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Volume 23, Number 5

September/October 2011

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LATIN AMERICAN AND CARIBBEAN AROMATIC PLANTS

SPECIAL ISSUE:

The Journal of Essential Oil Research is reviewed by Chemical Abstracts Service for referencing in CAS abstract literature. The Journal of Essential Oil Research is published as January/February, March/April, May/June, July/August, September/October, and November/December issues by Allured Business Media, 336 Gundersen Drive, Suite A, Carol Stream, IL 60188-2403, USA. Periodical postage paid at Carol Stream, IL and additional mailing offices. POSTMASTER: Send address changes to The Journal of Essential Oil Research, PO Box 27, Congers, NY 10920 USA. Subscriptions: For subscription inquiries, please contact us at, Tel: 1-866-616-3008 in US or 1-845-267-3008, Email: [email protected] Subscribe Online! www.JEORonline.com/subscribe United States and Canada—US$899.00 one year; all other countries—US$999.00 one year, shipped by air. See the subscription card elsewhere in this issue.

Copyright ©2011 Allured Business Media. Authorization to photocopy items is granted by Allured Business Media provided that the fee of US$14.00 per copy per item is paid directly to the Copyright Clearance Center, Transactional Reporting Service, 21 Congress St., Salem, MA 01970, 1041-2905/00. Missing issues: Claims submitted for the replacement of missing issues must be made within three months of the date of issue for subscribers in the U.S. and Canada and within six months of the date of issue for all other countries. Abstracting and Indexing: The Journal of Essential Oil Research is covered by the following abstracting and indexing services: Biological Abstracts (BIOSIS), BIOSIS Pre-views, CAB Abstracts, Chemical Abstracts Services, Current Contents/Agriculture, Biology & Environmental Service (ISI), Elsevier BIOBASE/Current Awareness in Biological Sciences, EMBASE/Excerpta Medica (Elsevier), Kosmet (IFSCC), Research Alert (ISI), SciSearch (also known as Science Citation Index-Expanded) (ISI). Allured Business Media makes all attempts to publish accurate information, however this publication may contain technical inaccuracies or typographical errors. The reader assumes all risks concerning the suitability and accuracy of the information within this publication. Allured Business Media assumes no responsibility for and disclaims all liability for any such inaccuracies, errors or omissions in this publication and in other documents referred to within or affiliated with this publication.

Robert P. AdamsBaylor UniversityGrover, Texas, USA

K. Husnu Can BaserAnadolu UniversityEskisehir, Turkey

Jose J. BarrosoUniversita de LisboaLisbon, Portugal

Joseph J. BrophyUniversity of NSWSidney, Australia

Gerhard BuchbauerUniversity of ViennaVienna, Austria

Robin CleryGivaudan Schweiz AGDubendorf, Switzer-land

Giovanni DugoUniversità di MessinaMessina, Italy

Xavier FernandezUniversité de Nice-Sophia-AntipolisNice, France

Karl Heinz KubeczkaUniversity of WurzburgMargetschochheim, Germany

Brian M. LawrenceConsultantWinstom-Salem, North Carolina, USA

Massimo MaffeiUniversity of TurinTurin, Italy

Julie L. MarkhamUniversity of West SidneyRichmond, Australia

Takayuki ShibamotoUniversity of CaliforniaDavis, California, USA

Felix TomiUniversité de CorseAjaccio, France

Ian SouthwellPhytoquestAstonville, NSW, Australia

Arthur O. TuckerDelaware State UniversityDover, Delaware, USA

Alvaro M. ViljoenTshwane University of TechnologyPretoria, South Africa

Editor in Chief — Luigi MondelloDipartimento Farmaco-chimico, Facoltà di FarmaciaUniversità degli Studi di Messina, Viale AnnunziataI- 98168 - Messina, ItalyTel: 39-090-6766536 • Fax: 39-090-358220 • E-mail: [email protected]

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AimsThe Journal of Essential Oil Research (JEOR) is a scientific journal devoted entirely to all facets of pure and applied studies on essential oils or plant volatiles, excluding those of a purely agricultural or horticultural nature. The main areas of emphasis of the journal are:

Analytical Chemistry Chemical Composition Microbiological ActivityBiological Activity Chemical Synthesis Plant Biochemistry/BiosynthesisBiotechnology Chemosystematics Toxicology

The Journal of Essential Oil Research is international in scope, as can be seen by the composition of the Editorial Board. The goal of this Board is the timely publication of papers of a high standard of technical merit and scientific quality. All manuscripts submitted for publication in the JEOR will be formally reviewed by no less than two members of the scientific community who are regarded as authorities in that field. To be considered as a subject for publication, the manuscript must contain information on the aromatic principles of a plant or its isolate, or must be directed toward furthering our knowledge of the aromatic plant and animal kingdoms. This journal will serve as a forum for the publication of formally refereed manuscripts devoted to the field of essential oils and plant volatiles. Consequently, concise contributions on the experimental or theoretical investigations of some facet of essential oils, aromatic plants, or plant and animal interactions are invited for publication.

(ISSN 1041-2905)

Volume 23, Number 5 www.JEORonline.com September/October 2011

The Journal of Essential Oil Research

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2/Journal of Essential Oil Research Vol. 22, September/October 2010

Calendar of Events

September 4–9—59th International Congress and Annual Meeting of the Society for Medici-nal Plant and Natural Product Research; Antalya, Turkey; contact: The Society for Medicinal Plant and Natural Product Research; www.ga2011.org

September 6–8—2011 PAM (Aromatic and Medicinal Plants) Congress and 30th International Days of Essential Oils & Extracts; Digne-les-Baines, France; www.pole-pass.fr

September 11–14—42nd International Symposium of Essential Oils; Antalya, Turkey; www.iseo2011.org

October 31–November 2—IFSCC Conference; Bangkok, Thailand; contact: International Federa-tion of Cosmetic Chemists; tel: 44-0-1582-726661; [email protected]; www.ifscc.org

November 3—RIFM’s 2011 Annual Meeting; contact: The Research Institute for Fragrance Mate-rials; www.rifm.org

November 6–10—IFEAT 2011; Barcelona, Spain; contact: International Federation of Essential Oils and Aroma Trades; www.ifeat.org

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(ISSN 1041-2905)

Volume 23, Number 5 www.JEORonline.com September/October 2011


Leaf Secretory Structure and Volatile Compounds of Eugenia copacabanensis Kiaersk. (Myrtaceae)

R. do Carmo de O. Arruda and C.P. Victório ......................1

Chemical Composition and Cytotoxic Activity of Essential Oil from Myrcia laruotteana Fruits

M.É.A. Stefanello, D. Riva, E.L. Simionatto, J.E. de Carvalho, A.L.T. Góis Ruiz and M.J. Salvador .......7

Chemotaxonomic Importance of Sesquiterpenes and Flavonoids in Five Argentinian Species of Polygonum Genus

M. Derita and S. Zacchino .................................................11

Seasonal Evaluation and Chemical Composition of Volatile Fractions from Piper claussenianum by Hydrodistillation and SPME

A.M. Marques and M.A.C. Kaplan ....................................15

Analysis of the Chemical Composition and Antimicrobial Activity of the Essential Oil from Lippia triplinervis Gardner (Verbenaceae)

S.G. Leitão, J.P.L. Damasceno, M.G. Martini, S.N. Miranda, P.M. Neufeld, F. Regina Salimena and H.R. Bizzo ....................................................................20

Activity against Streptococcus pneumoniae of the Essential Oil and d-Cadinene Isolated from Schinus molle Fruit

A. Pérez-López, A.T. Cirio, V.M. Rivas-Galindo, R.S. Aranda and N.W. de Torres ........................................25

Chemical Composition of Leaf Essential Oils of Calyptranthes microphylla B. Holts & M.L., Myrcia aff fosteri Croat and Eugenia octopleura Krug & Urb from Panama

A.I.S. Tenorio, D. Vargas, A. Espinosa, A. Díaz and M.P. Gupta ..........................................................................29

Essential Oil from the Leaves of Campomanesia guaviroba (DC.) Kiaersk. (Myrtaceae): Chemical Composition, Antioxidant and Cytotoxic Activity

A.C.R.F. Pascoal, C.C. Lourenço, L. Sodek, J.Y. Tamashiro, G.C. Franchi, Jr., A.E. Nowill, M.É.A. Stefanello and M. José Salvador .................................................................34

Contents

Anti-inflammatory Activity of Some Essential OilsS.Pérez G., M. Zavala S., L. García A. and M. Ramos L..................................................................38

Chemical Composition and Antibacterial Activity of Origanum majorana L. Essential Oil from the Venezuelan Andes

S. Ramos, L.B. Rojas, M. Eugenia Lucena,G. Meccia and Alfredo Usubillaga .....................................45

Anti-inflammatory and Antioxidant Activity of a Methanolic Extract of Phyllanthus orbicularis and its Derived Flavonols

Y.I. Gutiérrez Gaitén, M.M. Martínez, A.B. Alarcón, M.M.Vázquez, J.L.F. Hernández, L.D. Roche and L. Rastrelli ...................................................................50

Chemical Composition of Essential Oils from Ripe and Unripe Fruits of Piper amalago L. var. medium (Jacq.) Yunck and Piper hispidum Sw.

M.L.F. Simeone, S. Bos Mikich, L.C. Côcco, F.A. Hansel and G.V. Bianconi ...........................................54

Chemical Composition and Larvicidal Effects of Essential Oil from Bauhinia acuruana (Moric) against Aedes aegypti

R.W. da Silva Gois, L.M. de Sousa, T.L.G. Lemos, A.M.C. Arriaga and M. Andrade-Neto, G.M.P. Santiago, Y.S. Ferreira, P.B. Alves and H.C.R. de Jesus ....................59

Chemical Composition and Biological Properties of the Leaf Essential Oil of Tagetes lucida Cav. from Cuba

E.L. Regalado and M.D. Fernández, J.A. Pino, J. Mendiola and O.A. Echemendia ....................................63

Analytical Characterization of Industrial Essential Oils from Fruits and Leaves of C. aurantifolia Tan. and C. latifolia Swing

I. Bonaccorsi, P. Dugo, L. Mondello, D. Sciarrone, G. Dugo and L. Haro-Guzman ..........................................68

Appendix: Submission Guidelines


Journal of EssEntial oil rEsEarch

The Italo-Latin American Society of Ethnomedicine (SILAE, www.silae.it) is an international nonprofit 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 in 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 has aimed to individualize new ways of collaboration between its member countries and other European nations, as well as Asiatic countries, to sign accords with intergovernmental organizations. SILAE proposes to establish contacts with scientific communities, universities, and research centers for the pursuit of medicinal and food plants knowledge and to encourage the exchange and mobility of professors, researchers, and PhD students. Moreover SILAE_live, the one-to-one live chat and messenger at www.silae.it, is the first scientific chat on the Web and is a tool developed to engage the interest and imagination of the public and for helping non-scientists to understand and enjoy scientific discoveries and 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.

In Latin America, aromatic plants are an essential part of traditional health care systems. This has recently attracted the attention of many scientists and encouraged them to screen plants to study the biological activities of their oils from chemical and pharmacological investigations to therapeutic aspects. Essential oils are valuable natural products used as raw materials in many fields, including perfumes, cosmetics, aromatherapy, phytotherapy, spices and nutrition. Although essential oils have been used therapeutically for centuries, there is little published research on many of them. Most of the chemical constituents of plant essential oils belong to terpenoid compounds, including monoterpenes, sesquiterpenes, and their oxygenated derivatives. These low molecular weight (most below 300 g/mol) compounds easily diffuse across cell membranes to induce biological reactions. In recent years, there has been a tendency for applied studies of essential oils to focus on antimicrobial and the mosquito larvicidal activities as well as anti-inflammatory bioactivity.

This special issue focused on Latin American and Caribbean Aromatic Plants, which you now hold, is comprised of 15 research articles related to different areas of aromatic plants: chemical composition and biological properties of the essential oil, novel techniques for analysis and characterization of secondary metabolites, and several of these papers are collaborative works between two or more countries.

Many papers present the compositions of essential oils from different aromatic plants using analytical techniques such as GC, GC/MS, esGC, and MDGC. Various authors report the in vitro activity of an essential oil against bacteria, yeasts, plasmodia and HHV 1/HHV 2 strains, leukemic cells lines, different human cancer cells and Aedes aegypti. Four papers present the compositions of essential oils from different Myrtaceae species; one paper covers reports published in the last five years on the anti-inflammatory activities of several essential oils isolated from 43 plants. Two papers deal with the GC and GC/MS analysis of more polar compounds in aromatic plant extracts as sesquiterpenes and flavonoids.

The editors would like to thank the contributors who gave so generously their time and experience and who made this publication a valuable tool for scientists in the field of essential oil and aromatic plants chemistry, analysis 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.

The editors are also very grateful to the Editorial Board of JEOR for embracing this project with interest and enthusiasm, and for the opportunity to publish this special issue. We hope that this will be the first in a long series in this attractive and interesting journal.

Luca RastrelliGuest EditorDipartimento di Scienze Farmaceutiche e Biomediche University of Salerno, Italy

Luigi MondelloEditor in Chief, JEOR

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Identifi cation of Essential Oil Components byGas Chromatography/Quadrupole Mass SpectroscopyBy Robert P. Adams

This is the third edition on mass spectra and retention times of common components in plant essential oils. It differs from the previous editions in two important areas: An additional 400 compounds have been added and all 1606 compounds have been carefully re-analyzed from their original sources on an HP5970 MSD mass spectrom-eter using HP Chemstation software. In addition, the library (including retention times) is now available for the most common mass spectrometer/computer systems.

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Vol. 23, September/October 2011 Journal of Essential Oil Research/1

Rec: May 2011

Acc: May 2011

Leaf Secretory Structure and Volatile Compounds of Eugenia copacabanensis Kiaersk. (Myrtaceae)

Rosani do Carmo de O. Arruda Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul (UFMS), Campo Grande, MS,

Brasil

Cristiane P. Victório*Centro Universitário Estadual da Zona Oeste (UEZO), Rio de Janeiro, Rio de Janeiro, 23070-200, Brasil

Abstract

Eugenia copacabanensis Kiaersk. (Myrtaceae) is restricted to salt marshes or restinga areas along the southeastern coast of Brazil. This work analyzes the leaf secretory structures and volatile compounds produced in E. copacaban-ensis plants collected in the Marambaia Restinga of Rio de Janeiro City. Simultaneous-distillation extraction (SDE), when combined with GC-FID and GC/MS analyses, revealed a-pinene (20.2%) and b-pinene (50.4%) as the major volatiles of this plant, proving that it is rich in monoterpenes. trans-Caryophyllene (10.3%) was the only sesquiterpene identified. Using light microscopy, E. copacabanensis leaves presented numerous randomly distributed oil secretory cavities in the mesophyll, while histochemical tests showed the presence of terpenoids and lipophilic substances ac-cumulated in secretory cavities and parenchyma cells.

Key Word Index

Eugenia copacabanensis Kiaersk., Myrtaceae, Atlantic Rainforest, a-pinene, b-pinene, caryophyllene, restinga, secretory activity, simultaneous-distillation extraction, volatile composition.

1041-2905/11/0001-05$14.00/0 —© 2011 Allured Business Media

J. Essent. Oil Res., 23 (September/October 2011)

*Address for correspondence: [email protected]

Introduction

Restingas comprise one of Brazil’s prime ecosystems. Restingas are characterized by areas of open vegetation within the coastal plains, and because of their abundant endemic species, both known and unknown, restingas are protected (1). Under the influence of the Atlantic Ocean, these ecosys-tems occur in areas between the inner dunes and the lowland altitudinal zone of the Atlantic Rainforest. Families such as Bromeliaceae, Leguminosae, Myrtaceae and Clusiaceae are among the most represented in this biome (2). The diversity of genera and endemic species provides both food and shelter to forest fauna.

Myrtaceae is an ecologically important family in Brazil’s Atlantic Rainforest, and it represents the largest number of species in the Brazilian restinga (3). This monophyletic family is characterized by the presence of entire leaves containing oil glands and internal phloem (3, 4). Secreted oil is accumulated in a spherical internal cavity bounded by a secretory epithelium. Against the light, the secretory cavities in leaves are visible to the naked eye as translucent dots.

Considered the largest of the New World Myrtaceae genera, Eugenia L. is currently estimated to contain 500 to

2,000 species, and their distribution ranges from southern Mexico to Cuba and the Antilles and then south to Uruguay and Argentina. A few species are found in Africa (5). Eugenia includes species with fleshy fruits used as food, and some are cultivated for ornamental purposes.

Eugenia copacabanensis Kiaersk. (subtribe Eugeniinae O. Berg, Myrtoideae) is known as “cambuí amarelo,” or yel-low cambuí, by the yellow color of its fruits (Figure 1A). Its specific name most likely derives from the Copacabana district of Rio de Janeiro City. The distribution of E. copacabanensis occurs sporadically in shoals surrounding Rio de Janeiro City. This plant’s natural habitat is restricted to salt marshes or restinga areas in Rio de Janeiro State (specifically, in the South Beach Reserve, Marambaia Restinga, Jacarepaguá, and state ecological reserves in Maricá Municipalities and Cabo Frio). Consequently, this species is considered endemic and rare (6). In the Marambaia Restinga, E. copacabanensis occurs in open and unflooded areas, where it is exposed to intense wind and light. The reduction in the number of individuals of this species is directly related to human occupation of these natural salt marshes, thus posing a threat to the entire tropical ecosystem. It is hoped that the dual goals of creating conservation units


Arruda et al.

Table I. The main leaf volatiles (%) of Eugenia copacabanensis Kiaersk. collected in the Marambaia Restinga, Rio de Janeiro.

Constituent RI lit.a RI calculated Relative area (%)b

a-pinene 939 936 20.2±7.5

b-pinene 979 980 50.4±14.81,8 cineole 1031 1034 2.4±0.5geijerene (n.d.)* 1150 1146 1.7±0.6menth-1,5-dien-8-ol (n.d.)* 1170 1168 1.0±0.2á-ylangene (n.d.)* 1375 1373 1.2±0.3trans-caryophyllene 1419 1414 10.3±2.8 Monoterpenes total 74.0 Sesquiterpenes total 13.2 Total identified 87.2

aRI= Retention Index (see Adams, 2007). bResults are given as the mean of triplicate extractions; n.d. = not detected by GC/MS analysis by low concentration. *Probably terpenes obtained by comparison with RI analysis of Nakamura et al. (27).

Figure 1. Light microscopy images showing secretory structures of Eugenia copacabanensis Kiaersk. A. Leaves and fruit of E. copacabanensis. B-C. Paradermic sections of leaf mesophyll showing the distribution of secretory cavities. Histochemical tests: (B) Sudan IV and (C-D, F) Red oil O. D. Cross-section of leaves: reaction of Red oil O showed terpenoids in secretory cavities, parenchyma and a thick cuticle; arrows indicate druses. E. Paradermic section, in detail, overlying cells – Nile blue test. F, H and I. Petiole. F. Red oil O test; arrows indicate the thick cuticle on epidermis. G-H. KOH (potassium hydroxide) test for flavonoids (phenolic compounds). I. Ferric chloride test for phenolic compounds. *Secretory cavity.


E. copacabanensis

in the State of Rio de Janeiro and protecting arbustive plants where this species grows will dramatically reduce the risk of extinction.

Leaf anatomy of many Eugenia species has been studied in order to identify diagnostic characters, morpho-anatomy and the histochemistry of secretory cavities. Since this genus is edible and has medicinal uses, research has also provided chemical identification of its volatile compounds (7). In the Myrtaceae family, Eugenia is the fourth most important genera in the production of essential oils, after Eucalyptus, Melaleuca and Psidium. The chemical composition of essential oils in the Eugenia species reveals the presence of various types of terpenoids that can be used in the pharmaceutical, cosmetic and agrochemical industries. Particularly, the boiling of E. uniflora and E. dysenterica leaves produces a liquid yielding anti-inflammatory, antimicrobial and antihypertensive effects (8, 9). Anti-rheumatic effect has been attributed to the leaves of E. brasiliensis (10), and hypoglycemic activity has been reported for E. punicifolia (11).

Despite the large number of Brazilian species of Eugenia and their wide distribution in several biomes, relatively few studies have addressed either the anatomical structure of their leaves or the chemical composition of their essential oils. Therefore, this study aimed to identify the composition of volatile secre-tions, characterize the leaf anatomy, as well as localize the leaf volatile secretory structures, of E. copacabanensis.

Experimental

Plant material: Leaves of Eugenia copacabanensis were collected between 10 and 11 h from three individual plants growing in open shrub formation in July 2010, in the Maram-baia Restinga (Municipality of Mangaratiba; 23º02’75’’S, 43º35’68’’W), altitude 7 m, Rio de Janeiro City, Brazil. At the collection site, the following environmental conditions are typically observed. The climate is tropical, with a wet season having an average monthly rainfall between 40 and 50 mm or less between July and August and a dry winter season with temperatures averaging 20.9ºC. The average annual rainfall is 1,239.7 mm, and the average annual temperature is 23.7ºC (Instituto Nacional de Meteorologia-INMET, Brazil).

Marcelo da Costa Souza (Rio de Janeiro Botanical Garden, Brazil) undertook the taxonomic identification of E. copaca-banensis. A voucher specimen is deposited at the Herbarium of Rio de Janeiro Botanical Garden under accession number RB 415728.

Leaf anatomy and histochemistry: Mature leaves of two to three individuals were taken from the third or fourth nodes from the apex of the branch. The leaves were fixed in FAA70 (12) and preserved in 70% ethanol. The leaves were sectioned in longitudinal and transverse planes by the free-hand method or by Ranvier microtome. The leaf sec-tions were clarified in sodium hypochlorite and rinsed in 1% acetic acid and distilled water. Afterwards, the samples were stained with Alcian Blue 1% and fucsin 0.1% and rinsed in distilled water. All samples were mounted in 50% glycerin on slides with cover slips. The epidermis was described using leaf segments separated by maceration in solution of acetic acid and hydrogen peroxide 1:1 for 12 h, rinsed in distilled

water, stained with 1% Alcian Blue in 50% ethanol, and mounted in 50% glycerin.

Histochemical tests were performed on fresh leaf blades and petioles, which were sectioned by the free-hand method and then submitted to the following staining reagents: Sudan III, Sudan IV (11) and Sudan black B for total lipids (13); Red oil O for terpenoid compounds (14); Nile blue for acid and neutral lipophilic compounds (15); aqueous solution contain-ing 5% KOH (potassium hydroxide) for flavonoids; and ferric chloride for phenolic compounds (12). Control procedures for histochemical tests were carried out. All samples were rinsed with distilled water and mounted in glycerin on slides with cover slips. Observations were carried out and captured on light microscopy using an Olympus (BX-41).

Volatile extraction and analyses: Fresh leaves (5 g) of E. copacabanensis were cut and submitted to simultaneous distillation-extraction (SDE) for 90 min using 2 mL of dichlo-romethane as an organic collecting solvent (16). Mineral oil bath under stirrer/heat plate was used to apply heat to flasks. The heating temperatures for the sample and solvent flasks were controlled to 110-130oC and 55-60oC, respectively. The vapors were condensed as a result of the circulation of cooling water pumped to the apparatus. SDE samples were introduced to GC-FID and GC/MS for analysis. Table I shows averages of three extractions collected in the same period, July 2010.

Analytical GC (gas chromatography) was carried out on a Varian Star 3400 gas chromatograph fitted with a DB-1-MS column (30 m × 0.25 mm i.d.; 0.25 μm film thickness) and equipped with flame ionization detection (FID). Temperature was programmed from 60-240ºC at 3°C/min. The injection consisted of 1 μL of samples. Hydrogen was used as the carrier gas at a flow rate of 1 mL/min. The injector temperature was 260°C, with interface of 200°C. Leaf volatile samples were analyzed in splitless mode.

GC/MS analyses were carried out on a Shimadzu Model GC/MS-QP 5000 fitted with a HP-5/MS fused silica capillary column (30 m x 0.25 mm i.d.; 0.25 μm film thickness). GC/MS conditions were the same as above, except for 1) He which was used as the carrier gas at a flow rate of 1 mL/min and 2) the mass spectrometer which was operated on electron impact mode at 70 eV. Quantification was performed from GC-FID profiles using relative areas (%). Injections were carried out in triplicate from three volatile extractions, and standard de-viations were considered. Identification of components in the volatiles was based on retention indices relative to n-alkanes (C8-C19) and computer matching with the National Institute of Standards and Technology (NIST 05) library, as well as comparison between mass fragmentation patterns and those reported in the literature (17).

Results and Discussion

E. copacabanensis can be classified as a shrub or tree that grows up to 6 m in height. The trunk has a laminated outer skin, exfoliating in papyraceous blades. The leaves are ellipti-cal, ovate or lanceolate and glabrous, with yellowish petiole. In addition, the leaves are shiny with midrib prominent adaxially, densely dotted; revolute leaf margin with yellowish thickening (3) (Figure 1A).


Arruda et al.

In frontal view, anatomical analyses showed that the epi-dermal cells of E. copacabanensis present straight and thick walls covered by a shiny, smooth and thick cuticle (Figure 1D-E). Numerous stomata occur only on the abaxial surface. According to Fontenelle et al. (18), the stomata are anomocytic, and the entire leaf surface is covered by a layer of wax flakes. Both the thick, shiny cuticle and the wax reduce the invasion of pathogens, aid in water conservation, and reflect incident light, thereby cooling the leaf (19). The cuticular layer showed an intense reaction to the presence of lipophilic substances and terpenoids, as detected by staining with Sudans and Red oil, indicating a possible relationship of this class of metabolites with protection against radiation and high air evaporative de-mand. Environments like restingas are subject to fluctuations in water availability; therefore, plants exhibit many attributes associated with resource conservation (20). On drier, nutrient-poor sites, many species have a strongly thickened cuticular layer and cutinization with protective function, reducing the loss of nutrients by leaching (21).

