophyllene
Germacra-
1688 4(15),5,10(14)-trien- 0.1 0.3±0.1 S
1α-ol
1690 2,3-Dihydrofarnesol tr S
Eudesma-4(15),7-dien-
1694 tr S
1β-ol
1697 (Z,Z)-2,6-Farnesol 0.1 S
1743 Mint sulfide tr S
1745 α-Oxobisabolene 0.1 S
1768 β-Costol tr S
1770 Benzyl benzoate tr O
14-Hydroxy-α-
1777 tr S Figure 1: Typical TIC chromatograms of the herein analyzed N. cataria,
muurolene
14-Hydroxy-δ- N. nuda and N. fasenii essential oil samples (for sample designation see
1797 0.1 S Table 2).
cadinene
Hexahydrofarnesyl
1845 tr 0.1±0.0 O
acetone Almost 200 different compounds, representing 95.9-99.1%
2067 Abietatriene tr O
2107 (E)-Phytol 0.1 tr O of the total oils, were identified in the 7 presently analyzed
2300 Tricosane tr tr tr O samples (Table 1). Typical TIC chromatograms of the
2364 (Z)-9-Octadecenamidef tr O analyzed samples are depicted in Figure 1. Chemical
2400 Tetracosanee tr O
2500 Pentacosanee tr tr O compositions of the analyzed N. nuda and N. cataria oils
2600 Hexacosanee tr tr O were in general agreement with the literature data [3a,g].
2800 Octacosanee tr O Major compounds identified in N. faassenii oil were
Total 95.9 98.5±1.1 98.0±1.5
Number of
4aα,7α,7aα-nepetalactone (67.8%), 1,8-cineole (6.6%) and
119 75±5 115±9 germacrene D (4.8%). Additional 4 compounds with the
components
Monoterpenoids (M) 85.0 94.2±7.1 66.8±5.1 relative amount higher than 1% of were: β-pinene (2.7%),
Hydrocarbons 7.5 1.0±0.3 9.4±3.1 (E)-β-ocimene (2.6%), 4aα,7β,7aα-nepetalactone (2.3%)
Oxygenated 77.5 93.8±4.3 57.4±5.5
Iridane (MI) 70.4 93.1±6.8 13.1±2.9
and (E)-β-farnesene (1.0%).
1018 Natural Product Communications Vol. 6 (7) 2011 Radulović et al.

Table 2: List of the essential oil samples used in the statistical analyses.
Sample Yielda
Dendrogram
Taxon Ref. 60000
designation (%)
N. faas1 p.s.b 0.2
N. faassenii Bergmans ex Stearn N. faas2 [3a] 0.1 50000
N. faas3 [3a] 0.2
N. muss1 [3b] -c
N. muss2 [3c] 0.2 40000
N. mussinii Spreng.
N. muss3 [3d] 0.2
(syn. N. racemosa Lam.)

Dissimilarity
N. muss4 [3e]d 0.7
N. muss5 [3e] 0.1 30000
N. nep1 [3c] -
N. nepetella L. N. nep2 [3a] 0.8
N. nep3 [3f] 1.2 20000
N. nuda1 p.s. 0.5
N. nuda2 p.s. 0.6
N. nuda L. N. nuda3 p.s. 0.6 10000
N. nuda4 [3a] 0.03
N. nuda5 [3a] 0.2
N. cat1 p.s. 0.5 0
N. cat2 p.s. 0.5

N. cadmea
N. cat2
N. cat1

N. muss1
N. rtanj
N. muss4
N. muss5
N. muss3

N. muss2
N.cat5
N.nep3
N.nep2
N. cat3
N. nep1
N.sib
N.cat6
N. faas1
N.fass2

N.nuda4
N.nuda5

N. cilicia

N.faas3

N. cat4
N. crispa
N. ispah
N. mahan

N. nuda2
N. nuda1
N. nuda3
N. ucr
N. erem
N. mac
N. sept
N. folio

N. rivul
N. oxy
N. cat3 p.s. 0.4
N. cataria L.
N. cat4 [3g] -
N. cat5 [3a] 0.1
N. cat6 [3a] 0.2 A
N. macrosiphon Boiss. N. mac [3h] 0.2
N. ucrainica L. ssp. kopetdaghensis N. ucr [3i] 0.04
N. oxydonata Boiss. N. oxy [3j] 0.1
N. foliosa Moris N. foli [3k] 0.1 Dendrogram
12000
N. septemcrenata Erenb N. sept [3l] 0.4
N. sibirica L. N.sib [3a] 1.0 11000
N. rtanjensis Diklic & Milojevic N. rtanj [3m] 1.0 10000
N. cadmea Boiss. N. cadmea [3n] 2.1
N. crispa Willd. N. crispa [3o] - 9000
N. mahanensis Jamzad & Simmonds N. mahan [3o] - 8000
N. ispahanica Boiss. N. ispah [3o] -
Dissimilarity