On both sides of the leaf cells, either isolated or pairs of polygonal cells overlap the internal secretory cavities (termed overlying cells) (Figure 1E). The overlying cells have low af-finity for dyes, and in fresh leaves of E. copacabanensis, they are translucent. The number of overlying cells among Eugenia species varies and, as such, represents a taxonomic feature (18, 22). Although optical microscopy does not show visible pores, it is possible that these cells are related to the elimination of volatile substances accumulated in the secretory cavities whose odor pervades the site collection areas of the plants analyzed. More detailed studies could clarify the function of these cells for the species of Eugenia.

In cross section, the leaf of E. copacabanensis presents a one-layered epidermis. The mesophyll is isobilateral (Figure 1D, G), a pattern very different from most Eugenia species (4, 23), but similar to the description of Fontenelle et al. (18) for E. copacabanensis found in the City of Maricá. Two layers of palisade parenchyma with many chloroplasts on each side of the epidermis can be observed. The spongy parenchyma is composed of six bulky layers and a few chloroplasts, con-stituting a watery tissue (Figure 1G).In E. copacabanensis, exposure to the intense light is reflected in the development of the palisade under both sides of the leaf. A positive reaction to tests for phenolics and flavonoids was found throughout the mesophyll and in cells of the epidermis. These two important substances reduce the absorption of wavelengths harmful to the sub-cellular structure (24). In restinga environments, plants are exposed to intense light, and, as such, the produc-tion of flavonoids shows an evolutionary correlation with the protection of plants against high levels of ultraviolet radiation found in these locations. Abundant calcium oxalate druses are often found in the ground parenchyma of E. copacabanensis (Figure 1D, G, H) (18).

The essential oil cavities appear to be randomly distributed in mesophyll, and they accumulate a yellowish-green secretion directed toward both sides of the leaf (Figure 1B-D). In the leaf blade of E. copacabanensis, secretory structures are located

adjacent to the leaf epidermis, and in the petiole, they more deeply embedded (Figure 1F). The location of the secretory cavities in the leaf seems to vary between species of Eugenia. In E. umbelliflora and E. brasiliensis, the secretory structures may be the deepest on the adaxial side and near the periphery on the abaxial surface, connecting with the epidermis through a set of cells that form a neck (25).

The secretory cavities are spherical, large and formed by a secretory epithelium that delimits a space which retains the material secreted (Figure 1B-C, G). Secretory cells have thin walls and react positively to tests for phenolic compounds. The histochemical tests showed that secretions, which accumulated in the cavities of E. copacabanensis, showed positive reaction to tests for the recognition of acidic and neutral lipophilic sub-stances, terpenoids and essential oils (Figure 1B-D). According to Metcalfe and Chalk (4), the presence of secretory cavities containing oil terpenoids and other aromatic compounds is a hallmark of Myrtaceae.

The number of secretory cavities can vary within the same species, depending on environmental factors. Accordingly, Donato and Morretes (26) found that individuals of E. brasil-iensis grown under conditions of high brightness, as well as high salinity and temperature, had a greater number of cavities compared to plants of the same species grown under low light conditions and more humidity.

The vascular system of E. copacabanensis consists of vascular bundles associated with fibers surrounded by a parenchymatous endoderm with chloroplasts. In the petiole and midrib region, the vascular system is organized in the form of an open arc, and the phloem completely surrounds the xylem characterizing the amphicrival bundles (Figure 1H) common to some Myrtaceae (4) and observed in some Myrtales. Pericyclic fibers bypass the vascular system of the petiole. Peripheral layers of collenchyma and internal parenchyma cells are observed to fill the cortical region of the petiole and midrib. Secretory cavities are located deep, and flavonoids and phenolic substances were identified in this region of the leaf (Figure 1G, I).

Morpho-physiological changes in plants in response to dif-ferent environmental and survival conditions are, collectively, an important component of tropical biodiversity. In this regard, the distribution of E. copacabanensis in both open and shaded sandbanks suggests the plasticity of this species in adapting to the environment. In fact, environmental conditions account for many phenotypic differences in the morpho-anatomical and chemical features seen in the Eugenia species.

Through histochemical testing, we verified the correlations among volatile composition, production and accumulation, especially in secretory cavities. The content of the cavities is composed of terpenoids, essential oils and lipophilic sub-stances, as confirmed by gas chromatography analysis. The main volatile components identified were a-pinene (20.22%), b-pinene (50.41%), 1,8-cineole (2.38%) monoterpenes, and trans-caryophyllene (10.34%). Such substances are also found in other species of Eugenia (26), being intensively volatilized at times of increased solar intensity.

Using SDE, this study revealed a higher concentration of


E. copacabanensis

a-pinene and b-pinene in E. copacabanensis when compared to data provided in other studies for E. candolleana DC., E. punicifolia (HBK) DC, or E. copacabanensis collected in Janu-ary using hydrodistillation (27). A high content of a-pinene and b-pinene was also found in E. rotundifolia collected in the Grumari Restinga, Rio de Janeiro (28). On the basis of other studies, many variations in volatile composition have been shown among Eugenia species as a consequence of ecosystem type, seasonal changes, including rainfall, and the intensity of sunlight (29, 30). As determined by SDE, caryophyllene, among all volatiles of E. copacabanensis, was the unique ses-quiterpene found in this study. Caryophyllene is one of the most recognizable sesquiterpenes in chemical profiles of the essential oils of Eugenia spp, as shown in the present study (27). It should be noted that detection by GC/MS may have been prevented by the low concentration of other sesquiter-penes in the volatile dichloromethane extract, particularly in view of the contradictory findings of Nakamura et al. (27), who reported the occurrence of about 81.15% of sesquiterpenes against 13.37% monoterpenes in leaf oils of E. copacabanensis using hydrodistillation. Differences in the composition of E. copacabanensis volatiles may be associated with differences in the ecosystem of origin. We collected this species in its natural habitat, the restinga ecosystem, where it is under strong environmental pressure caused by high solar intensity, temperature and salinity when compared with the specimen used by Nakamura et al. (27). Their specimen was cultivated in a canopied area with low light intensity and low temperature. Other factors can also influence volatile composition, such as seasonality and the method of volatile extraction.

Numerous secretory cavities with spherical lumen were observed to be randomly distributed in mesophyll. The use of histochemical reagents, such as Sudans, Red oil and Nile blue, showed that these cavities are responsible for the production and accumulation of lipophilic substances, terpenoids and essential oils. This finding agrees with volatile composition obtained by gas chromatography, in which it was found that the main compounds were a- and b-pinenes and 1,8-cineole, representing more than 70%, and trans-caryophyllene, consisting of 10.34%, of volatiles. Overall, the anatomical and phytochemical results of this study could help researchers engaged in species identification and could be applied to phylogenetic approaches. Moreover, the volatile composition of E. copacabanensis, as demonstrated in this work, supports the pharmacological effects indicated for the essential oils of some Eugenia species (8, 9). Finally, in addition to medicinal uses, research has demonstrated that the essential oils of some species of neotropical Myrtaceae can be used as natural insecticides, suggesting that the volatile substances in E. copacabanensis, as determined in this study, might also serve as safe insect control agents.

Acknowledgements

We especially acknowledge the FAPERJ for financial support, the taxonomist Marcelo da Costa Souza for species identification and the Brazilian Army, in particular those responsible for the administra-tion of the Marambaia Restinga, for their stewardship of the area and granting us access to the material used in this study. We also thank

Anestor Mezzomo and Marco Lacerda, who lent us their photos of E. copacabanensis habits, and David Martin who edited the English version.

References

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A. Kelecom, G.L. Reis, P.C.A. Fevereiro, J.G. Silva, M.G. Santos, 2. C.B.M. Neto, M.S. Gonzalez, R.C.S. Gouvea and G.S.S. Almeida, A multidisciplinary approach to the study of the fluminense vegetation. An. Acad. Bras. Ciênc., 74, 171-181 (2002).

M.C. Souza and M.P. Morim, Subtribos Eugeniinae O. Berg e Myrtinae 3. O. Berg (Myrtaceae) na Restinga da Marambaia, RJ, Brasil. Acta Bot. Bras., 22(3), 652-683 (2008).

C.R. Metcalfe and L. Chalk, 4. Anatomy of the Dicotyledons -Leaves, stem and wood in relation to Taxonomy with notes on economic uses. Clarendon, Oxford UP (1950).

F.F. Mazine and V.C. Souza, New species of 5. Eugenia sect. Racemosae (Myrtaceae) from Brazilian Amazon Rainforest. Kew Bull., 64, 147–153 (2009).

D.S.D. Araujo, 6. Análise florística e fitogeográfica das restingas do Estado do Rio de Janeiro. Ph.D. Thesis, Universidade Federal do Rio de Janeiro, Rio de Janeiro (2000).

M.F.S. Ramos, S.S. Monteiro, V.P. da Silva, M.J. Nakamura, A.C. Siani, 7. Essential oils from Myrtaceae species of the Brazilian Southeastern Forest (Restinga), J Essent Oil Res, 22, 109-112 (2010).

M.E.A. Stefanello, A.C. Cervi, I.Y. Ito, M.J. Salvador, A. Wisniewski Jr 8. and E.L. Simionatto, Chemical composition and antimicrobial activity of essential oils of Eugenia chlorophylla (Myrtaceae). J. Essent. Oil Res., 20, 75-78 (2008).

A.E. Consolini, O.A. 9. Baldini and A.G. Amat, Pharmacological basis for the empirical use of Eugenia uniflora L. (Myrtaceae) as antihypertensive. J. Ethnopharmacol., 66, 33-39 (1999).

P. Corrêa, 10. Dicionário das Plantas Úteis do Brasil e das Exóticas cultivadas. Imprensa Nacional. Ministério da Agricultura, Rio de Janeiro, vol. III. (1984).

L.I.F. Jorge, J.P.L. Aguiar and M.L.P. Silva, Anatomia foliar de pedra 11. hume-caá (Myrcia sphaerocarpa, M. guianensis e E. punicifolia - Myrtaceae). Acta Amaz., 30, 49-57 (2000).

D.A. Johansen, 12. Plant Microtechnique. McGraw-Hill, New York (1940).

A.G.E. Pearse, 13. Histochemistry Theoretical and Applied, 4th ed. Longman Group Limited, London (1980).

G. Clark, 14. Staining Procedures. Williams and Wilkins, Baltimore (1981).

A.J. Cain, The use of Nile blue in the examination of lipids. 15. Q. J. Microsc. Sci., 88, 383-392 (1947).

M. Godefroot, P. Sandra and M. Verzele, New method for quantitative 16. essential oil analysis. J. Chromatogr., 203, 322 (1981).

R.P. Adams, 17. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed. Allured Publ. Corp., Carol Stream, IL (2007).

G.B. Fontenelle, C.G. Costa and R.D. Machado, Foliar anatomy and 18. micromorphology of eleven species of Eugenia L. (Myrtaceae). Bot. J. Linn. Soc., 115, 111-133 (1994).

V.P. Gutschick, Biotic and abiotic consequences of differences in 19. leaf structure. New Phytol., 143, 3-18 (1999).

B.H.P. Rosado and E.A. Mattos, Variação temporal de características 20. morfológicas de folhas em dez espécies do Parque Nacional da Restinga de Jurubatiba, Macaé, RJ, Brasil. Acta Bot. Bras., 21, 741-752 (2007).

I.M. Turner, Sclerophylly: primarily protective? Func. Ecol., 21. 8, 669-675 (1994).

C.M.V. Cardoso, S.L. Proença and M.G. Sajo, Foliar anatomy of 22. the subfamily Myrtoideae (Myrtaceae). Aust. J. Bot., 57, 148-161 (2009).


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S.M. Gomes, N.S.D.N. Somavilla, K.M. Gomes-Bezerra, S.C. 23. Miranda, O.S. De-Carvalho and D. Graciano-Ribeiro, Anatomia foliar de espécies de Myrtaceae: contribuições à taxonomia e filogenia. Acta Bot. Bras., 23, 223-238 (2009).

J.D. Mauseth, 24. Plant anatomy. Benjamin Cummings Publ. Co., Menlo Park, California (1988).

A.M. Donato and B.L. Morretes, Foliar anatomy of 25. Eugenia brasiliensis Lam. (Myrtaceae) from restinga and forest areas. Braz. J. Pharmacong., 17, 426-443 (2007).

R.A. Cole, W.A. Haber and W.N. Setzer, Chemical composition of 26. essential oils of seven species of Eugenia from Monteverde, Costa Rica. Biochem. Syst. Ecol., 35, 877-886 (2007).

M.J. Nakamura , S.S. Monteiro, C.H.B. Bizarri , A.C. Siani, M.F.S. 27. Ramos, Essential oils of four Myrtaceae species from the Brazilian southeast. Biochem. Syst. Ecol., 38, 1170-1175 (2010).

A.C.A. Defaveri, A. Sato, L.B. Borré, R.A.S. San Gil, R.C.O. Arruda 28. and C.A.S. Riehl, Eugenia neonitida Sobral and Eugenia rotundifolia Casar. (Myrtaceae) essential oils: composition, seasonality influence, antioxidant activity and leaf histochemistry. J. Braz. Chem. Soc., in press (2011).

D.C.H. Fischer, R.P. Limberger, A.T. Henriques and P.R.H. Moreno, 29. Essential oils from leaves of two Eugenia brasiliensis specimens from Southeastern Brazil. J. Essent. Oil Res., 17, 499-500 (2005).

N.P. Lima, S.H.F. Cerqueira, O.A. Fávero, P. Romoff and J.H.G. Lago, 30. Composition and chemical variation of the essential oil from leaves of Eugenia brasiliensis Lam. and Eugenia sp. (Myrtaceae). J. Essent. Oil Res., 20, 223-225 (2008).


Rec: Feb 2011

Acc: June 2011

Chemical Composition and Cytotoxic Activity of Essential Oil from Myrcia laruotteana Fruits

Maria Élida A. Stefanello and Dilamara RivaDepartamento de Química, Universidade Federal do Paraná, Caixa Postal 19081, 81531-900, Curitiba, PR, Brazil

Edésio L. SimionattoInstituto de Pesquisas Tecnológicas de Blumenau, Universidade Regional de Blumenau, 89030-080, Blumenau, SC, Brazil

João E. de Carvalho and Ana Lucia T. Góis Ruiz Divisão de Farmacologia e Toxicologia, CPQBA, UNICAMP, Caixa Postal 6109, 13083-970, Campinas, SP, Brazil

Marcos J. Salvador*Instituto de Biologia, Departamento de Biologia Vegetal,Curso de Farmácia, UNICAMP, Caixa Postal 6109, 13083-970,

Campinas, SP, Brazil

Abstract

The essential oil isolated by hydrodistillation from unripe fruits of Myrcia laruotteana Camb. (Myrtaceae) was analyzed by GC and CG/MS. Forty-four components were identified, representing around 83% of total oil. The major components were a-bisabolol (23.6%) and a-bisabolol oxide B (11.5%). The cytotoxicity of the oil and of a fraction rich in a-bisabolol was tested in vitro against U251 (glioma), UACC-62 (melanoma), MCF-7 (breast), NC1-ADR/RES (ovarian-resistant), 786.0 (kidney), NCI-H460 (lung), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon) and K562 (leukemia) human cancer cells and against VERO (no cancer cell). The oil exhibited antiproliferative activity against all cell lines (TGI < 100 mg/mL), with exception of NCI-H460 cell line (TGI > 125 mg/mL). The highest activity of the oil was observed against U251 (TGI 20.46 mg/mL), 786.0 (TGI 20.74 mg/mL), UACC-62 (TGI 26.98 mg/mL) and PC-3 (TGI 27.63 mg/mL) cell lines. The fraction rich in a-bisabol showed a similar activity profile. It was most active against OVCAR-3 (TGI 8.58 mg/mL) and 786.0 (TGI 8.74 mg/mL).

Key Word Index

Myrcia laruotteana, Myrtaceae, essential oil composition, a-bisabolol, a-bisabolol oxide B, antiproliferative activity.



Introduction

Myrcia DC (Myrtaceae) is a large genus represented in Brazil by more than 300 species (1). Myrcia laruotteana Camb., known as “cambui” is a shrub or treelet, growing wild in the Central, Southeastern and Southern Brazil, Paraguay and Northern Argentine. Its leaves are oblong (6 cm long and 2 cm wide), with a weak aroma. The flowers are white and, in contrast with leaves, strongly aromatic. The unripe fruits are bright-green, containing essential oil that decrease during the ripening, giving edible deep purple small berries (5-10 mm), almost without smell (2). The chemical composition of essen-tial oils of leaves and flowers was previously reported (3). The present work describes the essential oil composition of unripe fruits and its antiproliferative activity against cell lines.

Experimental

Plant material: Unripe fruits of M. laruotteana (2-5 mm long) were collected in November 2009 in Curitiba, Paraná State, Brazil (S 25o 25´ 48´´, W 49o 16´ 15´´, 934 m). The plant was identified by Dr. Armando C. Cervi (Departamento de Botânica, UFPR) and a voucher was deposited in the Her-barium of Universidade Federal do Paraná (UPCB) under code number 53303.

Fresh unripe fruits were submitted to hydrodistillation in a Clevenger-type apparatus for 4 h. The oil was recovered with diethyl ether and dried over anhydrous Na2SO4. The solvent was removed under vacuum. The oil was kept under refrigera-tion for further analysis.

Analysis of the essential oils: Oil sample analyses were performed on a Shimadzu GC-17A gas chromatograph (FID)

M. laruotteana


equipped with a DB-5 fused silica capillary column (30 m ´ 0.25 mm, 0.25 mm film thickness), temperature programmed as follows: 50ºC for 3 min, and then programmed from 50-240ºC at 3ºC/min, after which it was kept isothermal at 240ºC for 5 min. The carrier gas was He at a flow of 1.2 mL/min; injector port and detector temperature were 240ºC and 250ºC, respectively. Samples were injected by splitting and the split ratio was 1:20. The relative percentage of components was based on the peak

Table I. Chemical composition (%) of essential oil from M. laruotteana unripe fruits.

Number Componenta R.I.b R.I.c %

1 perillene 1102 1092 0.4 ± 0.12 endo-fenchol 1114 1112 tr3 4-hydroxycryptone 1315 1306 0.6 ± 0.14 ethyl nerolate 1354 1359 0.2 ± 0.05 E-b-damascone 1414 1418 tr6 dehydrosesquicineole 1471 1466 0.6 ± 0.17 trans-cadina-1(6),4-diene 1476 1473 0.6 ± 0.18 a-muurolene 1500 1497 0.6 ± 0.19 d-amorphene 1512 1512 0.4 ± 0.110 g-cadinene 1513 1517 0.4 ± 0.111 trans-calamenene 1522 1521 0.1 ± 0.012 a-calacorene 1545 1541 0.4 ± 0.013 selina-3,7(11)-diene 1546 1550 0.6 ± 0.114 trans-dauca-4(11),7-diene 1557 1556 tr15 germacrene B 1561 1563 2.1 ± 0.116 maaliol 1567 1569 tr17 spathulenol 1578 1579 5.4 ± 0.118 caryophyllene oxide 1583 1581 tr19 globulol 1590 1587 6.3 ± 0.220 viridiflorol 1592 1595 4.6 ± 0.121 guaiol 1600 1605 0.6 ± 0.222 rosifoliol 1600 1608 0.6 ± 0.223 ∆,10-di epi-cubenol 1619 1615 tr24 10-epi-g-eudesmol 1623 1622 tr25 1-epi-cubenol 1628 1630 0.9 ± 0.126 muurola-4,10(14)-dien-1b-ol 1630 1631 0.9 ± 0.127 cis-cadin-4-en-7-ol 1636 1638 tr28 epi-a-cadinol 1640 1644 2.4 ± 0.029 epi-a-muurolol 1642 1647 2.2 ± 0.030 a-muurolol 1646 1650 tr31 a-bisabolol oxide B 1658 1657 11.5 ± 0.532 a-cadinol 1654 1659 5.8 ± 0.533 khusinol 1680 1681 tr34 a-bisabolol 1685 1690 23.6 ± 0.535 10-nor-calamenen-10-one 1702 1705 tr36 oplopanone 1740 1737 tr37 E, E-farnesol 1741 1739 1.4 ± 0.138 a-bisabolol oxide A 1749 1758 0.6 ± 0.039 b-bisabolen-12-ol 1760 1765 1.3 ± 0.140 2E, 6E-methylfarnesoate 1784 1781 5.8 ± 0.641 b-bisabolenol 1789 1789 tr42 eudesm-11-en-4a,6a-diol 1808 1810 0.5 ± 0.143 iso-acorone 1811 1815 tr44 2Z, 6E-farnesyl acetate 1822 1820 1.4 ± 0.1 Total identified 82.8 Monoterpenes 1.2 Sesquiterpene hydrocarbons 5.8 Oxygenated sesquiterpenes 75.8

Compounds are listed in order of their elution from a CP-Sil-8CB column; tr = trace < 0.09%;a Identification based on mass spectra and RI published (5) and computer matching of the mass spectra with NIST 1998 library (quality level more than 90%); b retention index published (5); c retention index experimental on a CP-Sil-8CB column.

areas obtained by electronic integration without FID response factor correction. The results are average of three analyses.

GC/MS analysis was performed on Varian Saturn 2000 apparatus using a CP-Sil-8CB fused silica capillary column (30 m x 0.25 mm; 0.25 mm film thickness) in the same condi-tions described above, with MS operating by electron impact ionization at 70 eV with scan mass range of 40-400 m/z at a sampling rate of 1.0 scan/s. Compounds were identified by

Stefanello et al.


a-bisabolol (78.4%). This fraction was analyzed by 1H and 13C NMR (Brucker AC200, 200 MHz, CDCl3). The spectra data were compatible with a-bisabolol (7).

Cytotoxicity assay: It was used the U251 (glioma, CNS), UACC-62 (melanoma), MCF-7 (breast), NCI-ADR/RES (ovarian-resistent), 786-0 (kidney), NCI-H460 (lung, no small cells), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon), K562 (leukemia) and VERO cell lines. The assay was done as

Table II. Antiproliferative activity of essential oil of Myrcia laruotteana and a fraction rich in a-bisabolol

Cell lines TGI (mg/mL) a

Essential Oil EOF b Doxorubicin

U251 20.46 16.89 6.24UACC-62 26.98 28.00 0.30MCF-7 88.31 20.84 >25 NCI-ADR/RES >125 >50 >25 786-0 20.74 8.74 25NCI-H460 79.83 33.50 >25 PC-3 27.63 - 6.32 OVCAR-3 52.36 8.58 3.30 HT-29 59.29 19.16 >25 K562 - 25.48 0.15

VERO 36.45 7.91 >25

-: No tested; a TGI – Total Growth Inhibition – concentration that inhibited cell growth by 100%. The coefficients of variation obtained in these analyses were below to 5%; b fraction of the essential oil containing a-bisabolol (78.4 ± 2.0%), a-bisabolol oxide B (7.8 ± 0.6%), viridiflorol (3.4 ± 0.5%), epi-a-muurolol (2.5± 0.2%) and 1-epi-cubenol (2.0± 0.1%) as determined by GC-FID.

computer search using digital libraries of mass spectral data (4) and by comparison of their retention indices and authen-tic mass spectra (5), relative to C8-C32 n-alkane series (6) in a temperature-programmed run.

Isolation of a fraction rich in a-bisabolol: A sample of oil (80 mg) was submitted to silica gel column chromatography eluted with pentane, dichlorometane and diethyl ether, giving 13 fractions of 3 mL each one. Fraction 6 (8.5 mg) was rich in

Figure 1. GC chromatogram of the essential oil from Myrcia laruotteana fruits showing the major components: spathulenol (17), globulol (19), viridiflorol (20), a-bisabolol oxide B (31), a-cadinol (32), a-bisabolol (34) and, 2E, 6E-methylfarnesoate (40).

M. laruotteana


described previously (8). Briefly, the cells were distributed in 96-well plates (100 mL cells/well) and exposed to various con-centrations of essential oil (0.25, 2.5, 25.0 and 250.0 mg/mL) in DMSO (0.1%) at 37oC, with 5% of CO2, for 48 h. The final concentration of DMSO did not affect the cell viability. A 50% trichloroacetic acid solution was added and after incubation for 30 min at 4oC, the cells were washed and dried. Cell prolifera-tion was determined by spectrophotometric quantification (at 540 nm) of the cellular protein content using sulforhodamine B. The experiments were carried, at least, in triplicate and the concentration necessary to total growth inhibition (TGI) was calculated in mg/mL. Doxorubicin was used as positive control. The data were analyzed using ANOVA and the F-test used to determine any difference among the groups.


The hydrodistillation of M. laruotteana unripe fruits fur-nished colorless oil, with yielding of 0.3%, which was higher than previously reported yields from leaves (0.05%) and flow-ers (0.07%) (3). The components of the oils, the percentage of each constituent and the retention indices are summarized in the Table I and the GC chromatogram is shown in the Figure 1. The oil was characterized by high content of oxygen-ated sesquiterpenes, mainly of bisabolane group. The major components were a-bisabolol (23.6%) and a-bisabolol oxide B (11.5%). This composition is very similar to those of leaves and flowers, except for presence of a-bisabolol oxide (3). The predominance of sesquiterpenes, mainly type bisabolane, in the essential oil has been reported for several species of Myrcia (9-11). The essential oil of Myrcia splendens contains almost 80% of a-bisabolene (10) while a-bisabolol is almost half of the oil of M. bracteata (11).

The oil exhibited antiproliferative activity for almost all cell lines evaluated, with TGI varying of 20.46-88.31 mg/mL, with exception of NCI-ADR/RES cell line, for which the TGI was higher than 125 mg/mL. The most significant activity was observed against U251 (glioma), 786-0 (kidney), UACC-62 (melanoma) and PC-3 (prostate), all with TGI lower than 30 mg/mL. The activity of the fraction rich in a-bisabolol was similar, with TGI varying of 8.58-33.50 mg/mL (Table II). These results suggest that the activity of oil is related to pres-ence of a-bisabolol.

The sesquiterpene a-bisabolol has been used in phar-maceutical products as drug permeation, anti-inflammatory, antispasmodic, anti-allergic and vermifuge. Besides, antimi-crobial, antiplasmodial, antioxidant and anticancer activities also have been reported for pure a-bisabolol or oil rich in this compound (12, 13). The antitumor activity was previ-ously observed against glioma and pancreatic carcinoma cells (13, 14). The present study show that, in addition to glioma, a-bisabolol is also effective against ovarian (OVCAR-3) and kidney (786.0) carcinoma cells, with TGI of 8.58 and 8.74 mg/mL, respectively. However, both oil and the fraction rich in a- bisabolol exhibited toxicity against VERO cell (no cancer

cell) with TGI of 36.45 and 7.91 mg/mL, respectively. This result with VERO cell was unexpected because a-bisabolol is considered a nontoxic compound (14). Thus, further investiga-tions are necessary to confirm the potential of the essential oil of M. laruotteana fruits as a citotoxic agent useful for in vivo applications in cancer treatment.

Acknowledgements

The authors thank the FAPESP for financial support. MJS and JEC are grateful to CNPq for research scholarships. D. Riva is grateful to CAPES for scholarship.

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M. E. A. Stefanello, A. C. Cervi, A. Wisniewski-Jr and E. L. Simionatto,3. Essential oil composition of Myrcia laruotteana Camb..J. Essent. Oil Res., 19, 466-467 (2007).

National Institute of Standards and Technology, 4. PC version of the NIST/EPA/NIH Mass Spectral Database. U.S. Department of Commerce, Gaithersburg, MD (1998).


H. van Den Dool, P.D.J.A. Kratz, 6. Generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr., 11, 463-471 (1963).

M. Miyazawa, H. Nankai and H. Kameoka, 7. Biotransformation of

(-)-a-bisabolol by plant pathogenic fungus, Glomerella cingulata. Phytochemistry, 39, 1077-1080 (1995).

P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. 8. Vistica, J. T. Warren, H. Bokesch, S. Kenney and M. R. Boyd, New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst., 82, 1107-1118 (1990).

M. E. A. Stefanello, A. C. R. F. Pascoal and M. J. Salvador, 9. Essential oils from Neotropical Myrtaceae: chemical diversity and biological properties. Chem. Biodiv., 8, 73-94 (2011).

M. J. Nakamura, S. S. Monteiro, C. H. B. Bizarri, A. C. Siani, 10. Essential oils of four Myrtaceae species from the Brazilian Southeast. Biochem. Syst. Ecol., 38, 1170-1175 (2010).

R. A. Pereira, M. G. B. Zoghbi and M. N. C. Bastos, 11. Essential oils of twelve species of Myrtaceae growing wild in the sandbank of the Resex Maracanã, State of Pará, Brazil. J. Essent. Oil-bearing Plant, 13, 440-450 (2010).

V. Popovic, S. Petrovic, M. Pavlovic, M. Milenkovic, M. Couladis, O. 12. Tzakou, S. Duraki and M. Niketic, Essential oil from the underground parts of Laserpitium zernyi: potential source of a-bisabolol and its antimicrobial activity. Nat. Prod. Comm., 5, 307-310 (2010).

G. P. P. Kamatou and A. M. Viljoen, 13. A review of the application and

pharmacological properties of a-bisabolol and a-bisabolol-rich oils. J. Am. Oil Chem. Soc., 87, 1-7 (2010).

E. Cavalieri, S. Mariotto, C. Fabrizi, A. C. Prati, R. Gottardo, S. Leone, 14.

L. V. Berra, G. M. Lauro, A. R. Ciampa and H. Suzuki, a-bisabolol, a nontoxic natural compound, strongly induces apoptosis in glioma cells. Biochem. Biophys. Res. Comm., 315, 589-594, 2004.

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Acc: April 2011

Chemotaxonomic Importance of Sesquiterpenes and Flavonoids in Five Argentinian Species of Polygonum

Genus

M. Derita* and S. ZacchinoPharmacognosy Area, Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario,

Rosario, Argentina

Abstract

Leaves of five species of Polygonum genus belonging to Persicaria section were collected from the northeast and central lowlands of Argentina and their dichloromethane extracts were analyzed by GC/EM. They were investigated for the presence of four drimane-type sesquiterpenes: polygodial (1), isopolygodial (2), drimenol (3) and confertifolin (4), previously isolated from P. acuminatum; and the presence of three flavonoids: pinostrobin (5), flavokawin B (6) and cardamonin (7), previously isolated from P. persicaria. Results showed that among the five species of Persicaria section studied, two species contained sesquiterpenes 1-4 but not flavonoids 5-7, other two species contained fla-vonoids 5-7 but not sesquiterpenes 1-4, and only one species contained compounds 1-7. These results add evidences to a previous proposal to sub-classify the Persicaria section of Polygonum genus from a chemotaxonomic point of view. [Editor’s note: This paper’s references are notated within the main body of the text in superscript, so as to avoid reader confusion with the discussed sesquiterpenes and flavonoids.]

Key Word Index

Polygonum genus, Persicaria section, sesquiterpenes, flavonoids, chemotaxonomic importance.



Introduction

The Polygonum genus (Polygonaceae) is well known for pro-ducing a variety of secondary metabolites including flavonoids,1 triterpenoids,2 anthraquinones,3 coumarins,4 phenylpropanoids,5 lignans,6 stilbenoids,7 tannins8 and sesquiterpenoids.9 It is rep-resented in Argentina by 21 species, which are divided into five sections: Echinocaulon, Amblygonum, Persicaria, Tiniaria and Polygonum.10

Polygonum punctatum Elliot, P. persicaria L., P. acumi-natum Kunth., P. lapathifolium L. and P. hydropiperoides Michux var. hydropiperoides, are five out of the 11 perennial herbs, belonging to the Persicaria section, which grow in the northeast and central lowlands of Argentina.

In a previous work, and considering that Gattuso11 and Cialdella10 suggested a delimitation of the Persicaria section to those species of Polygonum genus containing some kind of irritant valves, we suggested that the presence of the sesquit-erpene polygodial (1) could be also of diagnostic value for the delimitation of the Persicaria section. Following this point of view, the inclusion of P. hydropiperoides var. hydropiperoides and P. lapathifolium (which do possess neither polygodial nor valvate glands) within the Persicaria section could be the

subject of a further revision.12

In this work we add evidence to that proposal, investigating the presence of three sesquiterpenes (in addition to polygo-dial) isolated from P. acuminatum [isopolygodial (2), drimenol (3) and confertifolin (4)], and three flavonoids isolated from P. persicaria [pinostrobin (5), flavokawin B (6) and carda-monin (7)], in DMC extracts of the species aforementioned. (Figure 1).

Experimental

Plant material: Polygonum hydropiperoides Michux var. hydropiperoides was collected during the flowering season (March 2005) in San Luis province, Merlo district (32º35´S Lat., 65º03´O Long. and 850 m elevation), identified by Elisa Petenatti and deposited at the Herbarium of the National University of San Luis (UNSL # 9256). P. punctatum Elliot, P. persicaria L., P. acuminatum Kunth. and P. lapathifolium L. were harvested in March 2005 in Santa Fe province, Puerto Gaboto district (32º27´S Lat., 60º48´O Long. and 25 m elevation), identified by Susana Gattuso and deposited at the Herbarium of the National University of Rosario, Argentina (UNR Gattuso, S. 97, 108, 94, and 115, respectively).

Polygonum


Dried and powdered leaves of the species mentioned above (30 g each) were macerated with dichloromethane (DCM) for 24 h (3X). The solvent was evaporated under reduced pres-sure to yield 0.86, 1.14, 1.05, 0.80 and 0.50 g of DCM soluble extracts, respectively.

Compounds isolation: Fractionation by column chroma-tography of P. acuminatum leaves DCM extract allowed us to isolate 70, 53, 35 and 27 mg of compounds 1-4 respectively. Fractionation by column chromatography of P. persicaria leaves DCM extract allowed us to isolate 40, 36 and 25 mg of compounds 5-7 respectively. Compounds 1-4 were previously isolated from Drymis spp.,13,14 P. punctatum15 and P. acumi-natum,9 while compounds 5-7 were previously isolated from Boesenbergia pandurata, Myrica pensilvanica, P. ferrugineum and Piper spp.1,16,17 All spectrums of the isolated compounds in this work, were compared with the literature cited to assure the identity of each structure.

Analysis of the DCM extracts: All the extracts were submitted to GC-MS using a Turbo Mass Perkin Elmer chro-matograph, equipped with a fused silica column (SE-30 25 m x 0.22 mm ID) with He as a carrier gas, coupled to a mass selective detector, film 0.25 μm, ionization energy 70 eV with a temperature programme of 70-200ºC at 10ºC/min; total time 30 min. Compounds 1-7 were identified by comparison of their retention time and their MS spectrum with the authentic samples obtained from our previous works. Chromatograms are shown in Figure 2.

Figure 1. A) Sesquiterpenes isolated from P. acuminatum: polygodial (1), isopolygodial (2), drimenol (3), confertifolin (4). B) Flavonoids isolated from P. persicaria: pinostrobin (5), flavokawin B (6), cardamonin (7).

Figure 2: Gas chromatograms of dichloromethane extracts of P. acuminatum (A), P. persicaria (B), P. punctatum (C), P. lapathifolium (D) and P. hydropiperoides var. hydropiperoides (E). Peaks at 14.74/14.36 min in (A), (B) and (C) belong to drimenol (3). Peaks at 15.59/15.38 min in (A), (B) and (C) belong to confertifolin (4). Peaks at 16.16/16.38 min in (A), (B) and (C) belong to isopolygodial (2). Peaks at 17.41/17.40 min in (A), (B) and (C) belong to polygodial (1). Peaks at 20.81, 20.93, 20.91 min in (B), (D) and (E) belong to pinostrobin (5). Peaks at 22.69, 22.83, 22.80 min in (B), (D) and (E) belong to flavokawin B (6). Peaks at 23.51, 23.19, 23.14 min in (B), (D) and (E) belong to cardamonin (7).

Derita et al.


Compound descriptions: Polygodial (1) MP: 48ºC. [a]D: -27º (c 1.00, CHCl3). IR

(KBr): 2927, 2850, 2726, 1722, 1680, 1642, cm-1. 1H NMR (300 MHz, CDCl3): 9.48 (1H, d, J= 4.2 Hz, H-11); 9.42 (1H, s, H-12); 7.11 (1H, m, H-7); 2.78 (1H, dddd, J= 6.2, 2.1, 2.1, 2.1 Hz, H-9); 2.55-2.40 (1H, m, H-6a); 2.35-2.20 (1H, m, H-6b); 1.82 (1H, m, H-1b); 1.54-1.43 (3H, m, H-2a, 2b, 3b); 1.34 (1H, td, J= 4.0 and 13.4 Hz, H-1a); 1.26-1.16 (2H, m, H-3a, H-5); 0.92; (3H, s, Me-15); 0.91 and 0.89 (6H, 2s, Me-14 and Me-15). 13C NMR (75 MHz, CDCl3): 201.9 (HC=O); 193.2 (HC=O); 154.4 (=CH); 138.1 (=C); 60.2 (CH); 48,8 (CH); 41.7 (CH2); 39.5 (CH2); 36.8 (C); 33.0 (C); 33.0 (CH3); 25.1 (CH2); 21.9 (CH3); 17.9 (CH2); 15.2 (CH3). MS (EI, 70 eV): m/z (%) = 234 [M+], 216 [M+ - H2O], 206 [M+ - CO], 191 [206 - Me].

Isopolygodial (2) [a]D: +30º (c 1.00, CHCl3). IR (KBr): 2927, 2850, 2726, 1722, 1680, 1642, cm-1. 1H NMR (300 MHz, CDCl3): 9.86 (1H, d, J= 2.7 Hz, H-11); 9.41 (1H, s, H-12); 7.11 (1H, dd, J= 2.7 and 4.9 Hz, H-7); 3.26 (1H, bm, H-9); 2.57 (1H, dt, J= 5.0, 5.0, 20.6 Hz, H-6a); 2.22 (1H, dddd, J= 1.8, 2.6, 11.6 and 20.6 Hz, H-6b); 1.80 (1H, m, H-1b); 1.62-1.52 (5H, m, H-1a, 2a, 2b, 3b, 5); 1.19 (1H, ddd, J= 0.62, 4.1 and 12.0 Hz, H-3a); 0.97 (3H, s, Me-15); 0.94 and 0.92 (6H, 2s, Me-13 and Me-14). 13C NMR (75 MHz, CDCl3): 202.2 (HC=O); 192.8 (HC=O); 153.5 (=CH); 137.3 (=C); 58.5 (CH); 44.2 (CH); 42.0 (CH2); 37.6 (CH2); 37.1 (C); 32.9 (C); 32.7 (CH3); 25.5 (CH2); 21.9 (CH3); 21.5 (CH2); 18.4 (CH3). MS (EI, 70 eV): m/z (%) = 234 [M+], 216 [M+ - H2O], 206 [M+ - CO], 191 [206 - Me].

Drimenol (3) MP: 98ºC. [a]D: -15º (c 1.00, CHCl3). IR (KBr): 3405, 2922, 1620 cm-1. 1H NMR (300 MHz, CDCl3): 5.54 (1H, m, H-7); 3.85 (1H, dd, J= 3.3, 11.3 Hz, H-11B); 3.73 (1H, dd, J= 3.3, 11.3 Hz, H-11A); 1.97 (1H, m, H-6a); 1.95 (1H, m, H-1b); 1.92 (1H, m, H-6b); 1.86 (1H, m, H-9); 1.79 (3H, s, Me-12); 1.48 (2H, m, H-2a and 2b); 1.41 (1H, m, H-3b); 1.20 (2H, m, H-3a and H-5); 1.07 (1H, m, H-1a); 0.89, 0.87 and 0.85 (9H, 3s, Me-13, 14 and 15). 13C NMR (75 MHz, CDCl3): 132.9 (=C); 124.0 (=CH); 60.9 (H2C-OH); 57.3 (CH); 49.9 (CH); 42.1 (CH2); 39.8 (CH2); 36.0 (C); 33.3 (CH3); 32.9 (C); 23.6 (CH2); 22.0 (CH3); 21.9 (CH3); 21.9 (CH3); 18.8 (CH2); 14.9 (CH3). MS (EI, 70 eV): m/z (%) = 234 [M+], 216 [M+ - H2O], 206 [M+ - CO], 191 [206 - Me].

Confertifolin (4) MP: 153ºC. [a]D: +70º (c 1.00, CHCl3). IR (KBr): 1769, 1677 cm-1. 1H NMR (300 MHz, CDCl3): 4.72 (1H, ddd, J= 2.8, 2.8, 16.9 Hz, H-1a); 4.62 (1H, ddd, J= 1.7, 3.5, 16.9 Hz, H-1b); 2.54-2.05 (2H, m, H-4a and b); 1.92-1.30 (9H, m, H-5a and b, H-5a, H-7a and b, H-8a and b, H-9a and b); 1.15 (3H, s, Me-9a); 0.93 and 0.89 (6H, 2s, Me-6a and b). 13C NMR (75 MHz, CDCl3): 174.5 (C=O); 170.8 (=C); 123.4 (=C); 68.2 (CH2); 51.2 (CH); 41.6 (CH2); 36.6 (CH2); 36.0 (C); 33.2 (C); 33.2 (CH3); 21.4 (CH2); 21.4 (CH3); 20.9 (CH3); 18.3 (CH2); 18.0 (CH2). MS (EI, 70 eV): m/z (%) = 234 [M+], 216 [M+ - H2O], 206 [M+ - CO], 191 [206 - Me].

Pinostrobin (5) MP: 87ºC. [a]D: -51º (c 1.00, CHCl3). IR (KBr): 3450, 2950, 1647, cm-1. 1H NMR (300 MHz, CDCl3): 12.04 (1H, s, OH); 7.49-7.40 (5H, m, H-2´-6´); 6.10 (1H, d, J= 2.3 Hz, H-8); 6.09 (1H, d, J= 2.3 Hz, H-6); 5.44 (1H, dd, J= 3.1 and 12.9 Hz, H-2); 3.83 (3H, s, O-Me); 3.11 (1H, dd, J= 12.9 and 17.2 Hz, H-3a); 2.84 (1H, dd, J= 3.1 and 17.2 Hz, H-3b). 13C NMR (75 MHz, CDCl3): 195.8 (C=O); 168.0

(=C); 164.1 (=C); 162.8 (=C); 138.4 (=C); 128.9 (=CH); 128.9 (=CH); 128.9 (=CH); 126.2 (=CH); 126.2 (=CH); 103.1 (=C); 95.1 (=CH); 94.2 (=CH); 79.2 (CH); 55.7 (OCH3); 43.4 (CH2). MS (EI, 70 eV): m/z (%) = 270 [M+].

Flavokawin B (6) MP: 95ºC. IR (KBr): 3450, 2940, 1630, cm-1. 1H NMR (300 MHz, CDCl3): 12.04 (1H, s, -OH); 7.92 (1H, d, J= 15.4 Hz, H-b); 7.79 (1H, d, J= 15.4 Hz, H-a); 7.64-7.28 (5H, m, H2-6); 6.12 (1H, d, J= 2.3 Hz, H-5´); 5.98 (1H, d, J= 2.3 Hz, H-3´); 3.93 and 3.85 (6H, 2s, 2 O-Me). 13C NMR (75 MHz, CDCl3): 192.6 (C=O); 168.4 (=C); 166.2 (=C); 162.5 (=C); 142.3 (=CH); 135.6 (=C); 130.0 (=CH); 128.9 (=CH); 128.9 (=CH); 128.4 (=CH); 128.4 (=CH); 127.5 (=CH); 106.3 (=C); 93.8 (=CH); 91.3 (=CH); 55.9 (O-CH3); 55.6 (O-CH3). MS (EI, 70 eV): m/z (%) = 284 [M+].

Cardamonin (7) MP: 98ºC. IR (KBr): 3400, 2924, 1638, cm-1. 1H NMR (300 MHz, DMSO-d6): 13.7 (1H, s, -OH); 7.83 (1H, d, J= 15.6 Hz, H-b); 7.73-7.70 (2H, m, H-2 and H-6); 7.68 (1H, d, J= 15.6 Hz, H-a); 7.46-7.44 (3H, m, H-3, 4 and 5); 6.01 (1H, d, J= 2.2 Hz, H-5´); 5.92 (1H, d, J= 2.2 Hz, H-3´); 3.88 (3H, s, O-Me). 13C NMR (75 MHz, DMSO-d6): 192.2 (C=O); 166.7 (=C); 165.6 (=C); 163.1 (=C); 142.3 (=CH); 135.4 (=C); 130.8 (=CH); 129.5 (=CH); 128.8 (=CH); 128.8 (=CH); 128.0 (=CH); 128.0 (=CH); 105.5 (=C); 96.3 (=CH); 92.2 (=CH); 55.5 (O-CH3). MS (EI, 70 eV): m/z (%) = 270 [M+].


The analysis of the GC spectra clearly showed that P. hydropiperoides var. hydropiperoides and P. lapathifolium do not possess sesquiterpenes 1-4 but contain flavonoids 5-7; meanwhile P. punctatum and P. acuminatum do not possess flavonoids 5-7 but contain sesquiterpenes 1-4. Surprisingly, P. persicaria (the specie that gives the name to the whole section) possesses compounds 1-7.

Hereby, taking into account our previous work12and these new results, we can propose that within the Persicaria section of Polygonum genus there are two kinds of species that could be sub-classified by a chemotaxonomic point of view: those that produce the sesquiterpenes (1-4) but not the flavonoids (5-7), and those that biosynthesize the flavonoids (5-7) but not the sesquiterpenes (1-4). To assure this proposal, we must go on studying other species that belong to the Persicaria section of Polygonum genus in terms of their content in sesquiterpenes and flavonoids.

Acknowledgements

The authors wish to acknowledge CONICET, ANPCyT, ERASMUS MUNDUS lot 18 ARBOPEUE, UNR.

References

S. López, M. González Sierra, S. Gattuso, R. Furlán, S. Zacchino, 1. An unusual homoisoflavanone and a structurally related dihydrochalcone from Polygonum ferrugineum. Phytochemistry, 67, 2152-2157 (2006).

M. Duwiejua, I. Zeitlin, A. Gray, P. Waterman, 2. The anti-inflammatory compounds of Polygonum bistorta: Isolation and characterisation. Planta Med., 65, 371-374 (1999).

T. Yim, W. Wu, D. Mak, K. Ko, 3. Myocardial protective effect of an

Polygonum


anthraquinone-containing extract of Polygonum multiflorum ex vivo. Planta Med., 64, 607-611 (1998).

X. Sun, A. Sneden, 4. Neoflavonoids from Polygonum perfoliatum. Planta Med., 65, 671-674 (1999).

M. Takasaki, T. Konoshima, S. Kuroki, H. Tokuda, H. Nishino, 5. Cancer chemopreventive activity of phenylpropanoid esters of sucrose, vanicoside B and lapathoside A, from Polygonum lapathifolium. Cancer Let., 173, 133-138 (2001).

H. Kim, E. Woo, H. Park, 6. A novel lignan and flavonoids from Polygonum aviculare. J. Nat. Prod., 57, 581-586 (1994).

G. Nonaka, N. Miwa, I. Nishioka, 7. Stilbene glycoside gallates and proanthocyanidins from Polygonum multiflorum. Phytochemistry, 21, 429-432 (1982).

K. Wang, Y. Zhang, C. Yang, 8. Antioxidant phenolic compounds from rhizomes of Polygonum paleaceum. J. Ethnopharmacol., 96, 483-487 (2005).

M. Derita, M. Leiva, S. Zacchino, 9. Influence of plant part, season of collection and content of the main active constituent, on the antifungal properties of Polygonum acuminatum Kunth. J. Ethnopharmacol., 124, 377-383 (2009).

A. Cialdella, 10. Revisión de las especies Argentina de Polygonum (Polygonaceae). Darwiniana, 29, 179-246 (1989).

S. Gattuso, 11. Structure and ultrastructure of the secretory glands in the genus Polygonum (L), section Persicaria (Polygonaceae). Biocell, 25, 229-233 (2001).

M. Derita, S. Gattuso, S. Zacchino, 12. Ocurrence of polygodial in species of Polygonum genus belonging to Persicaria section. Biochem. Syst. Ecol., 36, 55-58 (2008).

V. Cechinel Filho, V. Schlemper, A. Santos, T. Pinheiro, R. Yunes, G. 13. Mendes, J. Calixto, F. Delle Monache, Isolation and identification of active compounds from Drymis winteri barks. J. Ethnopharmacol., 62, 223-227 (1998).

D. Muñoz-Concha, H. Vogel, R. Yunes, I. Razmilic, L. Bresciani, A. 14. Malheiros, Presence of polygodial and drimenol in Drymis populations from Chile. Biochem. Syst. Ecol., 35, 434-438 (2007).

T. De Almeida Alves, F. Lacerda Ribeiro, H. Kloos, C. Zani, 15. Polygodial, the fungitoxic component from the Brazilian medicinal plant Polygonum punctatum. Mem. do Inst. Oswaldo Cruz, 96, 831-833 (2001).

K. Hodgetts, 16. Approaches to 2-substituted chroman-4-ones: synthesis of (-)-pinostrobin. Tetrahedron Let., 42, 3763-3766 (2001).

B. Burke, M. Nair, 17. Phenylpropene, benzoic acid and flavonoids derivatives from fruits of Jamaican Piper species. Phytochemistry, 25, 1427-1430 (1986).

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Acc: June 2011

Seasonal Evaluation and Chemical Composition of Volatile Fractions from Piper claussenianum by

Hydrodistillation and SPME

André Mesquita Marques* and Maria Auxiliadora Coelho KaplanNúcleo de Pesquisas de Produtos Naturais (NPPN), Centro de Ciências da Saúde, Bloco H . Universidade Federal do Rio

de Janeiro (UFRJ), Brasil. CEP: 21941-590

Abstract

The essential oils from Piper species are chemically diverse, with several biological activities as well as commercial and pharmacological values reported. This work aims to compare the volatile constituents from leaves and inflores-cences obtained by two different extraction techniques and show the seasonal variation of nerolidol in the essential oil from leaves of Piper claussenianum. The P. claussenianum volatile components were obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation, coupled to gas chromatography/mass spectrometry (GC/MS) analysis. Leaf and inflorescence volatile fractions were analyzed in fresh and dried conditions. In total, 40 compounds were identified, accounting for 88.8-97.7% of the constituents. The high percentage of nerolidol in the leaves and linalool in the inflorescences were remarkable in this species, accounting for up to 77.0% and up to 50.0%, respectively, collected monthly. Results of (E)-nerolidol seasonal variation study carried out in the growth period of January/December 2009, suggest the plant leaves which provide an essential oil with high content of (E)-nerolidol over 88.0% are primarily observed in the September/December collection period.

Key Word Index

Piper claussenianum, nerolidol, essential oil, SPME, hydrodistillation.



Introduction

The essential oils (EOs) from aromatic herbs traditionally ob-tained by hydrodistillation are of increasing use in aromatherapy due to the popular interest for natural compounds with curative properties and economical value, such as scents in perfumes, cosmetics and cleaning agents. Various novel techniques have been developed for EO extraction from plants. Among these, headspace solid phase microextraction (HS-SPME) allows the rapid fingerprinting of a plant headspace (1). Over the last several years, solid-phase microextraction (SPME) has gained acceptance in many fields as an accurate, rapid, sensitive and solvent-free sampling method. Many researchers report its ap-plication as a useful approach in sample preparation of volatile compounds from complex matrices (2). Chemically diversified essential oils from Piper species have noteworthy biological activities as well as commercial and pharmacological values. Terpenes such as linalool and nerolidol have been used for scent composition bouquet in perfumes and as antimicrobial products (3-7). In a previous work, we reported, for the first time, the chemical compositions of essential oils from the leaves and flowers of Piper claussenianum, in addition to their anti-parasitic activity against a strain of Leishamania amazonensis.