N. eremophila Hausskn. & Bornm. N. erem [3o] - 7000


N. rivularis Bornm. N. rivul [3o] - 6000
N. cilicia Boiss. N. cilicia [3p] -
a b 5000
Given as in the original references (%, w/w or v/w); p.s.-present study;
c
Yield not given in the original references; d Reference [3e] plus personal 4000
correspondence with K. H. C. Baser; syn.-synonym. 3000

2000
As already mentioned, monoterpene profiles of hybrids 1000
and their parent taxa could serve as a mirror of the gene
0
flow [5]. We wanted to verify this hypothesis in the case of
N. cadmea
N. cat2
N. cat1

N. muss1
N. rtanj
N. muss4
N. muss5
N. muss3

N. muss2
N.cat5
N.nep3
N.nep2
N. cat3
N. nep1
N.sib
N.cat6
N. faas1
N.fass2

N.nuda4
N.nuda5

N. cilicia

N.faas3
N. crispa
N. ispah
N. mahan
N. cat4
N. nuda2
N. nuda1
N. nuda3
N. ucr
N. erem
N. mac
N. sept
N. folio

N. rivul
N. faassenii, and decided to use essential oil composition N. oxy

as a reflection of the inheritance of the biosynthetic


apparatus of this hybrid taxon.
B
Figure 2: A-Dendrogram (AHC1 analysis; original variables)
For that reason, chemical compositions of the herein representing chemical composition dissimilarity relationships of 36
analyzed samples and 29 other oils originating from Nepeta essential oil samples (observations) obtained by Euclidian
different Nepeta taxa (Table 2), including the two distance dissimilarity (dissimilarity within the interval [0, 50300], using
aggregation criterion-Ward's method). Three groups of samples (C1-C3)
previously studied oils of N. faassenii and 8 oils of the were found (from left to right); B-Expanded section of dendrogram A,
parent species N. nepetella and N. mussinii, were showing the dissimilarity interval [0, 12000].
compared using multivariate statistical analysis (MVA:
AHC and PCA), Figures 2-5. The exact identity of the The dendrogram depicted in Figure 2, obtained as the
Nepeta taxa other than N. faassenii, N. nepetella and N. result of AHC analysis performed using original variables
mussinii was unimportant for the present study and these (percentage composition of the oils, AHC1), delimitates
were chosen randomly (either from the literature or three statistically different classes of samples, C1-C3.
analyzed in parallel with N. faassenii) to provide a Class C1 (4aα,7α,7aα-nepetalactone class) groups all
representative data set, large enough to be suitable for samples with a high relative amount of 4aα,7α,7aα-
MVAs. Moreover, several different populations of N. nuda nepetalactone (more than 65%).
and N. cataria were included as a sort of a “control group”,
to estimate the potential influence of environmental and Oils obtained from N. nepetella (N. nep1-N. nep3), as well
geographical factors, and the possibility of the existence of as the two N. faassenii samples (N. faas1 and N. faas2)
different chemotypes of one taxon. were also grouped within this class. The mutual feature of
the oils, including two N. mussinii samples, clustered
within the C2 ((4aα,7α,7aβ-nepetalactone class)) was the
Nepeta x faassenii essential oil Natural Product Communications Vol. 6 (7) 2011 1019

Dendrogram Dendrogram
60000
35000

50000 30000

25000
40000
Dissimilarity

Dissimilarity
20000
30000

15000
20000
10000

10000
5000

0
0

N. ucr
N. muss2

N. muss1
N. muss4
N. muss3
N. rtanj

N. muss5
N.nuda4
N. faas1
N. cat2
N. cat1
N.cat5
N.cat6

N.fass2
N.nep2
N.sib
N. cat3
N. nep1
N. cadmea
N.nep3

N. mahan
N. crispa
N. ispah
N. cat4
N. nuda3
N. nuda1
N. nuda2
N.nuda5

N. cilicia

N.faas3
N. erem

N. sept

N. mac
N. rivul

N. folio

N. oxy

N. rtanj

N. ucr
N. muss4

N. muss5

N. muss2

N. muss3
N. nuda2
N. nuda1
N. nuda3

N.faas3

N.nuda4
N.faas1
N.cat6

N.cat5
N. faas2

N.nuda5
N. cadmea
N. cat3
N. cat1
N. cat2
N. nep2
N. nep3
N.sib
N. sept
N. mac

N. folio

N. oxy
A A

Dendrogram Dendrogram

10000
5000
9000

4000 8000

7000
Dissimilarity

Dissimilarity

3000 6000

5000

2000 4000

3000

1000 2000

1000

0 0
N. ucr
N. muss2

N. muss1
N. muss4
N. muss3
N. rtanj

N. muss5
N.nuda4
N. faas1
N. cat2
N. cat1
N.cat5
N.cat6

N.fass2
N.nep2
N.sib
N. cat3
N. nep1
N. cadmea
N.nep3
N. crispa
N. ispah
N. mahan
N. cat4
N. nuda3
N. nuda1
N. nuda2
N.nuda5

N. cilicia

N.faas3
N. erem

N. sept

N. mac
N. rivul

N. folio

N. oxy

N. rtanj

N. ucr
N. muss4

N. muss5

N. muss2

N. muss3
N. nuda2