The high percentage of the sesquiterpene (E)-nerolidol in the leaves and the monoterpene linalool in the inflorescences was remarkable to this species. Due to this fact and previous reports concerning about leishmanicidal properties of terpenes we were encouraged to carry out the investigation of these oils on this parasite. Both assayed EOs extracted by hydrodistillation have inhibited the growth of parasites. MIC and IC50 values of the leaf EO were respectively 57.6 mg/mL and 30.4 mg/mL while the EO from inflorescences were 664.0 mg/mL and 1328.0 mg/mL. These results suggest the higher percentage of nerolidol in leaf EO to be the responsible compound for the more effective growth inhibition (3).

This work aims to investigate the chemical composition of these piperaceous essential oils extracted using hydrodistilla-tion (HD) from separated organs (leaves and flowers) as well as the volatile fractions extracted using HS-SPME from P. claussenianum. Seasonal monitoring of nerolidol in the leaf essential oils was also performed. In both procedures, analy-ses were carried out using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). SPME consti-tutes a suitable alternative to extract volatile and semivolatile chemicals in a wide range of matrices. A major advantage

P. claussenianum


includes the capability to combine volatile fraction extrac-tion and preconcentration in a single step (8). Some physical factors such as sample agitation, headspace equilibration temperature, extraction time, and analyte diffusion rate from the vapour phase to the fibre surface also contribute to high SPME efficacy. (9). The evaluation of aromatic fractions from natural compounds is important in agricultural science and food chemistry. The determination of essential oil composi-tion is necessary for quality control of breeding parameters, cultivation, and production of aromatic plants. Quality control of raw material, intermediates, and end products is further-more required in the food industry for production of spices, essential oils, or flavored food (10). The most usual method for the determination of the essential oil content is by hydrodistil-lation. However, its use for the subsequent determination of the aroma compound compositions has often been discussed

Table I. Identified Compounds in the Essential Oil from Leaves of P. claussenianum.

Compounds aRILit bRI FRESH HD % DRY HD % SPME % Identification

1 a-pinene 939 936 0.2 - 0.6 RI, GCMS2 b-pinene 979 976 0.2 0.1 0.6 RI, GCMS3 b-myrcene 991 987 - 0.2 - RI, GCMS4 limonene 1029 1027 - - 0.3 RI, GCMS5 (Z)-b-ocimene 1037 1033 0.6 - 3.4 RI, GCMS6 (E)-b-ocimene 1050 1043 0.9 - 4.1 RI, GCMS7 cis Linalool oxide 1087 1083 0.6 - - RI, GCMS8 linalool 1097 1101 5.2 2.2 4.6 RI, GCMS, STD9 g-elemene 1338 1340 0.1 1.8 5.4 RI, GCMS10 b-elemene 1391 1387 0.5 0.8 - RI, GCMS11 (E)-caryophyllene 1419 1415 0.6 1.4 - RI, GCMS12 (Z)-b-farnesene 1443 1439 - - 6.9 RI, GCMS13 a-humulene 1455 1451 0.6 1.1 1.3 RI, GCMS14 g-muurolene 1480 1477 1.1 3.2 15.9 RI, GCMS15 a-selinene 1498 1497 0.4 - - RI, GCMS16 (Z)-a-bisabolene 1507 1508 - - 0.3 RI, GCMS17 d-cadinene 1523 1520 0.4 0.8 4.1 RI, GCMS18 (E)-g-bisabolene 1531 1531 - - 1.2 RI, GCMS19 a-cadinene 1539 1545 - - 0.6 RI, GCMS20 germacrene B 1561 1558 - - 0.5 RI, GCMS21 (E)-nerolidol 1563 1563 81.4 83.2 42.1 RI, GCMS, STD22 caryophyllene oxide 1583 1577 - - 1.1 RI, GCMS23 gleenol 1587 1586 - - 1.0 RI, GCMS24 a-eudesmol 1654 1654 0.5 2.9 - RI, GCMS% PEAK SUM OF IDENTIFIED COMPOUNDS: 93.3 97.7 94.0

a RI Lit: Literature Retention Indices16; b RI: Experimental Retention Indices; H.D: Hydrodistillation;

Table II. Monoterpene and Sesquiterpene fractions of the analyzed Essential Oils.

Fresh HD % Dry HD % SPME %

Leaves Inflorescences Leaves Inflorescences Leaves Inflorescences

Monoterpene 7.6 52.9 2.5 56.5 13.0 61.9Sesquiterpene 85.7 35.9 95.2 35.5 81.0 31.2Total 93.3 88.8 97.7 92.0 94.0 93.1

very critically since transformation processes of genuine aroma-active compounds due to the influence of heat, steam, and pH may occur (11). On the other hand, highly volatile components as well as water-soluble components can get lost during hydrodistillation. As with hydrodistillation, non-volatile substances are not detected with SPME, and thus do not affect the evaluation of the essential oils and related compounds. Similarly hydrodistillation is a very time-consuming method and therefore not useful, especially for the screening of very large quantities of plant samples for their aromatic composi-tion. Solid-phase microextraction (SPME) is a comparatively new, very simple and efficient method for routine laboratory analysis of organic compounds (12-15). Furthermore, SPME is the most time-saving sample preparation method for the subsequent gas chromatographic determination of the com-position of aroma-active compounds.

Marques et al.


lished data and computer matching with WILEY 275 and the National Institute of Standards and Technology (NIST 3.0) libraries provided by a computer-controlled GC/MS system. The results were also confirmed by comparing the compounds’ elution order with their relative retention indices reported in the literature (16). The retention indices were calculated for all the volatile constituents using the retention data of linear n-alkanes C8–C24.

Nuclear magnetic resonance spectroscopy: The crude EO obtained from leaves of P. claussenianum was analyzed by 1H and 13C NMR and recorded on a Brüker DRX 400 spec-trometer in order to confirm the presence of the sesquiterpene and to show the 13C NMR spectra value on characterization of terpenes. The chemical shifts were determined in CDCl3, using TMS as the internal standard. The spectrometric data for the 1H NMR spectra are organized in agreement with the convention: chemical shift (number of protons and multiplicity), and the data for 13C NMR spectra are organized by chemical shift. The signals of the NMR analyses were compared to the literature data (3, 16).

Head-space/Solid phase microextraction (HS-SPME): Fresh leaves and inflorescences of P. claussenianum C.DC. (500 mg) were reduced to small pieces and powdered one by one in separated way. Four headspaces with these materials were separately extracted by HS-SPME using

Figure 1. Seasonal evaluation of Nerolidol in EO of P. claussenianum leaves monthly harvested in 2009. The percentages of the nerolidol remains always over 77.0% (April) achieving the maximum content level in October (94.0%) during the Brazilian spring.

Experimental

Plant material and essential oil extraction: Leaves (100 g) of Piper claussenianum were collected in Castelo, ES in January 2010. The botanical vouchers were identified by Dr. Elsie Franklin Guimarães and kept at the Herbarium (HB) of the Rio de Janeiro Botanical Garden (JBRJ), registered under number RB 489043. The fresh and dried plant materials were submitted for hydrodistillation for 2 h in a modified Clevenger-type apparatus. The obtained essential oils (EO) were dried over anhydrous sodium sulphate, yielding 1.0% w/w in both studied specimens.

GC-FID analysis: The gas chromatography analyses were carried out on a GC 2010 Shimadzu with a ZB-1MS fused silica capillary column (30 m x 0.25 mm x 0.25 mm film thickness). The operating temperatures used were as follows: injector 260°C, detector 290°C and column oven 60°C up to 290°C (10°C/min). Hydrogen at 1.0 mL min-1 was used as a carrier gas. The percentages of the compounds were obtained by GC-FID analysis.

GC/MS analysis: Qualitative analyses were carried out on a GC-QP2010 PLUS Shimadzu with a ZB-5MS fused silica capillary column (30 m x 0.25 mm x 0.25 mm film thickness) under the experimental conditions reported for GC-FID analysis. The essential oil components were identified by comparing their retention indices and mass spectra to pub-

P. claussenianum


Table III. Identified Compounds in the Essential Oils from Inflorescences of P. claussenianum.

Compounds aRILit bRI FRESH HD % DRY HD % SPME % Identification

1 a-pinene 939 936 0.2 - 0.6 RI, GCMS2 b-pinene 979 976 0.2 - 0.6 RI, GCMS3 b-myrcene 991 987 0.1 - 0.3 RI, GCMS4 a-phellandrene 1003 1006 0.1 - 0.5 RI, GCMS5 l imonene 1029 1027 0.3 0.3 1.5 RI, GCMS6 b-phellandrene 1030 1029 0.3 - - RI, GCMS7 cineole 1031 1030 0.3 0.3 - RI, GCMS8 (Z)-b-ocimene 1037 1033 - - 2.6 RI, GCMS9 (E)-b-ocimene 1050 1043 0.3 0.5 5.3 RI, GCMS10 a-pinene oxide 1099 1101 - 0.5 - RI, GCMS11 linalool 1097 1102 50.2 54.5 50.6 RI, GCMS, STD.12 a-terpineol 1189 1195 0.3 - - RI, GCMS13 piperitone 1253 1257 0.6 0.5 - RI, GCMS14 g-elemene 1338 1340 - 0.3 1.3 RI, GCMS15 a-copaene 1377 1380 0.1 - - RI, GCMS16 b-cubebene 1388 1385 0.1 - - RI, GCMS17 (E)-caryophyllene 1419 1414 2.0 2.0 - RI, GCMS18 (Z)-b-farnesene 1443 1439 - - 4.4 RI, GCMS19 a-humulene 1455 1451 2.6 2.6 4.9 RI, GCMS20 g-muurolene 1480 1476 0.3 0.4 - RI, GCMS21 germacrene D 1485 1483 - - 0.5 RI, GCMS22 b-selinene 1490 1484 0.6 4.0 0.3 RI, GCMS23 a-selinene 1498 1497 - 0.4 - RI, GCMS24 trans guaiene 1503 1501 0.7 - - RI, GCMS25 (Z)-a-bisabolene 1507 1508 - - 1.7 RI, GCMS26 a-amorphene 1512 1514 0.3 0.2 1.0 RI, GCMS27 d-cadinene 1523 1527 0.9 0.9 2.4 RI, GCMS28 (E)-nerolidol 1563 1564 22.7 24.3 14.6 RI, GCMS, STD.29 t-cadinol 1640 1635 0.3 - - RI, GCMS30 eudesmol <7-epi-alpha> 1664 1666 5.0 - - RI, GCMS31 eudesm-7(11)-en-4-ol 1700 1707 0.3 0.5 - RI, GCMS

% PEAK SUM OF IDENTIFIED COMPOUNDS: 88.8 92.2 93.1

a RI Lit: Literature Retention Indices16; b RI: Experimental Retention Indices; H.D: Hydrodistillation;

a carboxen-divinylbenzene 75 mm fiber (CAR-DVB) at 80°C, and sample/ headspace equilibration time for 15 min. The extracted materials were immediately desorbed and analyzed by GC/MS.


Analyses of the essential oils from leaves and inflores-cences collected in Castelo, ES, Brazil, led to the identifica-tion of 40 components, corresponding to 89.0-97.7% of the constituents.

The identification of the compounds was performed by comparing their EI-MS and retention indices with those re-ported in the literature libraries. The structure of nerolidol was confirmed by 1H NMR and 13C NMR spectra obtained by the crude leaf EOs. Both oils, from leaves and inflorescences showed rich monoterpene and sesquiterpene fractions. Sesquiterpenes were identified as the main volatile fraction of the leaves EOs while monoterpenes were found in great amount in the inflo-rescences. The essential oils obtained by hydrodistillation from fresh and dried material of P. claussenianum aerial parts yielded about 1.0% (w/v) for both cases. The high percentage of the

sesquiterpene (E)-nerolidol in the leaves and the monoterpene linalool in the inflorescences was remarkable to this species. The oil from leaves of P. claussenianum was characterized by its high content in (E)-nerolidol (81.4%, 83.3%, 42.1%) as well as in linalool (5.2%, 2.2%, 4.6%), g-muurolene (1.1%, 3.2%, 15.9%), (E)-caryophyllene (0.6%, 1.4%, - ) and g-elemene (0.5%, 0.8%, 5.4%) in fresh HD, dry HD and SPME analyses, respectively (Table I). (E)-nerolidol, the main volatile compound from the leaf EOs, represents (81.0%) of the volatile fractions in the hydroditillation of fresh and (83.0%) in dried samples while SPME analysis has shown only a half percent of this compound of fresh leaf EO composition. As was expected, the EO from fresh leaf HD displays a higher content of monoterpenes: 6 (7.6%), than dried material: 3 (2.5%). On the other hand the sesquiterpene percentage in the EO from dried leaves showed the highest value, accounting for 95.2% while in the fresh EO it was found to be 85.7%. (Table II).

Although the dried material displays a higher percentage of sesquiterpenes in its composition the content did not differ that much from that of the fresh material. The relative composition of HS-SPME volatile fractions obtained from leaves shows a

Marques et al.


duce high quality of data. Furthermore, this study emphasizes the importance of choosing the appropriate harvest period in order to obtain the richest nerolidol content EO from leaves of P. claussenianum.

Acknowledgements

The authors thank Conselho Nacional de Desenvolvimento Cientí-fico Tecnológico (CNPq) for financial support and Mark English for English correction.

References

M.A. Dib, N. Djabou, J.M. Dejobert, H. Allali, 1. Characterization of volatile compounds of Daucus crinitus Desf. Headspace Solid Phase Microextraction as alternative technique to Hydrodistillation. Chem. Centr. J., 4, 1-15. (2010)

J. Fiori, M. Naldi, R. Gotti, 2. HS-SPME-CG-MS for the Quantification and Chiral Characterization of Camphor and Menthol in Creams. Chromatographia, 72, 941-947 (2010).

A.M. Marques, A.L.S. Barreto, E.M. Batista, L.S.M. Velozo, E.F. 3. Guimarães, R.M.A. Soares, M.A.C. Kaplan, Chemistry and Biological Activity of Essential Oils from Piper claussenianum (Piperaceae). Natural Product Communications. 5, 1837-1840 (2010).

Mookherjee, R.W. Trenkle, B.J. Chant, A.V. Vanouwerke, V. Kamath, 4. Perfume compsn. with white flower aroma - contg. farnesene isomers obtd. by dehydrating nerolidol isomers. FR2511244-A1; US4376068-A; FR2511244-A. 1983.

S. Koryo, 5. Fragrance composition, useful in foodstuffs/beverages and cosmetics, contains nerolidol oxide as main component. JP2005054062-A; JP4092380-B2. 2005.

Kuraray CO LTD (KURS-C), 6. High yield farnesene(s) prepn. - by dehydration of nerolidol in presence of sulphuric acid in hydrocarbon(s). JP61100532-A. 1986.

K. Asano, T Hisamitsu, S. Mihara, O. Okawa, 7. Composition useful in perfume or fragrance related product for increasing beta-endorphin production in brain, comprises essential oil containing linalool, I-alpha-terpineol, nerolidol, I-menthol, benzyl acetate, santalol or muscone. JP2009029761-A. 2009.

A.R. Lafuente, 8. Dertermination of fifteen active compounds released from paraffin-based active packaging in tomato samples via microextraction techniques. Anal Bioanal Chem. 395, 203-211 (2009).

Development of a headspace-solid phase micro extraction method 9. to monitor changes in volatile profile of rose (Rosa hybrida, cv David Austin) petals during processing. J. Chromatogr. A, 1150, 190–197 (2007).

E.E. Stashenko, J.R. Martinez, 10. Sampling volatile compounds from natural products with headspace/solid-phase micro-extraction. J. Biochem. Biophys. Methods., 70, 235–242 (2007).

J. Richter, I. Schellenberg, 11. Comparison of different extraction methods for the determination of essential oils and related compounds from aromatic plants and optimization of solid-phase microextraction/gas chromatography. Anal Bioanal Chem., 387, 2207–2217 (2007).

L. Ling, S. Guoxin, H. Yaoming, 12. GC/MS Analysis of the Essential Oils of Piper nigrum L. and Piper longum L. Chromatographia, 66, 785–790 (2007).

Z.Q. Fan, S.B. Wang, R.M. Mu, X.R. Wang, S.X. Liu, 13. A Simple, Fast, Solvent-Free Method for the Determination of Volatile Compounds in Magnolia grandiflora Linn. J. Anal. Chem., 64, 289-294 (2009).

C. Shufen, T. Shuo, O. Gangfeng, J. Shihong, P. Janusz, 14. Headspace solid-phase microextraction gas chromatography/mass spectrometry analysis of Eupatorium odoratum extract as an oviposition repellent. J. Chromatogr. B, 877, 1901–1906 (2009).

G. Özek, F. Demirci, T. Özek, N. Tabanca, D.E. Wedge, S.I. Khan, 15. Gas chromatographic/mass spectrometric analysis of volatiles obtained by four different techniques from Salvia rosifolia Sm., and evaluation for biological activity. J. Chromatogr. A, 1217, 741–748 (2010).


higher diversity and/or quantity of monoterpenes (13.0%) and sesquiterpenes (81.0%) on the essential oil profile comparing to hydrodistillation. These results suggest most of the highly volatile components absent in fresh and dried hydrodistillation EOs were lost due to lengthy times at a high temperature dur-ing the extraction process. In the inflorescences, the consituent found in the highest concentration was identified as linalool.

It was present in concentrations of up to 50.0% in the volatile fractions produced by each extraction technique. The highest concentration of the major terpenes (linalool and nerolidol) were found in the dried HD essential oil. Linalool was found as the main constituent (50.2%, 54.5%, 50.6%), fol-lowed by (E)-nerolidol (22.7%, 24.3%, 14.6%), a-humulene (2.6%, 2.6%, 4.9%), (E)-caryophyllene (2.0%, 2.0%, - ) and b-selinene (0.6%, 4.0%, 0.3%) in fresh HD, dried HD and SPME analysis, respectively (Table III).

The HS-SPME was a useful tool in qualitative analysis of highly volatile fractions, mainly the fresh material due the pres-ence of a higher quantity of monoterpenes. In these analyses, a low percentage of monoterpenes was obtained from the leaves, but higher when compared to HD for fresh or dried material. However, HS-SPME analysis of inflorescence showed a high percentage (61.9%) of the monoterpenes, mainly linalool con-tent (50.6%). This was expected since HS-SPME is suitable for volatile compounds. The sesquiterpene (E)-nerolidol was identified in lower amounts in the HS-SPME of leaves and inflorescences. In fact, this sesquiterpene was identified in approximately half of headspaces when compared to the hy-drodistillation of fresh and dried leaves. Once again, this is also expected, since HS-SPME is not a suitable technique for the analysis of semi-volatile compounds. As a result the nerolidol quantification was performed with the crude EO extracted by hydrodistillation. In HS-SPME procedure the samples remained at 80∞C during a time span of 15 min, which was not sufficient for the release of all the nerolidol content present in the leaves. Similarly, HS-SPME was more sensible for the determination and analysis of the presence of linalool in the inflorescences compared with nerolidol in the leaves. The seasonal monitor-ing of the EO from leaves of P. claussenianum was performed in order to determine the optimal time of harvest. In order to choose the appropriate harvest period for the specimen, about 100 g of fresh plant aerial parts were collected monthly. The EOs extracted by hydrodistillation were analyzed and no variation in the chromatographic profile was found. Thoughout the entire year, the leaf specimens displayed a marked higher essential oil rich in nerolidol, always up to 77.0%.

The highest nerolidol content was observed during the Brazilian spring collection period: Sept/Oct/Nov (87.0%, 94.0%, 92.0% respectively). The minimum level recorded was found during the autumn collection period Mar/Apr/May (78.0%; 77.0%; 80.0%) respectively (Figure 1). We have enhanced the importance of SPME as a useful technique to analyze the endangered species from the Brazilian Tropical Rain Forest, due to its simplicity, speed, low cost and solventless operation, needing just a small quantity of material for the analysis to pro-

P. claussenianum


Rec: Feb 2011

Acc: July 2011

Analysis of the Chemical Composition and Antimicrobial Activity of the Essential Oil from

Lippia triplinervis Gardner (Verbenaceae)

Suzana G. Leitão*, João Paulo L. Damasceno, Márcia G. Martini, Simone N. Miranda and Paulo M. Neufeld

Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, CCS, Bloco A, 2o andar, Ilha do Fundão, Rio de Janeiro, RJ, 21941-590 Brazil

Fátima Regina SalimenaInstituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Juiz de Fora-MG, Brazil

Humberto R. BizzoEmbrapa Agroindustria de Alimentos, Avenida das Américas 29501, Rio de Janeiro, RJ, 23020-470 Brazil

Abstract

The chemical composition of the essential oil from Lippia triplinervis (Verbenaceae), as well as its antimicrobial activity against Candida albicans and other clinical isolates, was analyzed. The major compounds identified in the oil obtained in April 2010 were myrcene (2.6%), ipsenone (11.4%), myrcenone (57.7%), (Z)-ocimenone (1.3%) and (E)-calamenene (4.8%). The essential oil of this same plant collected in September 2010, showed a quite similar chemical composition, but the relative percentage of myrcenone was reduced from 57.7% to 13.5%, whereas limonene (8.0%), (E)-tagetone (2.0%) and (Z)-tagetone (19.4%) were now major constituents of this oil. The minimum inhibitory con-centration (MIC) activity against C. albicans as well as other clinical isolates (C. glabrata, C. krusei, C. parapsilosis and C. tropicalis) ranged from 156 to 1,250 mg/mL. This is the first report of the chemical composition and antimicrobial activity of L. triplinervis essential oil.

Key Word Index

Lippia triplinervis, Verbenaceae, myrcene, myrcenone, limonene, ipsenone, (E)-tagetone, myrcenone, (Z)-tagetone.

Presented in part at the 41st International Symposium on Essential Oils, Wroclaw, Poland.



Introduction

The genus Lippia (Verbenaceae) comprises about 200 species occurring mainly in Central and South America, as well as in some areas of Tropical Africa (1). One of the main diversity centers of the genus Lippia is located at the “Cadeia do Espinhaço” Mountains, in the State of Minas Gerais, Brazil

(2). During one of our field trips for plant collecting in the mountains of Aiuruoca (Minas Gerais State), Brazil, we found a strongly aromatic shrub, growing above the height of 1800 m. This plant was identified as L. triplinervis Gardner (syn. Lip-pia iodophylla Schauer), a species with no previous chemical investigation reports and thus, was collected for investigation. This species is a shrub to 1.5 m tall, with coriaceous leaves, and the flowers are fragrant, rose or violet. The first specimen of this rarity, endemic of the “campos de altitude” species, has been

collected between stones at an altitude of 2000 meters, in Serra dos Órgãos, Itatiaia and Mantiqueira (Rio de Janeiro, Minas Gerais and São Paulo States) flowering in January, February, April and May (3). As of 1998, it has been included in the list of endangered species of the State of São Paulo, Brazil (4). Due to the scarce data about this plant in the literature, we decided to analyze the chemical composition of its essential oil, as well as to evaluate the antimicrobial activity against Candida albicans and other clinical isolates: C. glabrata, C. krusei, C. parapsilosis and C. tropicalis.

Experimental

Plant material and essential oil extraction: Lippia triplinervis Gardner was collected in flower above 1800 m,

Leitão et al.


Table I. Chemical composition (relative % peak area) of the essential oils from Lippia triplinervis (leaves) collected in April and September 2010.

No. Constituents RI lit 5,6 RIcalc April % September %

1 camphene 954 944 - 0.22 myrcene 991 992 2.6 2.43 n.i. - 1020 0.3 0.34 limonene 1029 1031 0.1 8.05 1,8 cineole 1031 1034 0.1 0.16 dihydro-tagetone 1053 1055 0.1 0.77 ipsenone 10836 1088 11.4 17.4 n.i. - 1093 0.1 0.28 linalool 1091 1100 0.4 0.39 n.i. - 1102 0.4 0.410 n.i. - 1115 0.2 0.1 n.i. - 1123 - 0.2 n.i. - 1132 - 0.1 n.i. - 1138 - 0.2 n.i. - 1141 - 0.111 (E)-tagetone 1144 1148 0.2 2.012 myrcenone 1150 1156 57.7 13.513 (Z)-tagetone 1152 1157 0.1 19.414 borneol 1169 1168 2.1 2.015 terpinen-4-ol 1177 1178 0.2 - n.i. - 1182 0.2 0.3

16 a-terpineol 1189 1191 1.4 0.9 n.i. - 1211 - 0.2 n.i. - 1218 - 0.317 (Z)-ocimenone 1229 1233 1.1 1.818 (E)-ocimenone 1238 1242 1.3 5.619 2-phenylethyl acetate 1258 1259 0.5 0.4 n.i. - 1273 - 0.220 bornyl acetate 1289 1285 0.6 0.821 (Z)-methyl cinnamate 1300 1306 t 0.522 piperitenone 1343 1342 - 3.6

23 a-cubene 1351 1350 0.2 -

24 a-copaene 1377 1374 0.5 0.725 (E)-methyl cinnamate 1378 1386 0.2 1.5

b-cubebene 1388 1388 0.3 -

a-gurjunene 1410 1406 0.2 0.2 (E)-caryophyllene 1419 1416 0.2 - n.i. - 1447 0.6 -

a-humulene 1455 1451 0.4 0.4 9-epi-(E)-caryophyllene 1466 1458 0.3 -

g-muurolene 1480 1478 0.1 0.4 (E)-muurola4(14),5diene 1494 1488 0.4 - n.i. - 1491 0.1 0.4 n.i. - 1500 0.1 0.4

g-cadinene 1514 1511 0.5 -26 (E)-calamenene 1529 1521 4.7 5.527 (E)-cadina1,4-diene 1535 1530 1.1 0.8

a-cadinene 1539 1535 0.2 - n.i. - 1541 0.2 0.228 germacrene B 1561 1553 0.8 0.7 caryophyllene oxide 1583 1579 0.2 - n.i. - 1584 0.6 - n.i. - 1591 0.3 0.3 guaiol 1601 1598 0.4 - humulene epoxide II 1608 1604 0.2 -29 1,10-di-epi-cubenol 1619 1611 1.1 1.5 n.i. - 1617 0.3 -

epi-a-cadinol 1640 1639 0.2 - (Z)-calamenen-10-ol 1661 1657 0.2 - (E)-calamenen-10-ol 1669 1665 0.2 - n.i. - 1675 0.4 -

L. triplinervis


at Aiuruoca, Minas Gerais, Brazil, in a place called “Campo dos Poejos” in April and September 2010. Plant material was identified by Dr. Fatima Salimena, from Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil, where a voucher specimen is deposited. The volatile oils from fresh leaves of L. triplinervis were obtained by hydrodistillation in a Clevenger type apparatus for 2 h, yielding 1.1% (April) and 1.0% (Sep-tember) of yellow essential oils, respectively.

Analysis of the essential oils: The oils were analyzed in an Agilent (Palo Alto, USA) 6890N gas chromatograph fitted with a 5% phenyl/95% methylsilicone (HP5, 25 m x 0.32 mm x 0.25 mm) fused silica capillary column. The oven temperature was programmed from 60ºC to 240ºC at 3ºC/min, and H2 was used as carrier gas (1.4 mL/min). It was injected 1.0 mL of a 1% solution of the oil in dichloromethane, in split mode (1:100). Injector was kept at 250ºC and detector (FID) at 280ºC. Mass spectra were obtained in an Agilent 5973N system operating in electronic ionization mode (EI) at 70 eV, with scan mass range of 40-500 m/z. Sampling rate was 3.15 scan/s. Ion source was kept at 230ºC, mass analyzer at 150ºC and transfer line at 260ºC. The mass detector was coupled to an Agilent 6890 gas chromatograph fitted with a low bleeding 5% phenyl/95% methylsilicone (HP-5 MS, 30 m x 0.25 mm x 0.25 mm) fused silica capillary column. Injection procedure and oven temperature program were the same as above. Helium was the carrier gas, at 1.0 mL/min. Linear retention indices (LRI) were measured by injection of a series of n-alkanes (C7-C26) in the same column and conditions as above for GC analyses. Identification of the oil components was based on computer

search using the Wiley 6th ed. library of mass spectral data and by comparison of their calculated linear retention indices with literature data (5,6).

Antimicrobial assay: Minimum inhibitory concentrations (MIC) were determined by broth microdilution method accord-ing to the document M27-A3 (Candida spp) of the Clinical and Laboratory Standard Institute (7), using resazurin as indicator for cell viability (8). All determinations were performed in triplicate and two independent experiments lead to concordant results. Positive (medium plus inoculum without essential oil) and negative (medium without inoculum or essential oil) growth controls were included in all assays. Fluconazole was used as reference antibiotic.


Fifty-five compounds were identified in the essential oil from the leaves of Lippia triplinervis collected in April 2010, of which the major components were myrcene (2.6%), ipsenone (11.4%), myrcenone (57.7%), (Z)-ocimenone (1.3%) and (E)-calamenene (4.8%) (Table I). The essential oil of this same plant collected in September 2010, showed a quite similar chemical composition, with 46 identified compounds (Figure 1). However, in this oil the relative percentage of myrcenone was drastically reduced from 57.7% to 13.5%, whereas limonene (8.0%), (E)-tagetone (2.0%) and (Z)-tagetone (19.4%) now figure as major constituents.

Myrcenone, one the major components of these essential oils, has been described as an important constituent of some

n.i. - 1764 0.9 0.2 n.i. - 1795 0.8 0.5 n.i. - 1804 0.4 - n.i. - 1829 0.5 - n.i. - 1908 1.0 0.4 Number of Identified Compounds 55 46

Total Identified Compounds % 92.5 96.3

n. i. – not identified , t – trace (< 0.1%).

Table I. Continued

No. Constituents RI lit 5,6 RIcalc April % September %

Table II. Minimum inhibitory concentration (MIC, ± SD) of the essential oila of Lippia triplinervis against Candida clinical strains.

Sample C. albicans C. parapsilosis C. krusei C. tropicalis C. glabata

MIC (mg. mL-1)

L. triplinervis 1,250.0 1,250.0 312.5 625.0 156.25 (721.6878) (0.0) (180.422) (0.0) (0.0)

Fluconazoleb 0.5 0.5 16.0 0.25 1.0

aSeptember, bonly one assay.

Leitão et al.


Figure 1. Gas Chromatograms (GC-FID) of the essential oils of Lippia triplinervis collected in April and September 2010.

L. triplinervis


other Lippia species such as L. lacunosa (64.2%), L. adoen-sis (0.81-14.89%); L. alba (38% to over 50%), L. multiflora

(54.6%), L. juneliana (17.2%), L. javanica and L. asperifolia (1). Ipsenone, a rarer monoterpene, has been described as one of the components of the volatile pheromone from Ips cembrae, the “large larch bark beetle” (9). It has been reported for the first time in the plant kingdom in the essential oil of Lippia multiflora from Congo (10). Later, it was also found to be the major component of an ipsenone rich-type (42-61%) of Lippia javanica (Burm. F.) Spreng. indigenous to South Africa (11), as well as in L. adoensis Hoechst ex Walp. var. adoensis

(12). The presence of high yields of (Z)-tagetone (19.4%) in the essential oil of L. triplinervis collected in September is noteworthy, since this substance has been reported to occur in high amounts (11.3% (Z)-tagetone; 30.2% (E)-tagetone) in the essential oil of another chemotype of L. multiflora (13). Apart from the presence of ipsenone, the chemical composition of these oils closely resembles that of L. lacunosa previously studied by our group, which has a strong mango-like aroma, due to the presence of myrcenone (1). In the same way, the essential oil from L. triplinervis displayed similar sensory characteristics, which was one of the features that guided the plant collection in the field.

The antimicrobial activity of the essential oil of L. triplin-ervis collected in September was evaluated by broth dilution method and the minimum inhibitory concentration (MIC) was determined to confirm the antimicrobial activity agaisnt Candida clinical strains. The MIC data are summarized in Table II. Candida glabrata was the most susceptible to the essential oil, followed by C. krusei and C. tropicalis, with MIC values below 625 mg/mL.

This is the first report of the chemical composition and antimicrobial activity of L. triplinervis essential oil.

Acknowledgements

The authors thank CNPq (Edital Universal) and FAPERJ for financial support.

References

S.G. Leitão, D.R. Oliveira, V. Sulsen, V. Martino, Y.G. Barbosa, H.R. 1. Bizzo, D. Lopes, L.F. Viccini, F.R.G. Salimena, P.H.P. Peixoto and G. Leitão, Analysis of the Chemical Composition of the Essential Oils Extracted from Lippia lacunosa Mart. & Schauer and Lippia rotundifolia Cham. (Verbenaceae) by Gas Chromatography. J. Braz. Chem. Society., 19, 1388-1393 (2008).

FRG Salimena, 2. PhD Thesis, Universidade de São Paulo, Brazil, 2000.

A.N. Caiafa and A.F. Silva, Composição florística e espectro biológico 3. de um campo de altitude no parque estadual da serra do brigadeiro, Minas Gerais – Brasil, Rodriguésia 56(87), 163-173 (2005).

Diario Oficial do Estado de São Paulo, Resolução da Secretaria de 4. Meio Ambiente de 09-03-1998, Brazil, 1998

RP Adams, Identification of Essential Oil Components by Gas 5. Chromatography/Quadrupole Mass Spectrometry, Allured Publ Corp, Carol Stream, IL, 2001.

L.S. Chagonda and J. C Chalchat. The essential oil of the fruit of 6. Garcinia huillensis Welw. & Oliv. from Zimbabwe. Flavour Fragr. J. 20, 313-315 (2005).

Clinical Laboratory Standard Institute. Reference Method for Broth 7. Dilution Antifungal Susceptibility Testing of Yeasts. Approved standard, M27-A3. Wayne, PA: Clinical Laboratory Standard Institute, 2008.

S.D. Sarker, L. Nahar and Y. Kumarasamy, Microtitre plate-based 8. antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods., 42(4), 321–324 (2007).

Q.H. Zhang, G. Birgersson, F. Schlyter, and G.F. Chen, Pheromone 9. components in the larch bark beetle, Ips cembrae, from China: Quantitative variation among attack phases and individuals. J. Chem. Ecol. 26, 841–858 (2000).

G. Lamaty, C. Menut, J.M. Bessierea, J.A. Ouambab and T. Siloub, 10. 2-Methyl-6-Methylene-7-Octen-4-One, a constituent of Lippia multiflora essential oil. Phytochemistry, 29(2), 521-522 (1990)

A.M. Viljoen, S. Subramoney, S.F. Van Vuuren, K.H.C. Baser and B. 11. Demirci, The composition, geographical variation and antimicrobial activity of Lippia javanica (Verbenaceae) leaf essential oils. J. of Ethnopharm. 96, 271–277 (2005)

K. Asres and F. Bucar, 12. Lippia adoensis var. adoensis: studies on the essential oil composition and antioxidant activity. Eth. Pharm.l J. 20, 32–38 (2002)

Y. Pélissier, C. Marion, J. Cassadebaig, M. Milhau, D. Kone, G. 13. Loukou, Y. Nanga and J.M. Bessiére, A Chemical, Bacteriological, Toxicological and Clinical Study of the Essential Oil of Lippia multiflora Mold. (Verbenaceae). J. Essent. Oil Research, 6, 623–630 (1994).

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Rec: Feb 2011

Acc: July 2011

Activity against Streptococcus pneumoniae of the Essential Oil and d-Cadinene Isolated from Schinus

molle Fruit

Alejandro Pérez-López*, Anabel Torres Cirio, Verónica M. Rivas-Galindo, Ricardo Salazar Aranda and Noemí Waksman de Torres

Departamento de Química Analítica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México., P.O. Box 2316 Sucursal Tecnológico, C.P. 64841 Monterrey, N.L; México

Abstract

Streptococcus pneumoniae is a major cause of respiratory infections. This study aims to test the activity of the essential oil obtained from the fruit of Schinus molle against S. pneumoniae resistant to conventional antibiotics and to identify the compounds responsible for the activity. A fraction showing antimicrobial activity (MIC 125 mg/mL) was obtained. The principal components were identified as: b-myrcene (39.7%), p-cymene (19.5%), d-cadinene (7.8%), a-phellandrene (7.1%) and limonene (4.1%). Bioassay-guided fractionation led to the identification of d-cadinene as the principal active constituent (MIC of 31.25 mg/mL). The findings reported here highlight and justify the global spread of the use of S. molle for the treatment of respiratory infections.

Key Word Index

Schinus molle, essential oil, antimicrobial, d-cadinene, Streptococcus pneumoniae.



Introduction

Streptococcus pneumoniae is the causal agent of infections in the respiratory tract such as pneumonia and sinusitis; this bacteria causes meningitis, septicemia, and otitis media in the general population as well as in hospital patients. Pneumonia has a high rate of annual mortality worldwide, mainly in chil-dren (1). Studies in Latin American countries show that most serious clinical cases of pneumonia are associated with bacte-rial infection with predominance of S. pneumoniae followed by Haemophilus influenzae type b (2,3). Multiple microbial resistances acquired over time is alarming; many species of infection-causing bacteria, which once seemed under control, are now difficult to treat (4-6). Furthermore, the number of strains of S. pneumoniae resistant to penicillin and other b-lactamics and to vancomycin is increasing (2, 7).

An alternative approach to fight these antibiotic-resistant strains of microorganisms is to use antimicrobially active in-gredients obtained from plants, in particular from those used in traditional medicine (8).

In México, as in other developing countries, traditional medicine is an important source of products for treating com-mon infections. About 25% of the Mexican population depends exclusively on the use of medicinal plants (9).

It is well known that plants containing essential oils, which

serve to protect and prolong the life of the plant because they often constitute a means of defense against predators, can act as insect repellents (10,11). The essential oils are used in natural therapies and alternative medicine as remedies for many infectious diseases, and the antimicrobial properties of essential oils have been long recognized and traditionally used for respiratory tract infections and as ethnic medicines for colds. In the medicinal field, inhalation therapy with es-sential oils has been used to treat acute and chronic bronchitis and acute sinusitis (12). Several studies have confirmed that essential oils have highly significant antimicrobial properties against bacteria, yeast, and fungi (13-15).

Schinus molle is a plant belonging to the Anacardiaceae family and is native to South America; however, it is found worldwide (16). In traditional medicine, the plant is used against coughs, colds, tuberculosis, bronchitis, and fever (9, 17). The antimicrobial efficacy of S. molle and related species has been previously reported (18-21).

In a previous work, we reported the antimicrobial activ-ity of a hexane extract obtained from S. molle fruit against S. pneumoniae (9). Following from these antecedents, the pres-ent work aimed to determine the chemical composition of the essential oil by GC/MS analysis, evaluate its activity against S. pneumoniae, and isolate the main active component.

S. pneumoniae


Experimental

Chemicals: The following chemicals and other materials were used in the study: hexane, ethyl acetate, and methanol (Fermont, Productos Químicos Monterrey, Monterrey, N.L. Mexico); dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA); alkane standard solution C8–C20 (Fluka, Sigma-Aldrich, Switzerland); oxacillin and vancomycin (Sigma-Aldrich); silica gel 60, 0.2–0.5 mm (Merck, Darmstadt, Germany); thin layer chromatography (TLC) silica gel 60 aluminum sheets 20 x 20 cm (Merck); agar supplemented with bovine blood (BBL, Becton Dickinson of Mexico, Estado de México, Mexico); and cation-adjusted Mueller-Hinton broth supplemented with 5% lysed horse blood (CAMHB-LHB; BBL, Becton Dickinson, Sparks, MD, USA).

Bacterial culture: Two isolates of S. pneumoniae resistant to oxacillin but sensitive to vancomycin (InDRE 24-CCpn-02; InDRE 49619) were obtained from the “Instituto Nacional de Diagnóstico y Referencia Epidemiológicos” (InDRE, México. D.F., Mexico). The microorganisms were maintained on agar supplemented with bovine blood (BBL, Becton Dickinson de México) until use.

Plant material: S. molle fruit was collected in Arteaga, Coahuila, Mexico, in November 2008. A voucher specimen (UNL 024166) was deposited in the herbarium of the Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León.

Isolation of essential oil: The oil was isolated by hydro-distillation of ground fresh fruit (100 g/1 L water) for 4 h, using a Clevenger-type apparatus. The oil obtained was collected in two fractions (according to as they distilled), one colorless and the other yellow, and conserved at -4°C until use.

Antimicrobial activity: S. pneumoniae strains resistant to b-lactamic antimicrobials were tested with microdilution assays according to the National Committee for Clinical Laboratory Standards (9, 22). In order to prepare the inocula, S. pneumo-niae strains were cultured in Petri dishes containing blood agar (Bacto, Becton Dickinson). Plates were incubated overnight at 37°C and suspensions were prepared by transferring colonies to 0.85% NaCl solution until the turbidity of the 0.5 McFarland standard was reached. The suspensions were diluted 1:50 with cation-adjusted Mueller–Hinton broth supplemented with 5% lysed horse blood (CAMHB-LHB; BBL, Becton Dickinson) to make the working suspensions of S. pneumoniae. The es-sential oil was prepared at a concentration of 2 mg/mL in 20% of DMSO in CAMHB-LHB. The antimicrobial activity assay was performed in flat-bottom 96-well polystyrene microplates covered with a low evaporation lid. The culture medium was CAMHB-LHB. The concentration of the essential oil ranged from 500,000 to 15.625 mg/mL. Oxacillin and vancomycin were used as antimicrobial drug controls (64–4 mg/mL). The final concentration of microorganisms was 1 x 104 UFC. Plates were incubated at 37°C for 24 h and bacterial growth was ex-amined. The MIC was defined as the minimum concentration of essential oil that stops growth. Every biological assay was conducted in duplicate.

GC analysis: GC analysis was carried out on a GC Perkin Elmer Autosystem XL equipped with a flame ionization detec-tor and a HP-5MS column (30 m x 0.25 mm i.d., 0.25 mm film thickness). Helium (99.999%) was used as carrier gas at a flow rate of 0.5 mL/min. Injector and detector temperature were

set at 220°C and 290°C respectively. Oven temperature was programmed to 35°C for 9 min, then from 35°C to 150°C at 3°C/min and held for 10 min, then at 10°C/min to 250°C, and finally at 3°C/min to 270°C and held for 10 min. The samples were injected using the splitless mode. The injection volume was 2 μL. Percentage composition was calculated using peak normalization method assuming equal detector response for all the compounds.

GC/MS analysis: GC/MS was performed using an Agilent Technologies 6890N gas chromatograph equipped with an HP-5MS column (30 m x 0.25 mm i.d., 0.25 mm film thickness) and a 5973 INERT selective mass spectrometer. The carrier gas was He (99.999%) at a flow rate of 0.5 mL/min; ionization energy was 70 eV. Data acquisition was scan mode. Ionization source temperature was 230°C, quadrupole temperature was 150°C, and the injector temperature was 220°C. Oven temperature was programmed to 35°C for 9 min, then from 35°C to 150°C at 3°C/min and held for 10 min, then at 10°C/min to 250°C, and finally at 3°C/min to 270°C and held for 10 min. The samples were injected using the splitless mode. The injection volume was 2 μL. Components were identified by comparison of their retention indices relative to C8–C20 n-alkanes, and their mass spectra were compared with mass spectra from the US National Institute of Standards and Technology (NIST) library and reference data (23).

Activity guided fractionation: Yellow oil (8 g) obtained from the S. molle hydrodistillation was subjected to column chromatography over silica gel. Elution was started with hexane, and then with eluents of increasing polarity including EtOAc and MeOH to give four fractions (F1 to F4). On the basis of the antimicrobial activity, F1 (1.1 g) was further fractioned with 97:3 hexane:EtOAc and MeOH to give 14 fractions (F1-1 to F1-14). According to its antimicrobial activity, fraction F1-2 was further analyzed by GC/MS. Fraction F2 (0.32 g) was further subjected to column chromatography over silica gel, eluted with 97:3 hexane:EtOAc; fractions with similar Rf were combined to give, finally, seven fractions (F2-1 to F2-7). F3 (0.22 g) was further purified by preparative TLC on silica gel eluted with 10:2 hexane:AcOEt to yield three fractions (F3-1 to F3-3), which were analyzed by GC/MS. Fraction 4 (0.5 g) was subjected to column chromatography over silica gel, eluted with 97:3 hexane:EtOAc to give 10 fractions (F4-1 to F4-10). Fraction F4-6 was further separated by prepara-tive TLC on silica gel and eluted with 10:3 hexane:EtOAc to give three fractions (F4-6-1 to F4-6-3). Fraction F4-6-1 was analyzed by GC/MS. All GC/MS analyses were conducted as previously described.


Several papers reporting the potential antimicrobial activity of the essential oil obtained from S. molle have been published; Gundiza showed the activity of the essential oil obtained from leaves against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli, among other bacteria (24). Fuselli et al. demonstrated the activity of aerial parts (fruits and leaves) against Paenibacillus larvae larvae, a casual agent of American foulbrood in honey bees (20). Dikshit et al. found that the aerial parts of the plant inhibited 100% of the growth of mycelia of

Pérez-López et al.


Table I. Minimal Inhibitory Concentration (MIC) of essential oil obtained from Schinus molle fruit and fractions against

Streptococcus pneumoniae strains.

Essential oil from MIC (mg/mL) Schinus molle oil fruit (fraction) Streptococcus pneumoniae

Strain ATCC 49619 Strain 24-ccpn-02

Colorless 250 250 Yellow 125 125 F1 31.25 31.25 F2 250 125 F3 62.5 62.5 F4 62.5 62.5

F1-2 (d-cadinene) 31.25 31.25 F2-5 to F2-7

(a-cadinene) 500 500 F3-1 Not active Not activeF3-2 Not active Not activeF3-3 Not active Not activeF4-6-1 (t-muurolol) Not active Not active Cephalotin 1 4

Oxacillin 1 8

Table II. Composition of the yellow fraction from the essential oil of the fruit of Schinus molle

Name Percentage RIa RIb

a-pinene 3.2 930 939

b-myrcene 39.7 992 990

a-phellandrene 7.1 1002 1002p-cymene 19.5 1023 1024limonene 4.1 1028 1029methyl octanoate 2.3 1126 1127unknown 1 FW 152 0.9 1200 - verbenyl acetate <trans> 1.33 1291 1291unknown 2 FW152 2.0 1317 -

b-elemene 0.2 1390 1389

a-gurjunene 1.2 1408 1409

b-caryophyllene 0.8 1422 1417

a-humulene 0.7 1452 1452

g-muurolene 0.5 1475 1478germacrene D 0.7 1480 1485

a-muurolene 1.6 1499 1500

g-cadinene 1.6 1511 1513

d-cadinene 7.8 1520 1522elemol 0.3 1543 1548gleenol 0.6 1589 1586viridiflorol 0.4 1591 1592ledol 0.21 1601 1602

t-muurolol 0.2 1636 1642

a-cadinol 3.1 1650 1652

a IR= Retention indices relative to n-alkanes on HP-5MS column.b IR= Retention indices from literature

Microsporum gypseum, Trichophyton mentagrophytes, and Trichophyton rubrum, pathogenic fungi of several animals; they also demonstrated inhibition (although to a lesser de-gree) of the storage fungi Alternaria alternata, Aspergillus flavus, and Penicillium italicum (18). Padin et al. found that the ethanolic extract obtained from the fruit of S. molle, was active against Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, E. coli, Salmonella enteritidis, P. aeruginosa, and K. pneumoniae, with MICs between 1 and 5 mg/mL (21). Belhamel et al. reported that an extract obtained from the aerial parts was active against S. aureus, S. pyogenes, E coli, and Vibrio vulnificus (25). Our research group demonstrated the activity of a hexane extract obtained from the S. molle fruit against S. pneumoniae, H. influenzae, and Mycobacterium tuberculosis (9). Considering these findings and taking into account the importance of S. pneumoniae as a causal agent of respiratory tract diseases, in the present contribution we investigated whether the essential oil was responsible for the reported activity.

During the hydrodistillation process, colorless oil was first obtained (2.3% w/w) and subsequently yellowish oil could be distilled (1.0% w/w). The fractions were assayed for antimicro-bial activity. The yellow fraction was found to be more active against both antibiotic resistant S. pneumoniae strains with a MIC of 125 mg/mL; the colorless fraction showed a MIC of 250 mg/mL (Table I).

In this way, the yellow fraction was further analyzed by GC/MS. The results demonstrate that this essential oil is very rich in terpenes and 24 major components were found, of which 22 were identified (97% from the total area). Unidentified com-pounds are minor components and none of them represents more than 3% of the total area (Table II). The oil was abundant in monoterpenes (76.8% from the total area), predominantly b-myrcene (39.7%), p-cymene (19.5%), a-phellandrene (7.1%), limonene (4.1%) and a-pinene (3.2%). d-cadinene (7.8%) and a-cadinol (3.1%) were the main sesquiterpene components. This composition is consistent with that of the essential oil obtained from the fruits of S. molle collected in Peru (26) and Tunisia (27).

Following a bioassay-guided fractionation, column chro-matography of the yellow essential oil obtained from S. molle fruit over silica gel, yielded four fractions (F1 to F4), and all fractions showed antimicrobial activity against both S. pneu-moniae strains; however, F1 showed the best activity, with an MIC of 31.25 mg/mL (Table I). Purification of F1 yielded 14 fractions (F1-1 to F1-14), with fraction F1-2 being the most abundant and the most active against S. pneumoniae, as its MIC was 31.25 mg/mL for both strains. This fraction was identified through GC/MS as d-cadinene by matching the mass spectrum with the NIST and Adams libraries and its retention index was consistent with that reported for d-cadinene in the literature (23).

F2 yielded seven fractions upon purification (F2-1 to F2-7). From F3, three fractions were obtained (F3-1 to F3-3); MIC values for F3-1 to F3-3 against S. pneumoniae were 500 mg/mL for both resistant strains. The principal component in these fractions was identified as a-cadinene. On the other hand, F4 yielded 10 fractions (F4-1 to F4-10). Fraction F4-6 was further separated to afford three fractions (F4-6-1 to F4-

6-3). t-Muurolol was identified as the principal component in fraction F4-6-1; however, no activity against either strain of S. pneumoniae could be found. The results of the activ-ity found strongly suggest that d-cadinene is the principal

S. pneumoniae


compound responsible for the antimicrobial activity against S. pneumoniae. The fractions named as F3 and F4 obtained from the yellow fraction also showed good activity (MICs 62.5 mg/mL); however, this antimicrobial activity was lost during fractionation (Table I). These findings were also reported by other authors investigating other plants, which suggests that the compounds must remain together to be active, perhaps through a synergic mechanism, or that the presence of minor components enhances the activity (28).

The antimicrobial activity displayed by S. molle essential oil against S. pneumoniae supports the traditional use of this plant for the treatment of infectious diseases. The results presented here are important in the search for new antimicrobial agents against bacteria responsible for respiratory diseases, especially those resistant to conventional antibiotics.

Acknowledgements

The authors are grateful to the biologists Marco Antonio Guzmán Lucio and M.C. María del Consuelo González de la Rosa for definitive taxonomic identification of species reported here. We thank Ivonne Carrera for her technical assistance in the extraction procedures and acknowledge grants 103.5/08/3125 and 103.5/09/4913 from PROMEP-Mexico and U.A.N.L. (PAICYT) SA 233-09.

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G.M. Molina-Salinas, A. Pérez-López, P. Beceril-Montes, R. Salazar-9. Aranda, S. Said-Fernández, N. Waksman de Torres, Evaluation of the flora of Northern Mexico for in vitro antimicrobial and anti-tuberculosis activity. J Ethnopharmacology, 109, 435-441 (2007).

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Rec: Jan 2011

Acc: July 2011

Chemical Composition of Leaf Essential Oils of Calyptranthes microphylla B. Holts & M.L., Myrcia

aff fosteri Croat and Eugenia octopleura Krug & Urb from Panama*

Ana I. Santana TenorioCentro de Investigaciones Farmacognosticas de la Flora Panameña (CIFLORPAN),Departamento de Quimica Organica,

Universidad de Panama, Panama, Republicade Panama

Deisy Vargas and Alex EspinosaCentro de Investigaciones Farmacognósticas de la Flora Panameña (CIFLORPAN), Universidad de Panamá, Panama,

Republica de Panama

Albano DíazInstituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), Panama, Republica de Panama

Mahabir P. Gupta**

Centro de Investigaciones Farmacognósticas de la Flora Panameña (CIFLORPAN), Universidad de Panamá, Panama, Republica de Panama

Abstract

The chemical compositions of essential oils obtained by hydrodistilation from leaves of Calyptranthes microphylla B. Holts & M.L., Myrcia aff fosteri Croat and Eugenia octopleura Krug & Urb, belonging to family Myrtaceae were studied by GC and GC/MS. Forty-three compounds, representing 93.5% of the total, were identified in C. microphylla, b-pinene (48.4%) and b-bisabolene (12-0%) being the major components, while the essential oils of M. aff fosteri contained 38 compounds, a-bisabolol (19.2%) and b-bisabolol oxide (19.2%) being the principal components. From the essential oil of E. octopleura 40 components, consisting principally of a-pinene (43.0%) and limonene (23.6%) were identified. The oil from Myrcia aff fosteri showed activity against Staphylococcus aureus and Bacillus subtilis, which was comparable to chloramphenicol.

Key Word Index

Myrtaceae, Calyptranthes microphylla, Myrcia aff fosteri, Eugenia octopleura, a-pinene, b-bisabolene, b-bisabolol oxide, a-bisabolol, limonene.

*Presented in XIII National Congress of Science and Technology, Panamanian Association for the Advancement of Science, City of Knowledge, Panama, 6-9 October 2010.



Introduction

The family Myrtaceae consists of 129 genera and 4620 species distributed throughout the world (1). In Panama, 72 species pertaining to 17 genera have been reported (2). This family is rich in essential oils. In a previous study on Plinia cerrocampanensis Barrier, an endemic species of Panama, an oxygenated sesquiterpene a-bisabolol, a highly valuable constituent in the pharmaceutical and cosmetic industries, has been reported in very high yields (3).

In our ongoing research on the aromatic flora of Panama, we herein report the chemical composition of essential oils of three species Calyptranthes microphylla B. Holst & M.L. Kawas, Eugenia octopleura Krug & Urb and Myrcia aff Fosteri Croat, of which the latter is endemic to Panama. No chemical data on essential oil compositions of these essential oils has been published previously. However, the infusion of the leaves from a related species Calyptranthes bipennis O. Berg has been used as a stimulant and analgesic (4). Eugenia uniflora, known as

C. microphylla


Brazilian cherry tree, has been investigated and determined to be effective in treatment against digestive disorders and com-monly used as an antifebrile, ant-irheumatic, anti-inflammatory, diuretic, to lower blood glucose levels (5, 6), and has been used in the cosmetic industry. Oil from this species contains a mixture of atractylone and 3-furanoesdesmene (7). Essential oil composition of seven species of Eugenia from Costa Rica has been investigated. E. austin-smithii and E. cartagensis contain trans-2-hexenal, while a-pinene from E. haberi and E. zuchowskiae, linalool from E. monteverdensi and 1,8-cineol and zingiberene from Eugenia sp have been reported (8).

Experimental

Plant material: Leaves from wild plants were collected from two national parks (Cerro Jefe, Chagres National Park and National Park of Altos de Campana) with the permission of the National Environment Authority of Panama. Taxonomi-cal identification of plants was established by Alex Espinosa Taxonomist CIFLORPAN. The voucher specimens are depos-ited in the Herbarium the University of Panama (PMA) under number FLORPAN 8543 for C. microphylla (N:09o13.444´; W:79o22.327´; altitude: 863.0 m), FLORPAN 8548 for E. octopleura (N: 08o40.994´; W: 779o55.588´; altitude: 845.0 m) and FLORPAN 8544 for M. aff fosteri (N: 09o13.532´; W: 79o22.320´; attitude: 860.0 m). Leaves from the three species were hydrodistilled with a Clevenger-type apparatus to obtain essential oils in yields of 0.55, 0.75 and 0.74 for C. microphylla, M. aff fosteri (endemic) and E. octopleura, respectively.

Chemicals: Numerous authentic standards were used to build the homemade MS library and to determine the RI as described in the experimental section on polar and apolar column (9). All chemicals were purchased from Fluka - Sigma Aldrich (Quifar International SA Panama City, Panama).

Analysis of the essential oils: Oils were analyzed by GC and GC/MS. The quantification of the chemical components was carried out by using Agilent Technologies, model 6890N Gas Chromatograph (FID) with HP-5 capillary column with a (5% phenyl)-methylpolysiloxane as a stationary phase (30 m x 0.32 mm i.d.; 0.25 μm film thickness). Carrier gas: high purity N2, flow rate 1.0 mL/min, temperature 250oC, oven temperature program from 50oC to 220oC at 4oC/min, for 5 min and then at 6oC/min to 250oC with detector temperature of 280oC. Samples were injected by splitting and the split ratio was 1:20.

GC/MS analysis was done using an Agilent System model 6890N Gas Chromatograph, a model 5973 mass selective de-tector (MSD), and an Agilent Chem Station data system. The GC column was an HP-5 MS fused silica capillary with a (5% phenyl)-methylpolysiloxane stationary phase (30 m x 0.25 mm id.. x 0.25 μm film thickness). Helium was the carrier gas with a flow rate of 1.0 mL/min. GC temperature program were the same as GC-FID. The inlet temperature was 250oC and the oven temperature program was as follows: 50oC to 220oC at 4oC/min, for 5 min and then at 6oC/min to 250oC with the inter-phase temperature of 280oC. The split injection mode (1:142), detector temperature 270oC. 0.1μL of the pure essential oil was injected. MS interface temperature 270°C, Ms mode, E.I. detector voltage 1300 V; mass range 40-400 u, 1 scan/s.

Identification of components was achieved based on their

retention indices, (determined with reference to a homolo-gous series of fatty acids methyl esters) as previously reported (9). Table I shows the values of RI experimentally obtained compared with references RI values either calculated within this research or available in our homemade library built with authentic standards or with naturally occurring components identified in previous studies by 13C NMR and analyzed on SE-30 capillary column. The identification was considered reliable with a difference between experimental and standard RI below 5 units. The MS spectra were also compared to the spectral fragmentation patterns available from literature [Adams (10) and MS library (NIST database, Adams, Wiley9, Wiley6, Wiley275) Chen Station Data System (11)].

Determination of antimicrobial activity: Antimicrobial activity was carried out according to the disc diffusion assay, Rondon et al. (12).

The bacterial strains used were Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeroginosa y Klebsiella sp.

The fresh cultures were seeded on TSA media and incu-bated at 37oC for 24 h. The bacterial inoculum was diluted in sterile 0.85% saline to obtain a turbidity visually comparable to a McFarland No. 5 standard (106-8 CFU/mL). The bacte-rial suspension was inoculated on LB agar plates and later 5 mm filter paper disc impregnated with the known quantity of chloramphenicol was placed on the plates. This served as a standard. The disc impregnated with 5 μg/mL of essential oil was used to test the activity. A disc impregnated with 0.85% sterile saline served as a negative control. The plates were incubated at 37oC for 24 h and the diameter of the zones of inhibition was read. Each experiment was done in duplicate.


The qualitative and quantitative analytical results of three essential oils are shown in Table I.

In the essential oil form leaves E. octopleura, forty com-ponents, corresponding to 92.5% of the total, were identified. These were mainly sesquiterpene hydrocarbons, oxygenated monoterpenes, and monoterpene hydrocarbons. a-Pinene (43.1%) was the principal component, followed by limonene (23.6%), b-ocimene (5.5%), viridiflorol (3.6%), linalool (3.0%), trans-caryophyllene (1.5%) and a-terpineol (1.5%). Essential oil from a related species Eugenia speciosa also contained a-pinene (47.3%) and limonene (23.0%) in similar proportions, while the oil form E. cuprea was constituted principally by sesquiterpenes spathulenol, b-caryophyllene and caryophyl-lene oxide; the oil from E. octopleura contained higher amount monoterpenes. (13).

The oil from the leaves of an endemic species Myrcia aff fosteri, had an intense yellow color. In it, 38 compounds cor-responding to the 76.7% of total were identified. These were mainly sesquiterpene hydrocarbons and oxygenated sesqui-terpenes, represented by a-bisabolol (19.2%), b-bisabolene oxide (19.3%), b-bisabolol oxide B (7.0%), caryophyllene oxide (3.5%), trans-6,11-dimethyl-3,8-oxoethane-bicyclo-(6,3,0)-undeca-4,6-diene (3.1%), 1-epicubenol (3.4%), grosonol (3.2%), trans-6,11-dimethy-3,8-oxomethane-bicyclo-(6,3,0)undeca-4,6-diene (2.3%), a-calacorene (2.1%), a-cadinene

Tenorio et al.


Table I.

COMPONENTS RIexp RIref C. microph. B. Holst M. aff fosteri E. Octopl. & M.L. Kawas Croat Krug & Urb

hexanal nd nd t b 2-hexenal nd nd 0.1 b 3-hexen-1-ol nd nd 0.3 b cyclohexanol nd nd t b

a-pinene 126 127 48.4 a,b 43.0 b

camphene 139 138 0.2 a,b

b-pinene 142 144 4.5 a,b 0.8 a,b

6-methyl-5-hepten-2-one 155 157 0.4 a,b 0.2 a,b

b-myrcene 160 165 0.8 a,b

p-cymene 201 204 0.1 a,b 0.8 a,b

limonene 211 213 1.1 a,b t a,b 23.6 a,b

1,8-cineol 213 214 0.8 a,b b-ocimene* 228 231 5.1 a,b

endo-fenchol 245 b 0.2 a,b

p-cymenene 301 299 0.1 a,b 0.8 a,b

terpinolene 292 293 - blinalol 308 311 3.0 a,b

6-methyl-3,5-heptadien-2-one 309 313 0.2 a,b

a–camphonelal 321 322 0.7 a,b 4-acetyl-1-metylcyclohexane 323 323 0.3 a,b cis-pinocarveol 330 330 3.7 a,b 0.1 a,b

pinocarvone 339 340 1.6 a,b endo-borneol 340 341 0.3 a,b 0.2 a,b

terpinen-4-ol 345 345 0.4 a,b

acetophenone 348 349 1.0 a,b

a–terpineol 351 353 1.1 a,b 1.5 a,b

verbenone 359 357 t a,b trans-carveol 362 362 0.4 a,b 0.1 a,b

cis-p-mentha-1(7),8-dien-2-ol 366 365 0.2 a,b

geraniol 376 374 0.2 a,b carvone 384 384 t a,b pinocarvyl acetate 388 390 t a,b bornyl acetate 392 394 0.2 a,b

trans-carvyl acetate 404 402 t a,b

a–copaene 431 431 0.4 a,b 0.3 a,b 0.6 a,b

b–bourbononene 436 440 0.1 a,b

a–cedrene 446 444 t a,b

a–gurjunene 449 448 0.2 a,b

cis-a-bergamotene 451 451 0.6 a,b 0.2 a,b

a–santalene 454 454 0.2 a,b trans-caryophyllene 455 453 3.4 a,b 1.5 a,b

germacrene D 458 457 0.1 a,b

b–copaene 459 460 0.1 a,b

b–gurjunene 461 461 0.2 a,b

trans-a-bergamotene 461 463 0.2 a,b 0.8 a,b

a–humulene 471 470 1.0 a,b 0.3 a,b

santalene stereoisomer* 473 473 0.7 a,b aromadendrene 464 465 b 0.5 a,b

allo-aromadendrene 474 474 0.3 a,b 0.4 a,b

g–curcumene 474 474 0.1 a,b trans-cadine-1-(6)-4-diene 481 480 0.3 a,b

a–amorphene 482 482 0.6 a,b ar-curcumene 483 484 3.0 a,b 0.5 a,b 0.2 a,b

trans-b-farnesene 485 485 t a,b 0.2 a,b viridiflorene 489 490 0.1 a,b

a–muurolene 491 492 0.3 a,b 0.5 a,b

b–bisabolene 495 495 12.0 a,b 0.4 a,b

b–curcumene 498 498 0.4 a,b 0.1 a,b

b-amorphene 502 501 0.5 a,b

d-cadinene 503 503 2.0 a,b

d-amorfene 503 504 0.8 a,b cadine-1,4-diene 507 509 0.5 a,b

C. microphylla


g-bisabolene 507 510 1.0 a,b

a-cadinene 512 512 0.2 a,b

a-calacorene 516 517 2.1 a,b 0.1 a,b

cis-a-bisabolene 514 514 t a,b nerolidol 525 526 0.5 a,b 0.3 a,b

ß-calacorene 528 528 0.6 a,b ar-tumerol 536 536 0.5 a,b caryophyllene oxide 537 537 1.4 a,b 3.5 a,b viridiflorol 537 538 3.6 a,b

viridiflorol isomer* 540 540 0.5 a,b

t-cadinol 546 546 0.5 a,b

iso-longifolene 549 549 0.2 a,b

trans-6,11-dimethyl-3, 8-oxomethane-bicyclo-(6,3,0) undeca-4.6-diene 553 554 5.4 a,b

1-epi-cubenol 561 562 3.4 a,b gossonorol 564 563 3.2 a,b cedreanol 567 566 1.4 a,b t–muurolol 565 566 1.0 a,b

a–cadinol 574 574 1.4 a,b 0.8 a,b

ß-bisabolol oxide 577 578 19.2 a,b bisabolol oxide B 578 578 7.0 a,b iso-italacene 580 580 0.2 a,b

cadalene 580 581 1.7 a,b

a-bisabolol 590 592 1.6 a,b 19.2 a,b melaleucol 594 596 t a,b cryptomerione 608 609 0.8 a,b phytol 793 793 0.3 a,b Total 93.5% 76.7% 92.5%Monoterpene Hydrocarbons 54.0% 0.2% 75.1%Oxygenated monterpenes 8.8% 2.0% 6.1%Sesquterpene hydrocarbons 24.5% 8.2% 5.4%Oxygenated sesquiterpenes 5.9% 65.9% 5.9%Other 0.3% 0.4% 0.0%

Number of compounds identified 43 38 40

RI: average retention rate calculated experimentallya: identified by GC-FID; b: identified by GC/MS; t: trace amount, standard deviation= 0.1%; *correct isomeric form not identified

(2.0%), cadelene (1.7%) and a-cubebene (1.4%).In the oil from the leaves of C. microphylla 43 compounds

corresponding to 93.5% of the total were identified. It was constituted principally of a-pinene (48.4%), b-bisabolene (12.0%), b-pinene (4.5%), trans-caryophyllene (3.4%), cis-pinocarveol (3.7%), Ar-curcumene (2.9%), a-cadinol (1.8%), caryophyllene oxide (1.4%), limonene (1.1%); endo-fenchol and aromadendrene were identified by GC/MS, but in GC-FID their amounts could not be quantified. It is noteworthy, that a- and b-pinene in this oil are lost easily, resulting in an increase in the amount of bisabolene.

Of the three essential oils tested, only the oil form Myrcia aff fosteri showed good antimicrobial activity against S. aureus and B. subtilis. This did not show activity against Gram negative bacteria P. aeroginosa and Klebsiella sp. Chloramphenicol was used as a positive control. Candino et al. (14) have also reported antimicrobial activity in essential oil form Myrcia ovata against Escherichia faecalis, E. coli, Salmonela cholorasies, Streptococ-cus pneumoniae and Candida parapsilosis.

Table I. Continued

COMPONENTS RIexp RIref C. microph. B. Holst M. aff fosteri E. Octopl. & M.L. Kawas Croat Krug & Urb

Results show that Myrtaceae is an important aromatic family, and its other species warrant further studies.

Acknowledgements

Thanks are due to the National Secretariat for Science, Technol-ogy, and Innovation of Panama (SENACYT), and Organization of American States for financial support. Thanks are due to Raineldo Urriola of Smithsoniam Tropical Research Institute for permintting the use of GC/MS.

References

D.J. Mabberley, 1. The Plant-book. Cambridge University Press, Cambridge, UK. (1997).

M. Correa, M. Staff, C. Galdames, 2. Catálogo de Plantas Vasculares. Editorial Novo Art. Panamá. pp 599, (2004).

R. Vila, A.I. Santana, R. Pérez-Rosés, A. Valderrama, M.V. Castelli, S. 3. Mendonca, S. Zacchino, M.P. Gupta and S. Cañigueral, Composition and Biological Activity of the Essential Oil from Leaves of Plinia

cerrocampanensis, a New Source of a-Bisabolol. Bioresource Technology, 10 (7): 2510–2514 (2010).

Tenorio et al.


Carol Stream, IL (2005).

National Institute of Standards and Technology, 11. PC version of the NIST/EPA/NIH Mass Spectral Database. U.S. Department of Commerce, Gaithersburg, MD (1998).

M. Rondón, J. Velazco, J. Hernández, M. Pecheneda, A. Morales, J. 12. Rojas, J. Carmona, T. Díaz, Chemical composition and antibacterial activity of essencial oil of Tagetes patula L. (Asteraceae) collected in the Venezuela Andes. Revista Latinoamericana de Química. 2006.

M.A. Apel, M. Sobral, E.E.S. Schapoval, A.T. Henriques, 13. Essential Oils from Eugenia species–Part VII: Sections Phyllocalyx and Stenocalyx. J. Essent. Oil Res. 16, 135–138, 191–192, 437-439, (2004).

C. Candino, C. Portella, B. Laranjeira, B. Da Silva, A. Arriaga, G. 14. Santiago, G. Gomes, C. Almeida, C. Carvalho, Effects of Myrcia ovata Cambess. Essential oil on planktonic growth of Gastrointestinal microorganisms and biofilm formation of Enterococcus faecalis. Brazilian J Microbiology 41, 621-627, (2010). In press.

J. Sanz-Biset, J. Campos de la Cruz, M.A. Epiquién-Rivera, S. 4. Cañigueral, A first survey on the medicinal plants of the Chazuta Valley (Peruvian Amazon). J Ethnopharmacol., 122, 333-362 (2009).

A. Kanazawa, A. Patin, A.E. Greene, 5. Efficient, highly enantioselective synthesis of selina-1,3,7(11)-triene-8-one, a major component of the essential oil of Eugenia uniflora. J Nat Prod. 63, 1292–1294 (2000).

I.A. Ogunwande, N.O. Olawore, O. Ekundayo, T.M. Walker, J.M. Setzer, 6. Studies on the essential oils composition, antibacterial and cytotoxicity of Eugenia uniflora L. Int. J. Aromather. 15, 147–152 (2005).

A.C.L. Amorim, C.K.F. Lima, A.M.C. Hovell, A.L.P. Miranda, C.M. 7. Rezende, Antinociceptive and hypothermic evaluation of the leaf essential oil and isolated terpenoids form Eugenia uniflora L. (Brazilian Pitanga). Phytomedicine 16, 923-928 (2009).

R.A. Cole, W.A. Haber, W.N. Setter, 8. Chemical Composition of Essential Oils of Seven Species of Eugenia from Monteverde, Costa Rica. Biochemical Systematics and Ecology 35, 877-886 (2009).

R. Vila, F. Tomi, M. Mundina, A.I. Santana, P.N. Solis, J.B. Lopez 9. Arce et al. Unusual composition of the essential oils from the leaves of Piper aduncum. Flav. Fragr. J. 20, 67-69 (2005).

R.P. Adams, 10. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed. Allured Publ. Corp.,

C. microphylla


Rec: Jan 2011

Acc: June 2011

Essential Oil from the Leaves of Campomanesia guaviroba (DC.) Kiaersk. (Myrtaceae): Chemical Composition, Antioxidant and Cytotoxic Activity

Aislan C. R. F. Pascoal, Caroline C. Lourenço, Ladaslav Sodek and Jorge Y. Tamashiro Departament of Plant Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP, 13083-970,

Brazil

Gilberto C. Franchi, Jr. and Alexandre E. NowillOnco-Hematological Child Research Center, Faculty of Medical Sciences, State University of Campinas (UNICAMP),

13083-970, Campinas, SP, Brazil

Maria Élida A. StefanelloDepartment of Chemistry, University of State of Parana, 81530-900, Curitiba, PR, Brazil

Marcos José Salvador*Pharmacy Course, Department of Plant Biology, Institute of Biology, State University of Campinas (UNICAMP),

Campinas, SP, cp6109, 13083-970, Brazil

Abstract

The essential oil from the leaves of Campomanesia guaviroba (DC.) Kiaersk., obtained by hydrodistillation, was analyzed by GC and GC/MS. Sixteen compounds could be identified, representing around 94% of the total oil. The major components were myrtenal (27.0%), myrtenol (24.7%) and trans-pinocarveol (15.7%). The essential oil was also evaluated for DPPH radical-scavenging activity by TLC autographic assay, antioxidant capacity by ORAC-FL assay and antiproliferative activity against leukemic cells lines.

Key Word Index

Campomanesia guaviroba, Myrtaceae, essential oil composition, myrtenal, myrtenol, trans-pinocarveol, antioxi-dant, cytotoxic.



Introduction

The genus Campomanesia (Myrtaceae) comprises around 30 species of shrubs or small trees, aromatic, distributed mainly in tropical and subtropical South America (1). Most of the species produce edible fruits that are widely used to make liqueurs, juices and sweets (2). Several species are considered medicinal and have been used in folk medicine against diges-tive problems, fever, cough, flu, diabetes, bladder, heart and liver diseases (3). Campomanesia guaviroba is a tree up to 11 m high, belonging to the family Myrtaceae, distributed in Minas Gerais to Santa Catarina from the coastal region to the eastern highlands (4).

The essential oils are used as alternative remedies for the treatment of many infectious diseases or the preservation of food from the toxic effects of oxidants (5). The antioxidants block the free radicals formation in different ways and establish important control functions in some oxidative stress (6) and in

food conservation (7). Then, new natural antioxidants, mainly those isolated from medicinal plants, acquire great pharmacologi-cal importance and the search for these compounds has been developed much in the last several years (8, 9). Besides these facts, essential oils have shown antiproliferative activity and induction of apoptosis in various tumor cell lines (10-12).

Previous studies have shown that Campomanesia species produce oils rich in terpenes. The oils of C. aurea (12), C. guazumifolia (12), C. rhombea (12), C. xanthocarpa (12-15), C. phaea (16) and C. sessiliflora (14, 17) are characterized by predominance of sesquiterpenes, along with variable amounts of monoterpenes. In C. pubescens the oils from fruits and flowers also are rich in sesquiterpenes (18, 19), while in the leaf oil monoterpenes are predominant (20). C. adamantium, the most studied species, exhibited great chemical variability, with some samples producing mainly sesquiterpenes and others accumulating monoterpenes (13, 21-24). The present

Pascoal et al.


work reports, for the first time, the chemical composition and biological activity of the essential oil of C. guaviroba.

Experimental

Plant material: Leaves from C. guaviroba were collected in June 2010 in the state of São Paulo, Brazil, in the city of Campinas. Specimens were identified by one of the authors (J.Y.T.) and vouchers were deposited in the Herbarium of the University of Campinas (Unicamp). Fresh leaves were submit-ted to hydrodistillation in a Clevenger-type apparatus for 4 h. At the end of each distillation the oils were collected, dried with anhydrous Na2SO4, measured, and transferred to glass flasks that were filled to the top and kept at a temperature of −18°C for further analysis.

Analysis of the essential oil: The GC/EIMS (70 eV) analysis was performed on a Shimadzu GC/MS spectrometer equipped A Durabond-DB5 capillary column (30 m x 0.32 mm, 0.25 μm film thickness; J&W Scientific) was operated at 60ºC for 3 min, and then programmed from 60-220ºC at 5ºC/min, after which it was kept isothermal at 220ºC for 5 min. The carrier gas was He (99.99 g%; 1 mL/min) and the injector temperature was 250ºC. The analyses were performed in split mode, with a split ratio of 1:20. The essential oil components were identified by comparison of their retention indices (relative to n-alkanes) and mass spectra with those found in the literature (25, 26) and stored on the spectrometer database (NIST 1998). The results are average of three analyses.

Evaluation of antioxidant properties ORACFL kinetic as-say: The antioxidant capacities of the essential oil of C. guavirova was assessed through the oxygen radical absorbance capacity (ORAC) assay. The ORAC assay is based upon the inhibition of the peroxylradical-induced oxidation initiated by decomposition of a biological relevant peroxyl radical (2,2’-azobis(2-amidino-propane) dihydrochloride (AAPH), Aldrich, Milwaukee, WI), using fluorescein (Aldrich, Milwaukee, WI) as the fluorescent probe (27). The ORAC assays were carried out on a Synergy 2 (Biotek, Winooski, VT) multidetection microplate reader system. The temperature of the incubator was set at 37°C. The procedure was carried out according to the method established by Ou et al. (28) with modifications (29). The data are expressed as μmol of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Aldrich, Milwaukee, WI) equivalents (TE) per gram of oil on a dry basis (μmol of TE/g). In these tests, quercetin, isoquercitrin, and caffeic acid were used as positive controls. The analyses were performed in triplicate.

TLC autographic assay for DPPH Radical-Scavenging: Ten microlitres of a 1:250 dilution of the essential oil in metha-nol were applied to TLC plates (silica gel 60 GF254, Fluka, AG, Switzerland). The TLC plates were sprayed with a 0.2% 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich, St Louis, MO) solution in MeOH and left at room temperature. Plates were observed 30 min after spraying. Active compounds are observed as yellow spots against a purple background. Relative radical-scavenging activity was assigned as “strong” (samples that produced an intense bright yellow zone), “medium” (samples that produced a clear yellow spot), “weak” (samples that produce only a weakly visible yellow spot), or “not active” (samples that produced no sign of any yellow spot) (30).

Antiproliferative assay: This test aims to evaluate the cell viability of 12 different types of leukemias and they are: K562, HL60, NB4 RAMOS, RAJI Burkit, Jurkat, CEM, MOLT4,

Table I. Chemical composition (%) of leaf essential oil of Campomanesia guaviroba

Number Compounda RIb RIc %

1 trans-pinocarveol 1133 1139 15.72 pinocarvone 1156 1164 3.73 myrtenal 1189 1195 27.04 myrtenol 1192 1195 24.7

5 d-elemene 1266 1335d 0.5

6 a-copaene 1285 1374d 0.2

7 b-bourbonene 1389 1387 0.6

8 b-elemene 1393 1389 0.59 iso-caryophyllene 1412 1408 0.3

10 a-muurolene 1494 1500 0.5

11 d-cadinene 1508 1513 0.412 selina-3,7(11)-diene 1547 1545 0.813 spathulenol 1572 1576 5.414 caryophyllene oxide 1576 1582 5.015 allo-aromadendrene oxide 1633 1639 7.5

16 a-cadinol 1651 1654 1.2 Total identified 94.0 Monoterpenes 71.1 Sesquiterpene hydrocarbons 3.7 Oxygenated sesquiterpenes 19.2

Compounds are listed in order of their elution from a DB-5 column; the coefficients of variation obtained in these analyses were below 5%.a Identification based on mass spectra and RI published (25) and computer matching of the mass spectra with NIST 1998 library (quality level more than 90%); b retention index published (5); c retention index experimental on a DB-5 column; d retention index experimental calculated was not similar to that described in literature, but the fragmentation confirmed these compounds.

Table II. Concentration of the essential oil of Campomanesia guaviroba leaves able to inhibit 50% of the proliferative activity

of leukemic cell lines

Cell line IC50 µg/mL oila IC50 µg/mL Vincristinea

K562 63.14 0.006HL60 19.31 0.008NB 4 22.65 0.003RAMOS 21.21 1.887RAJI 57.94 0.042Jurkat 19.64 0.008CEM 19.63 0.004MOLT 4 80.00 0.014NALM 16 20.48 >100NALM 16 33.28 >100B15 26.80 0.009

RS4 38.09 >100

The coefficients of variation obtained in these analyses were below 5%.a *Leukemic cell lines: K562, HL60, NB4 human Myeloid Leukemia cell line, RAMOS, RAJI human lymphoma cell line Burkit, Jurkat, CEM, MOLT4 Lymphoid human leukemia cell line T, NALM6, NALM16, B15, RS4 B human leukemia cell line lymphoid.

C. guaviroba


Figure 1. GC chromatogram of the essential oil from the leaves of Campomanesia guaviroba showing the major components: trans-pinocarveol (1), myrtenal (3), and myrtenol (4).

NALM6, NALM16, B15, RS4 B. Cellular viability was deter-mined by the MTT reduction assay using a tetrazolium salt (3-[4,5 - dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, MTT (31). Briefly, the cells were distributed in 96-well plates (100 μL cells/well) and exposed to various concentrations of essential oil (100, 10, 0.1, 0.01, 0.001, 0.0001 and 0.00001 μg/mL) in DMSO (0.1%) at 37ºC, with 5% of CO2, for 48 h. The final concentration of DMSO did not affect the cell vi-ability. The selected method for assessing cell viability is the MTT (Sigma M2128) and the optical density was measured by spectrophotometry at 570 nm (Bio-Tek Power Wave XS). The experiments were carried out, at least, in triplicate and the median inhibitory concentration (IC50) was calculated in mg/mL as the concentration of the sample that decreased 50% of the viable cells compared with that of the control us-ing OD values of viable cells. Vincristine Sulfate was used as positive control.


The hydrodistillation of the leaves of C. guaviroba provided oil slightly yellowish, with pleasant and persistent aroma and yielding 0.016% (m/m). GC analyses showed a few prominent peaks (Figure 1). Sixteen compounds could be identified, representing around 94% of the total oil (Table I). The oil was dominated by monoterpenes (71.1%), followed by sesquiter-penes. The major components were myrtenal (27.0%), myrtenol (24.7%) and trans-pinocarveol (15.7%). The sesquiterpenes alloaromadendrene oxide, spathulenol and caryophyllene oxide were identified as minor constituents. A similar profile was previously reported for Campomanesia pubescens (20) and C. adamantium (23). The oils of these species also were characterized by predominance of monoterpenes along with a large number of minor constituents, including myrtenol. Monoterpenes with pinane skeleton, such as a-pinene and b-

pinene were present in significant amounts. The sesquiterpene spathulenol was identified in all analyzed Campomanesia oils, while caryophyllene oxide was a major component in the oils of C. guazumifolia (12) and C. phaea (16).

The antioxidant activity was evaluated by two methods. In the ORAC-FL assay, the essential oil result was 511 μmol TE/g, while TLC produced a yellow spot where the essential oil was applied, due to DPPH reduction. The essential oil was able to capture the radicals but the antioxidant activity by DPPH assay was lower in essential oil. Similar results were obtained with essential oil from leaves of C. adamantium that showed low activity (9.91% reduction of DPPH with 2270 μg/mL) compared to the flavonoid quercetin (90% reduction of DPPH with 20 μg/mL) (24). ORAC-FL and DPPH assays show differences of sensibility and on the basis of the chemical reactions involved in each test: ORAC is a hydrogen atom transfer reaction based assay (HAT) and DPPH is a single electron transfer reaction based assay (ET). It is apparent that the hydrogen atom transfer reaction is a key step in the radical chain reaction. Therefore, the HAT based method is more relevant to the radical chain-breaking antioxidant capacity (28, 29). The essential oil of C. guaviroba is rich in monoterpenes and with or without minor compounds with donor groups of the electron in ortho position in relation to phenolic hydroxyl (32, 33).

Cytotoxic activity of essential oil of C. guaviroba was investigated on a series of leukemia human cells by the MTT assay. The results are shown in Table II. The cytotoxic effect on JURKART, HL60, NB4, CEM, RAMOS B15 and cell lines was significantly stronger than that on the other six cell lines (K562, RS4, RAJI, MOLT4, NALM6 and NALM16). Of all leukemia lines, HL 60 was the most vulnerable to the essential oil with an IC50 of 19.31 μg/mL. Cytotoxic activities have been reported for several essential oils and their components (34). In particular, myrtenal and caryophyllene oxide showed activ-

Pascoal et al.


ity against human leukaemia cells (HL-60), with IC50 values of 114.85 μg/mL and 37.88 μg/mL, respectively (35). Therefore, the presence of these compounds in the essential oil of C. guaviroba may be responsible, at least in part, for observed antileukaemic activity.

Acknowledgments

The authors thank FAPESP, CNPq and FAEPEX-UNICAMP for fi-nancial support. MJS is grateful to CNPq for research scholarships.

References

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C. guaviroba


Rec: Feb 2011

Acc: May 2011

Anti-inflammatory Activity of Some Essential Oils

Salud Pérez G., Miguel Zavala S., Lucina García A. and Miguel Ramos L.*Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana-Xochimilco, Calzada del Hueso 1100, Col.

Villa Quietud, Del. Coyoacán, C.P. 04960 México D.F.

Abstract

There are many diseases that are associated with inflammation, such as infections by bacteria, virus and protozoa, autoimmune diseases such as arthritis and diabetes, Alzheimer’s disease, and cancer. There are many medications available to prevent or minimize the progression of the inflammation; they include non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids, but they have some secondary effects. Traditional medicine has been used to address the health demands of the population and nowadays it presents many opportunities in health care. Essential oils are use in this medicine to treat many diseases.

In a review of the last five years it was found that several essential oils with anti-inflammatory activity were isolated from 43 plants. In some cases, oils of the same genus but different species have this activity, such as the essential oils obtained from three species of genus Origanum, as well as three oils from three species of the Citrus genus, and three from the Pimpinella genus. In many cases the essential oil composition obtained has been determined, and in some cases the anti-inflammatory activity of the main compounds of these essential oils has been evaluated, such as carvacrol and isoeugenol, which showed an important anti-inflammatory activity. On the basis of this review, we can say that some essential oils could be an important source for the treatment of inflammatory diseases.

Key Word Index

Anti-inflammatory activity, essential oils, inflammatory diseases, review.



Introduction

Inflammation is a physiological response to a variety of agents including infectious microorganisms, toxic chemical compounds and physical injury. There are many diseases that are associated with the inflammation process, such as skin inflammation (1, 2), autoimmune diseases such as arthritis and diabetes, Alzheimer’s disease and cancer.

Many medications are available to prevent or minimize the progression of inflammation, includuing non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. NSAIDs such as acetyl salicylic acid, ibuprofen, diclofenac and their new related compounds are mainly selective COX-2 inhibitors; cyclooxygenase-2 is involved in the inflammation pathway. The regular use of NSAIDs can cause a number of side effects, some of which may be very serious. The most common are increases in the development of ulcers in the stomach and duodenum, as well as inhibition of uterine motility and hypersensitivity reaction (3), nausea, vomiting, indigestion, diarrhea, heartburn, headache, dizziness, rapid weight gain and breathing problems (4). The lengthy use of corticosteroids could produce the sup-pression of the function of pituitary-adrenal, hyperglycemia and increase susceptibility to infections (5).

The biological activities of many plants have been long known in ethnomedicine to treat inflammatory diseases. These

biological properties are often due to essential oils contained in plants which are used as herbal remedy in traditional medicine. It has been found that these essential oils possess different activities such anti-inflammatory and antiradical properties (6-8). For this reason, we did a review of the last five years and in this period we found that several essential oils with anti-inflammatory activity were isolated from 43 plants.

Essential oils with anti-inflammatory properties

Afromomum danielli (Hook f.) Schum and A. meleg-ueta Schum (Zingiberaceae): The analysis of the chemical composition of A. melengueta seed essential oil indicated that it is rich in sesquiterpenes. The other samples were rich in monoterpenes like limonene, 1,8-cineole, a- and b-pinenes, linalool and (E)-b-ocimene as the major components. The anti-inflammatory activity of A. daniellii seed essential oil was measured and gave an inhibition concentration 50 (IC50) of 237 ppm against 0.7 ppm for nordihydroguiaretic acid (NDGA). The results achieved highlight the potential of essential oils to be developed against inflammatory disorders (9).

Ageratum fastigiatum R. M. King et H. Rob. (Asteraceae): This plant is used in folk medicine as an anti-inflammatory, analgesic and antimicrobial. The main compounds found

Pérez et al.


in the essential oil were germacrene D, a-humulene and b-cedrene. The oil, with LD50 of 2.50 g/kg, inhibited the acetic acid-induced writhing at the dose of 200 mg/kg in the formalin test. In the hot plate test, after 30 min and 60 min of treatment, doses of 100 and 200 mg/kg increased the reaction time. The anti-edematogenic effect, reduction on the exudate volume and leukocyte mobilization were observed at doses of 100 and 200 mg/kg. A. fastigiatum possessed analgesic and anti-inflammatory properties (10).

Aucoumea klaineana Pierre (Burseraceae): a-Pinene, a-phelandrene, p-cymene and 1,8-cineole were the major components of the essential oil. The anti-inflammatory activ-ity was carried out by lipoxygenase method and the oil was not active (11).

Canarium scheinfurthii Engl. (Burseaceae): This plant grows in Cameroon; the main components of the essential oil obtained by hydrodistillation were p-cymene, limonene and a-terpineol. The oil had anti-inflammatory activity in lipoxy-genase method with an IC50 of 62.6 ppm (11).

Calycorectes sellowianus O. Berg (Myrtaceae): It is endemic to Brazil. The major constituents of 37 compounds of the leaves’ essential oil (GC/MS) were guaiol (13.1%) and b-caryophyllene (8.6%). The anti-inflammatory activity of this oil was investigated in vitro and in vivo. It reduced the treated neutrophils chemotaxis with 91% inhibition and had no effect on the carrageenan-induced paw edema (12).

Cinnamomum insularimontanum Hayata (Lauraceae): It has a strong fragrance and has been used as a folk medicine in Taiwan for a long time. The fruit essential oil was analyzed by GC/MS; the main constituents were a-pinene (9.45%), camphene (1.70%), b-pinene (4.30%), limonene (1.76%), cit-ronellal (24.64%), citronellol (16.78%) and citral (35.89%). The results obtained from nitric oxide (NO) inhibitory activity assay, essential oil and its dominant compound (citral) presented the significant NO production inhibitory activity, IC50 of essential oil and citral were 18.68 and 13.18 μg/mL, respectively. More-over, based on the results obtained from the protein expression assay, the expression of IKK, iNOS, and nuclear NF-kB was decreased and I Ba was increased in dose dependent man-ners. It proved that the anti-inflammatory mechanism of citral was blocked via the NFjB pathway, but it could not efficiently suppress the activity on COX-2. In addition, citral exhibited a potent anti-inflammatory activity on croton oil-induced mice ear edema, at doses of 0.1 and 0.3 mg per ear. The inhibition was 22% and 83%, respectively. The results presented that the fruit essential oil of C. insularimontanum and citral have anti-inflammatory effect. (13).

Cinnamomum osmophloeum Kaneh (Lauraceae): It is an endemic tree that grows in natural hardwood forest of Taiwan. The leaf essential oil components showed inhibitory effects as anti-bacterial, anti-termite, anti-mites, anti-mildew, anti-mosquito larvae, and anti-fungal. The chemical constituents of the essential oil were analyzed by GC/MS and they were found to be L-bornyl acetate (15.89%), caryophyllene oxide (12.98%), g-eudesmol (8.03%), b-caryophyllene (6.60%), T-cadinol (5.49%), ð-cadinene (4.79%), trans-b-elemenone (4.25%), cadalene (4.19%), and trans-cinnamaldehyde (4.07%). The effects of essential oil on oxide NO and prostaglandin E2 production in lipopolysaccharide (LPS)-activated RAW 264.7

macrophages were also examined. Results of nitric oxide tests indicated that the essential oil and its major constituents such as trans-cinnamaldehyde, caryophyllene oxide, L-borneol, L-bornyl acetate, eugenol, b-caryophyllene, E-nerolidol, and cinnamyl acetate have anti-inflammatory activity (14).

Citrus aurantium L. var bergamia (Rutaceae): The es-sential oil is extracted from the peel of the fruit, whose main components are limonene (40%), linalool (8%) and linalyl acetate (28%) (15). The anti-inflammatory activity of essential oil of Bergamot (BO) was tested on carrageenan-induced rat paw edema at different doses: 0.025, 0.05 and 0.1 mL/kg; the reduction in paw edema was 27.56%, 30.77% and 63.93% respectively, and indomethacin used as a reference produced an inhibition of 95.7%. These results showed that BO possesses anti-inflammatory effect (16).

Citrus sinensis L. (Rutaceae): Orange essential oil can be attributed to its properties like anti-inflammatory, antide-pressant, anti-spasmodic, antiseptic, aphrodisiac, carminative, diuretic, tonic, sedative and cholagogue. The anti-inflammatory activity of the oil was tested using the lipoxygenase enzymatic method; the IC50 was 20.3 mg/L (17).

Citrus sunki (Hayata) Tanaka (Rutaceae): C. sunki is used in traditional medicine for digestion, cold, and fever. The analysis of the essential oil of this plant by GC/MS showed that the major components were dl-limonene (68.18%) and b-myrcene (4.36%). The oil reduced the LPS-induced secretion of NO in RAW 264.7 cells. This result suggests that the essential oil has anti-inflammatory activity (18).

Cleistocalyx operculatus Roxb. (Myrtaceae): In folk medicine in China, Vietnam and some other tropical countries, it is widely used for the treatment of gastric ailments and as an antiseptic agent. The anti-inflammatory activity of the es-sential oil of C. operculatus buds inhibited lipopolysaccharide induced secretion of pro-inflammatory cytokines, including tumor necrosis factor-a (TNF-a) and interleukin-1 b (IL-1b) in RAW 264.7 cells, a mouse macrophage-like cell line. Also the mRNA expression of TNF-a and IL-1b was suppressed. Moreover, reporter gene analysis revealed that the oil blocked LPS-induced transcriptional activation of NF-kB in RAW 264.7 cells. Besides, the essential oil inhibited the ear edema induced by TPA (19).

Cordia verbenacea DC (Boraginaceae): This plant is a medicinal plant popularly used in Brazil as anti-inflammatory, antiulcer and anti-rheumatic agent without detailed pharmaco-logical and toxicological studies (20). a-Humulene and trans-caryophyllene were identified in C. verbenacea essential oil and the anti-inflammatory activity of the both compounds was evaluated in a model of acute inflammation in rat paw, induced by LPS. The treatment with a-humulene or trans-caryophyllene inhibited the LPS-induced NF-kB activation and neutrophil migration; however, only a-humulene prevented the production of cytokines TNF-a and IL-1b and the in vivo up-regulation of kinin B1 receptors, so that both sesquiterpenes might be used as agents to treat inflammatory diseases (21).

Cyperus esculentus L. and C. rotundus Linn. (Cyper-aceae): Anti-inflammatory, anti-arthritic, analgesic and an-ticonvulsant activities of the oils of both plants were study. Phytochemical tests of the oil are positive for flavonoids, trit-erpenoids, carbohydrates and proteins. The effects of the oils



were evaluated in the models of carrageenan-induced edema, formaldehyde induced arthritis, formalin induced writhing and MES induced convulsion. It was found that both essential oils posses good anti-inflammatory, anti-arthitic, analgesic and anticonvulsant activities (22).

Chenopodium album L. (Chenopodiaceae): This plant is commonly known as pigweed and in folk medicine is used as laxative, antihelmitic, against round and hook worms, and as a blood purifier. Also it is used for the treatment of hepatic disorders, spleen enlargement, intestinal ulcers and burns (23). The analysis of the essential oil of C. album leaves revealed that the main constituents were p-cymene (40.9%), ascaridole (15.5%), pinane-2-ol (9.9%), a-pinene (7.0%), b-pinene (6.2%) and a-terpineol. The oil had strong anti-inflammatory activity against TPA-induced ear edema in mice (24).

Dennettia tripetala G. Baker (Annonaceae): The fruit, bark, leaves and roots are used as spices and condiments. The leaves are used in combination with other medicinal plants to treat fever, typhoid, cough, worm infestation, vomiting and stomach upset (25). Atinociceptive and anti-inflammatory activity of the essential oil were tested in mice using the hot plate, acetic acid-induced writhings and formalin tests, while carrageenan-induced paw edema as anti-inflammatory model. The anti-inflammatory activity of the oil was comparable to dexamethasone (1 mg/kg) (26).

Drimys brasiliensis Miers (Winteraceae): This species has been used in folk medicine as analgesic and anti-inflammatory. The essential oils from leaves and stem barks were character-ized by GC-FID and GC/MS. The main components were monoterpenes (leaves 4.31% and stem barks 90.02%) and sesquiterpenes (leaves 52.31% and stem barks 6.35%). The evaluation of antinociceptive and anti-inflammatory potential of the essential oils and the sesquiterpene polygodial were evaluated in paw edema induced by carrageenan and formalin test in mice. The essential oil obtained from the stem barks significantly reduced the edema induced by carrageenan. The anti-inflammatory effect of stem barks oil (at 200 mg kg-1) was similar to that observed with indomethacin (at 10 mg kg-1) 30 and 60 min after the administration of essential oils. The effect of polygodial (at 200 mg kg-1) was lower than the oils (27).

Fortunella japonica (Thunb.) Swingle (Rutaceae): F. japonica is also known as round kumquat or Marumi kumquat. The fruit is rich in vitamins A and C. The main components of the essential oil are dl-limonene (61.58%) and carvone (6.36%). The oil significantly reduced LPS-induced NO re-lease in RAW 264.7 cells. This fact indicates that this oil has anti-inflammatory effect (18).

Hedychium coronarium Koen. (Zingiberaceae): It is commonly known as butterfly ginger, cinnamon jasmine, gargland flower and ginger lily. The rhizome has been used for the treatment of headache, diabetes, contusion inflammation and sharp pain due to rheumatism. Twenty-nine components were identified in the flowers essential oil and the main com-ponents were b-trans-ocimenone (28.05%), linalool (18.52%), 1,8-cineole (11.35%), a-terpineol (7.11%), 10-epi-g-eudesmol (6.06%), sabinene (4.59%) and terpinen-4-ol (3.17%). At doses of 100 mg/kg p.o. the oil produced significant inhibition of carrageenan-induced hind paw edema in rats (28).

Illicium anisatum Hayata (Illiciaceae): It is widely used

for treatment of some skin problems in traditional Chinese medicine. The fruit is an important source of essential and volatile oil. Fifty-two components were identified in the es-sential oil and the main components were eucalyptol (21.8%), sabinene (5.3%), a-terpinenyl acetate (4.9%), kaurene (4.5%), isopimaradiene (3.2%), safrol (2.7%), b-linalool (2.6%), g-cadinene (2.2%), a-cadinol (2.2%) and terpinen-4-ol (1.9%). The mechanism of the anti-inflammatory activities of I. anisatum essential oil (IAE) was evaluated whether it could modulate the production of nitric oxide (NO) and prostaglandin E2 (PGE2) by activated macrophages. The results indicate that IAE is an effective inhibitor of LPS-induced NO and PGE2 production in RAW 264.7 cells. These inhibitory activities were accompa-nied by dose-dependent decreases in the expression of iNOS and COX-2 proteins and iNOS and COX-2 mRNA. Also was evaluated the cytotoxic effects of the oil, It was found that IAE exhibited low cytotoxicity at 100 mg mL–1 (29).

Lippia sidoides Cham. (Verbenaceae): It is mainly used as an antiseptic (30). It was found that the topical application of leaf essential oil at doses of 1 and 10 mg/ear, respectively, reduced 45.93% and 32.26% the acute ear edema induced by 12-O-tetradecanoylforbol 13-acetate (TPA).

Melaleuca alternifolia Maiden et Betche (Myrtaceae): It has well established traditional and folk uses in Australia, specially as an antiseptic. The major constituent from the essential oil was terpinen-4-ol. This compound is considered, together with a-terpinene, g-terpinene, and a-terpineol, the main responsible for the anti-inflammatory activity from this essential oil. The oil showed anti-inflammatory activity on edema-induced by histamine in mice. Several clinical studies and observations, endorse the clinical external use of the oil for the treatment of vulvovaginitis, mainly candidiasic cases (31).

Mezoneuron benthamianum Baill. (Caesalpinoideae): This plant is used for the treatment of dermal infection, healing of refractory sores, blood disorders, as a laxative, for stomach troubles, eye treatments, genital stimulants/depres-sants, hemorrhoids, pain-killers, pulmonary troubles and as a chewing stick. The oil contained 15 compounds and the main components were 3-carene, pinene (11.8%), trans-nerolidol (13.5%), farnesene (11.6%) and thujene (6.7%). The essential oil was tested at different concentrations for its anti-inflammatory activity evaluated as inhibition of TPA induced ear edema in mice. The oil at 5.0 mg and 2.5 mg dose levels exhibited a significant anti-inflammatory activity with percentage edema reduction of 92.3% and 76.9%, respectively (32).

Myrciaria tenella (DC.) Berg. (Myrtaceae): It is known as Cambuí. The GC/MS analysis revealed that the main constitu-ents of the leaf essential oil were b-caryophyllene (25.1%) and spathulenol (9.7%). The oil reduced significantly the treated neutrophil chemotaxis with 93% inhibition, and in the systemic treatment at doses of 50 mg/kg p.o. reduced the carrageenan-induced paw edema with a similar effect for indomethacin (10 mg/kg), the positive control (33).

Nepeta cataria L. var. citriodora (Becker) (Lamiaceae): It is used as anti-tussive, expectorant and antiathmatic (34). The essential oil was analyzed by gas chromatography-flame ionization detector (GC-FID), four major components were identified trans,trans-nepetalactone, cis,trans-nepetalactone, trans,cis-nepetalactone and nepetalactol. At doses of 0.0005

Pérez et al.


mL/kg the oil presented peripheric anti-inflammatory proper-ties by reducing the induced edema after carrageenan injec-tion (35).

Ocotea quixos Lam. (Lauraceae): The main components of the essential oil were trans-cinnamaldehyde (27.9%) and methyl cinnamate (21.6%) (36). The anti-inflammatory activity of the essential oil and these two compounds were investigated in in vitro and in vivo models. The oil and trans-cinnamaldehyde, but not methyl cinnamate, significantly reduced LPS-induced NO release from J774 macrophages, inhibited LPS-induced COX-2 expression, and increased forskolin-induced cAMP production. At doses of 30-100 mg/kg of essential oil and 10 mg/kg of trans-cinnamaldehyde showed anti-inflammatory activity against paw edema in rats carrageenan-induced without damaging gastric mucosa (37).

Olea europea L. (Oleaceae): In Tunisian folk medicine this plant is used in the treatment of inflammatory diseases and bacterial infections. The analysis of the essential oil resulted in the identification of 32 compounds and the major com-pounds were a-pinene (52.7%), 2,6-dimethyloctane (16.57%) and 2-methoxy-3-isopropylpyrazine (6.01%). Intraperitoneal administration of O. europea essential oil at doses of 100, 200 and 300 mg/kg reduced acetic acid-induced abdominal constrictions and paw edema (38).

Origanum ehrenbergii Boiss and O. syriacum L. (La-miaceae): In the essential oil of O. ehrenbergii was found 37 components of which thymol (19%) and p-cymene (16.1%) were the main abundant compounds. Thirty-six components were found in the O. syriacum essential oil and the main compounds were thymol (24%) and carvacrol (17.6%). O. ehrenbergii oil inhibited NO production in the murine monocytic macrophage cell line RAW 264.7 with an IC50 value of 66.4 μg/mL (39).

Origanum vulgare L. (Labiatae): It is an aromatic plant of the Mediterranean flora that has been commonly used to treat diarrhea and pain. Identified in the essential oil were trans-sabinene hydrate, thymol and carvacrol. THP-1 macrophages were used as cellular model of atherogenesis and the release/secretion of cytokines (TNT-a, IL-1b, IL-6 and IL-10) and their respective mRNA expressions were quantified both in pres-ence or absence of supercritical oregano extracts. The results showed a decrease in pro-inflammatory TNF-a, IL-1b and IL-6 cytokines synthesis, as well as an increase in the production of anti-inflammatory cytokine IL-10. These results may sug-gest an anti-inflammatory effect of oregano extracts and their compounds in a cellular model of atherosclerosis (40).

Pelargonium graveolens L’Hér (Geraniaceae): This plant is commonly known as geranium. For many years in traditional medicine it has been used as an anti-asthmatic, anti-allergic, antioxidant, anti-diarrheic, antihepatotoxic, diuretic, tonic, haemostatic, stomachic and anti-diabetic (41). The main com-ponents of the essential oil were citronellol (26%), citronellyl formate (16%), linalool (10%), geraniol (8%), isomenthone (6%) and menthone (4%). It was found that this essential oil could inhibit the LPS-elicited expression of the induced proinflam-matory enzymes COX-2 and iNOS, as well as the NO produced by LPS-activated microglial cells. This inhibition did not result from a cytotoxic effect of the oil. Although high concentrations of citronellol could inhibit NO production from the cells, when administered at their natural relative concentrations in the oil,

neither citronellol nor the other constituents of the oil were effective at inhibiting NO production (42).

Pimpinella corymbosa Boiss, P. tragium Vill. and P. rhodanta Bois (Apiaceae): Pimpinella species have been used as animal feed to increase milk secretion (43), also the estrogenic activity of some isolated compounds and essential oils of different Pimpinella species were reported. The oils of these three species were effective in inhibiting NF-kB medi-ated transcription. The roots showed notably potent activities with IC50 values of 2, 3 and 6 μg/mL, respectively (44).

Rosmarinus officinalis L. (Labiatae): It is known as a common herb and household plant broadly used all around the world for different medicinal purposes, being a component of various established anti-inflammatory plant drug preparations, and having a long tradition of use for treating headaches, colds and colic, as well as other diseases (45). The effect of R. of-ficinalis essential oil dietary administration at concentrations of 1250, 2500 and 5000 ppm in carrageenan paw edema and trinitrobenzene sulfonic acid (TNBS) colitis was studied (46). Dietary supplementation with 5000 ppm of the oil initially increased after 2 h, but after 24 h suppressed the extent of paw edema, and in the TNBS model exhibited protective ef-fects on colonic mucosa and decreased macroscopic scores for colonic inflammation.

Sabina virginiana L. Antoine (Cupressaceae): It is com-monly known as eastern west cedar and has been used in the treatment of psoriasis, dermatitis, hemorrhoids and varicose veins. The leaves are found to exert effects on emmenagogue, as a stimulant, and as a diaphoretic in rheumatism (47). The leaves’ essential oil was analyzed by GC/MS; 31 compounds were identified, and the major constituents were limonene (32.9%), safrole (23.0%), asarone (15.9%) and a-pinene (5.2%). The essential oil was tested at different concentrations (0.075, 1.25, 2.5 and 5.0 mg/ear) for its anti-inflammatory assay evalu-ated as inhibition of TPA induced ear edema in mice. At doses of 5.0 mg/ear the inhibition was 66.7%. This effect was similar to that obtained with indomethacin (57.7%) (48).

Thymus vulgaris L. (Labiatae): Thyme has been used for respiratory ailments for its infection-fighting and cough suppressive qualities. Thyme tea is an old time favorite cough and cold remedy. The essential oils of thyme are grouped into three main types: thyme oil, which contains 42–60% phenols and is mainly thymol; origanum oil, which contains 63–74% phenols and is mainly carvacrol; and lemon thyme oil, which contains citral. The dietary addition of thyme essential oils to the diet at 3 concentrations (5000, 2500 and 1250 ppm) and fed to Balb/c mice. The extent of ear swelling in DTH/CHS reaction, paw edema induced by carrageenan administration and TNBS-induced colitis were evaluated. Dietary supplementation with 5000 ppm of oil decreased paw edema and ear swelling and the microscopic and macroscopic scores of colitis (49).

Zanthoxylum piperitum AP DC (Rutaceae): The major constituents of the essential oil were limonene and geranyl acetate. The oil decreased approximately 38% of nitrite production, as compared to LPS-induced nitrite production. However, the essential oil and its components did not suppress NO chemically in a cell-free system and inhibited iNOS mRNA transcription. The inhibition of E-selectin gene transcription by the oil caused the suppression of cellular adhesion. These



results suggest that the essential oil of this plant might have anti-immunological anti-inflammatory activity (50).

Zanthoxylum schnifolium Sieb. et Zucc.(Rutaceae): This plant has been used in traditional medicine for treatment of the common cold, stomachache and diarrhea (51, 52). The chemical composition of the essential oil was found by GC/MS and 55 compounds were detected. The main constituents were b-phellandrene (22.54%), citronellal (16.48%), and geranyl acetate (11.39%). The oil and its constituents (b-phellandrene, citronellal and geranyl acetate) significantly suppressed gene transcription of iNOS, the COX-2 gene, and biosynthesis of IL-1b by LPS-stimulated macrophage cells. This result suggests that the essential oil may be useful to relief and retardation of immunological inflammatory responses (53).

Zingiber officinale Roscoe (Zingiberaceae): This plant is commonly known as “ginger.” It is used in folk medicine to treat pain, inflammation, arthritis, urinary infections, and gastrointestinal disorders. The ginger essential oil at doses of 50, 100 and 200 mg/kg, p.o. significantly suppressed the acetic acid-induced writhing response in a dose-dependent manner. Maximum inhibition of the oil was observed at 200 mg/kg. GEO was found to contain monoterpenes and sesquiterpenes as principal compounds, suggesting that the anti-inflammatory and analgesic effects could be correlated to these essential oil constituents (54).

Zingiber zerumbet (L) Sm. (Zingiberaceae): It is locally known as lempoyang or wild ginger. In traditional medicine is used to cure swelling and loss of appetite. The juice of the boiled rhizomes has also been used as a medicine for worm and ascaris in children (55). The rhizomes’ essential oil was evaluated in acute and chronic inflammatory models, using carrageenan-induced paw edema and cotton pellet-induced granuloma, respectively; non-inflammatory-mediated pain was also assessed using a formalin test. The oil exhibited significant anti-inflammatory activity both in acute and chronic inflamma-tion, and also had anti-nociceptive activity (56).

Zizyphus jujube Miller (Rhamanaceae): In traditional medicine it is used in the treatment of diabetes and anti-fertility (57, 58), diarrhea and insomnia. The anti-inflammatory activity of the essential oil obtained from the seeds of Z .jujube was evaluated on ear edema induced with TPA in mice. The treat-ment with 1% and 10% of the essential oil caused significant decreased in ear thicknesses. Furthermore, histological analysis confirmed that this oil inhibited the inflammatory responses of skin inflammation in mice (59).

Discussion

Inflammatory diseases are generally treated with steroidal and non-steroidal anti-inflammatory drugs (60). However, both of them have significant negative side effects, reducing their use in certain segments of the population (61). Hence, there is a need to develop new drugs with novel modes of action and fewer side effects. The use of herbal therapy or alterna-tive medicine constituents is an attractive approach for the treatment of several inflammatory disorders (62). Essential oils are plant secondary metabolites that are used extensively in aromatherapy and various traditional medicinal systems and many of these oils possesses different pharmacological proper-

ties, one of which is the anti-inflammatory effects on several different models of inflammation as shown in this review. A large number of the essential oils contain various bioactive compounds, some of which have potent anti-inflammatory effect including carvacrol, limonene, citronellal, and cinnam-aldehyde, among others.

The results presented in this report suggest the applica-tions of essential oils or their components as anti-inflammatory agents and might accelerate the development of new drugs for different inflammatory diseases.

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Rec: Feb 2011

Acc: June 2011

Chemical Composition and Antibacterial Activity of Origanum majorana L. Essential Oil from the

Venezuelan Andes

Sulymar Ramos1, Luis B. Rojas1, Maria Eugenia Lucena2, Gina Meccia1* and Alfredo Usubillaga1

1Research Institute, Faculty of Pharmacy and Bioanalysis, University of Los Andes, Mérida, Venezuela 2Bioanalysis Clinic Department, Faculty of Pharmacy and Bioanalysis, University of Los Andes, Mérida, Venezuela

Abstract

Origanum majorana L. (Lamiaceae) is a plant that is used in gastronomy and natural medicine. The plant mate-rial used in this study was collected at San Isidro de Apartaderos, Mérida State. A yield of 0.6% of essential oil was obtained by hydro-distillation using a Clevenger trap. The main constituents found were: cis-sabinene hydrate (30.2%), terpinen-4-ol (28.8%), g-terpinene (7.2%), a-terpineol (6.9%), trans-sabinene hydrate (4.4%), linalyl acetate (3.8%), and a-terpinene (3.6%). The essential oil was also fractionated over a silica gel dry column. Two main fractions were isolated, the first containing 67.6% of cis-sabinene hydrate and the second 72.8% of terpinen-4-ol. The antibacterial activity of the oil was determined using the agar diffusion method and it was found that it was active against Staphylo-coccus aureus, Enterococcus faecalis, Escherichia coli, and Klebsiella pneumoniae. Antibacterial activity of the fractions obtained by dry column chromatography was also tested. It was found that the fraction rich in cis-sabinene hydrate was more active than the one rich in terpinen-4-ol. It was concluded that in the essential oil of Origanum majorana, cis-sabinene hydrate is the more important compound responsible for inhibition of bacterial growth.

Key Word Index

Origanum majorana L., Lamiaceae, essential oil composition, antibacterial activity, cis-sabinene hydrate, terpinen-4-ol.



Introduction

The Lamiaceae family has about 3,000 species, most of them aromatic plants. They are distributed around the world, but they are especially abundant in the Mediterranean region and Eastern Asiatic countries. In Venezuela there are around 90 species that belong to 19 genuses (1). The genus Origanum has about 20 species which are aromatic herbs. Origanum majorana L., popularly known as sweet marjoram, is an aro-matic herbaceous plant with small leaves and white or pink flowers native to the Middle East. It is used to treat digestive disorders, as well as appetizer, carminative, aphrodisiac, dia-phoretic hypotensor, expectorant, and sudorific (1-2). Its active constituents are used in the manufacture of anti-rheumatic ointments. In the food industry it is used to season meats and sausages (1). Its essential oil and alcoholic extracts are used in pharmaceuticals, perfumes and cosmetics (3).

The essential oil obtained by steam distillation contains

mainly terpinen-4-ol, which along with cis-sabinene hydrate is responsible for the characteristic flavor and fragrance of mar-joram oil. In addition to these compounds, a- and g-terpinene and terpinolene are the other major components (4-14); only the oil from Turkey (15) contains carvacrol as a main constitu-ent. Antioxidant activity of marjoram essential oil (4, 14) has been reported. Its volatile oil possesses antimicrobial properties against foodborne bacteria and mycotoxigenic fungi (4, 6, 8, 16). Previous studies from Brazil, Hungary and Tunisia (17-19) reported antibacterial activity against Gram-positive and Gram-negative microorganisms. In Venezuela, activity against Escherichia coli, Staphylococcus aureus y Pseudomonas sp. (20) has been reported.

In the present report, the essential oil of O. majorana, collected at San Isidro de Apartaderos, was analyzed and its antibacterial activity was determined. The oil was fractionated in an attempt to identify the components that were responsible for such antibacterial activity.

O. majorana


Experimental

Plant collection and oil extraction: Origanum majorana was collected at San Isidro de Apartaderos, a town located 3,340 meters above sea level at Mérida State, Venezuela. It was identified by Ing. Juan Carmona, and voucher specimen No. LR062 was deposited at the Merf Herbarium. Fresh leaves (500 g) were hydrodistilled for 3 h to obtain 3.1 mL of oil which was stored at 4ºC in the dark.

Essential oil fractionation: The oil (2 g) was treated over a silica dry column. A plastic 1.0 inch wide column was filled with silica gel (Merck, 230-400 mesh). Hexane was added until the solvent reached the lower part of the column which was then cut lengthwise in five equal parts. The pieces were labeled from the top to the bottom (A-E). The silica of each portion was extracted twice with 10 mL of diethyl ether and filtered. Each portion was then treated for 12 h with 20 mL of ethyl acetate. All fractions were then concentrated to a volume of 1.0 mL and stored for later analysis.

Analysis of the essential oil: GC-FID analysis was per-formed on a Perkin-Elmer Auto System gas chromatograph equipped with a 5% phenylmethylpolysiloxane fused-silica capillary column (AT-5, Alltech Associates Inc., Deerfield, IL, 60 m x 0.25 mm, film thickness 0.25 mm). The initial oven temperature was 60°C, it was then heated to 260°C at 4°C/min, and the final temperature kept for 20 min. The column injector and detector temperatures were 200°C and 250°C, respectively, and the carrier gas was He at 1.0 mL/min. A 1.0 mL sample was injected using a split ratio of 1:10. Retention indices were calculated relative to C8-C24 n-alkanes, and com-

Table I. Percentage composition of the essential oil of Origanum majorana L.

Peak Constituents % Oil Fr. A Fr. C RI RIlit

1 Butyl acetate t 1.2 805 812

2 a-Thujene 0.2 928 9313 Sabinene 1.4 974 9764 Myrcene 0.5 989 991

5 a-Terpinene 3.6 1017 10186 p-Cymene 2.4 1025 1026

7 b-Phellandrene 1.5 1030 1031

8 g-Terpinene 7.2 1060 10629 trans-Sabinene hydrate 4.4 5.1 3.2 1069 1065

10 a-Terpinolene 2.0 1089 108811 cis-Sabinene hydrate 30.2 67.6 9.9 1100 109812 cis-para-Menth-2-en-1-ol 2.0 2.1 1.7 1125 112113 trans-para-Menth-2-en-1-ol 1.1 2.0 1144 114014 endo-Borneol 0.4 1172 116515 Terpinen-4-ol 28.8 9.5 72.8 1185 1177

16 a-Terpineol 6.9 11.4 2.6 1196 118917 cis-Piperitol 0.5 1200 119318 trans-Piperitol 0.5 1213 120519 Geraniol t 1.1 1260 125520 Linalool acetate 3.9 8.2 1262 125721 Eugenol 0.3 1362 135622 Neryl acetate 0.3 1369 136523 Geranyl acetate 0.6 1.6 1387 138324 trans-Caryophyllene 0.6 1427 141825 Bicyclogermacrene 1.0 1508 1494

RI: Retention Indices were determined by GC on a HP-5 column.t: traces (< de 0,1 %)

pared with values reported in the literature (21). The GC/MS analysis was done on a Hewlett Packard GC-

MS system, Model 5973, fitted with a 30 m long, cross-linked 5% phenylmethylpolisiloxane (HP-5MS, Hewlett Packard, USA) fused-silica column (0.25 mm, film thickness 0.25 mm). The oven temperature conditions were the same used for GC-FID analysis. Source temperature 230°C; quadrupole temperature, 150°C; carrier gas He adjusted to a linear velocity of 34 cm/s; ionization energy, 70 eV; scan range, 40-500 amu; 3.9 scans/s. The injected volume was 1.0 mL of 2% solution of oil in n-heptane. A Hewlett-Packard ALS injector was used with split ratio 1:100. The identification of the oil components was based on a Wiley MS Data Library (sixth ed.), followed by comparison of MS data with published literature (21, 22).

Antibacterial activity: The essential oil and its fractions were assayed for their antibacterial activity. The microorganisms used were Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 19433), Escherichia coli (ATCC 25992) and Klebsiella pneumoniae (ATCC 23357).

The antibacterial activity was carried out according to the disc diffusion assay described by Rondón et al. (23). The strains were maintained in agar at room temperature. 0.5 mL of every bacteria inoculum was diluted in sterile 0.85% saline solution to obtain a turbidity visually comparable of a McFarland Nº 0.5 standard (106-8 CFU/mL). Every inoculum was spread over plates containing Mueller-Hinton agar and a paper filter disc (4 mm) saturated with both 10 μL of essential oil and the two fractions. The plates were left for 30 min at room temperature and then incubated at 37ºC for 24 h. The inhibitory zone around

Ramos et al.


Figure 1. Chromatogram of Marjoram essential oil obtained by hydrodistillation.

the disc was measured and expressed in mm. A positive control was also assayed to check the sensitivity of the tested organ-isms using the following antibiotics: Ampicillin, Piperaciline and Amikacine. A negative control was also included in the test using a filter paper disc saturated with DMSO to check possible activity of this solvent against the bacteria assayed. The experiments were repeated twice.


Origanum majorana leaves produced 3.1 mL of oil which was equivalent to 0.6% yield. It was possible to identify 25 components. Table I shows the percentage composition of

the essential oil. The major constituents of the oil were: cis-sabinene hydrate (30.2%), terpinen-4-ol (28.8%), g-terpinene (7.2%), a-terpineol (6.9%), trans-sabinene hydrate (4.4%), linalool acetate (3.8%) and a-terpinene (3.6%). The essential oil from Germany was reported to contain cis-sabinene hydrate, linalool, sabinene and b-caryophyllene as main constituents. French and Italian studies reported similar results (5), but the oil from Turkey (15) was reported to have a completely different composition, because O. majorana from Turkey contained 78% carvacrol. On the other hand, essential oils from Cuba (12), Brazil (17), Hungary (18), and Tunisia (19) were reported to have terpinen-4-ol, g-terpinene and linalool as main components.

Figure 2. Chromatogram of Marjoram essential oil (fraction A).

O. majorana


Figure 3. Chromatogram of Marjoram essential oil (fraction C).

A previous study performed on O. majorana collected at the Medicinal Plants Garden at the Faculty of Pharmacy (20) reported that the oil contained 3-ciclohexen-1-ol (41.7%) and cis-sabinene-hydrate (14.8%) as major components. Different results obtained in this study could be attributed to climatic differences since the Medicinal Plant Garden is located at an altitude of 1400 m above sea level.

The oil fractionation on the silica gel dry column permit-ted to isolate cis-sabinene-hydrate (67.6%) on fraction A (Fr. A) and terpinen-4-ol (72.8%) on fraction C (Fr. C) (Figures 1, 2, and 3).

Antibacterial activity was measured using the agar diffu-sion method. ATCC Gram positive and Gram negative strains were used. These results are shown on Table II. Our results confirm the activity found on previous studies. The antibacterial activity of fractions A and C were also assayed.

Fraction A and the total oil were found to be active against all assayed bacteria, but fraction A was found to be twice as ac-tive against E. faecalis ATCC 19433 and K. pneumoniae ATCC 23357. On the other hand fraction C was found to be active only against E. faecalis ATCC 19433 and E. coli ATCC 25992

and in general it was less active than the total oil. Fraction A, which was enriched chromatographycally in cis-sabinene hydrate, is more active possibly because the OH group is next to a methyl while terpinen-4-ol, which is the main component of fraction C, has an isopropyl group next to the OH. Since fraction A showed the largest activity, it was concluded that cis-sabinene-hydrate was the substance responsible for such antibacterial activity.

References

Font Quer, 1. Plantas Medicinales. El Dioscórides Renovado. Editorial Labor, Barcelona (1988).

A. Albornoz, 2. Medicina Tradicional Herbaria . Instituto Farmacoterapeutico Latino S.A, Caracas (1993).

K. Bauer, D. Garbe and H. Surburg, 3. Common fragrance and flavor materials, pp 204, Wiley-VCH. Weinheim (2001)

M.T. Baratta, H.J.D. Dorman, S.G. Deans, A.C. Figueiredo, J.G. 4. Barroso, G. Ruberto, Antimicrobial and antioxidant properties of some commercial essential oils. Flavour Fragr. J., 13(4), 235-244 (1998).

J. Novak, F. Pank, J. Langbehn, W.D. Blüthner, C. Vender, L.V. 5. Niekerk, W. Junghanns, C. Franz, Determination of growing location

Table II. Antibacterial activity of the essential oil of Origanum majorana L.

Microorganisms Inhibition zone (mm)

Controls

Oil 1:10 Fr. A Fr. C AN AM PRL DMSO

Staphylococcus aureus ATCC 25923 16 - 13 - 34 -Enterococcus faecalis ATCC 19433 12 - 23 13 30 -Escherichia coli ATCC 25992 15 9 13 10 32 -Klebsiella pneumoniae ATCC 23357 13 - 28 - 32 -

Inhibition zone, diameter measured in mm, disc diameter 4 mm. average of two consecutive trial.AN: Ampicillin (10 µg); AM: Amikacine (30 µg); PRL: Piperaciline (8 µg)

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of marjoram (Origanum majorana L.) samples by comparison of essential oil profiles. Flavour Fragr. J., 19, 263-267 (2004).

D.J. Daferea, B.N. Ziogas and M.G. Polissiou, GC–MS analysis 6. of essential oils from some Greek aromatic plants and their fungitoxicity on Penicillium digitatum. J. Agr. Food Chem., 48, 2576-2581 (2000).

A.E. Edris, A. Shalaby and H.M. Fadel, Effect of organic agriculture 7. practices on the volatile aroma components of some essential oil plants growing in Egypt II: sweet marjoram (Origanum majorana L.) essential oil. Flavour Fragr. J., 18, 345-351 (2003).

N.B. Ezzeddine, M.M. Abdelkefi, R. Ben Aissa, M.M. Chaabouni, 8. Antibacterial screening of Origanum majorana L., oil from Tunisia. J. Essent. Oil Res., 13, 295–297 (2001).

M.B. Lawrence, Progress in essential oil: marjoram oil. 9. Perfum Flavor, 19, 39-40 (1994).

J. Novak, C. Bitsch, J. Langbehn, F. Pank, M. Skoula, Y. Gotsiou et 10. al. Ratios of cis- and trans-sabinene hydrate in Origanum majorana L. and Origanum microphyllum (Bentham) Vogel. Biochem. Syst. Ecol., 28, 697-704 (2000).

J. Novak, J. Langbehn, F. Pank, C.M. Franz, Essential oil compounds 11. in historical sample of marjoram (Origanum majorana L. Lamiaceae). Flavour Fragr. J., 17, 175-180 (2002).

J.A. Pino, A. Rosado, M. Estarron, V. Fuentes, Essential oil of 12. Marjoram (Origanum majorana L.) grown in Cuba. J. Essent. Oil Res., 9, 481-482 (1997).

R.R. Vera and M.J. Chane, Chemical composition of the essential 13. oil of marjoram (Origanum majorana L.) from Reunion Island. Food Chem., 66, 143-145 (1999).

W.J. Jun, B.K. Han, K.W. Yu, M.S. Kim, I.S. Chang, H.Y. Kim, H.Y. 14. Cho, Antioxidant effects of Origanum majorana L. on superoxide anion radicals. Food Chem., 75, 439-444 (2001).

K.H.C. Baser, N. Kirimer and G. Tümen, Composition of the essential 15. oil of Origanum majorana L. from Turkey. J. Essent. Oil Res., 5, 577-579 (1993).

S.G. Deans and K.P. Svoboda, The antimicrobial properties of 16. Marjoram (Origanum majorana L.) volatile oil. Flavour Fragr. J., 5, 187-190 (1990).

C. Busattaa, R.S. Vidala, A.S. Popiolski, A.S. Mossi, C. Dariva, M.R.A. 17. Rodrigues, F.C. Corazza, M.L. Corazza, J.V. Oliveira, R.L. Cansian, Application of Origanum majorana L. essential oil as an antimicrobial agent in sausage. Food Microbiol., 25, 207-211 (2008).

E. Vági, B. Simándi, A. Suhajda, E. Héthelyi, Essential oil composition 18. and antimicrobial activity of Origanum majorana L. extracts obtained with ethyl alcohol and supercritical carbon dioxide. Food Res. Int., 38, 51-57 (2005).

N. Ben Hamida-Ben Ezzeddine, M.M. Abdelkéfi, R. Ben Aissa, M.M. 19. Chaabouni, Antibacterial screening of Origanum majorana L. oil from Tunisia. J. Essent. Oil Res., 13, 295-297 (2001).

M. Meza, N. González and A. Usubillaga, Composición del aceite 20. esencial de Origanum majorana L. extraído por diferentes técnicas y su actividad biológica. Rev. Fac. Agron., 24, 725-738 (2007).

R.P. Adams, 21. Identification of essential oils components by gas chromatography/mass spectroscopy. 4th ed. Allured Publ. Corp., Carol Stream, IL, 1-499 (2007).

N.W. Davies, Gas Chromatographic retention indices of monoterpenes 22. and sesquiterpenes on methyl silicona and carbowax 20 M Phases. J. Chromatogr, 503, 1-24 (1990).

M. Rondón, J. Velasco, A. Morales, J. Rojas, J. Carmona, M. Gualtieri, 23. V. Hernández, Composition and antibacterial activity of the essential oil of Salvia leucantha Cav. cultivated in Venezuela Andes. Rev. Latinoam. Quim., 33, 55-59 (2005).

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Rec: Mar 2011

Acc: July 2011

Anti-inflammatory and Antioxidant Activity of a Methanolic Extract of Phyllanthus orbicularis and its

Derived Flavonols

Yamilet Irene Gutiérrez Gaitén*1, Migdalia Miranda Martínez1, Adonis Bello Alarcón1, Mariano Martínez Vázquez2, Jose Luis Figueroa Hernández3, Liván Delgado Roche1 and Luca Rastrelli41Pharmacy and Foods Institute, University of Havana, 222 and 23 Ave., La Coronela, La Lisa, Cod, 13600, Havana, Cuba

2Institute of Chemical, Nacional Autonomous University of Mexico, Coyoacan, Cod, 04510, Mexico. D.F. 3 Faculty of Medicine, Nacional Autonomous University of Mexico, Coyoacan, Cod, 04510, Mexico. D.F.

4Departimento di Scienze Farmaceutiche e Biomediche, University of Salerno, Via Ponte Don Melillo, 84084, Fisciano-Salerno, Italy

Abstract

In order to validate the use of Phyllanthus orbicularis (Phyllantaceae) in the traditional medicine of Cuba as an anti-inflammatory remedy, the methanolic (MeOH) extract has been evaluated in vivo for anti-inflammatory activity on 12-O-tetradecanoyl-13-acetate phrobol (TPA) assay in mouse and in vitro for the antioxidant activity on Ferric Reduction Antioxidant Power assay. This extract exerted in vivo a significant anti-inflammatory activity. Subsequent fractionation and analysis of the extract has led to the isolation and characterization as major constituents of apigenin (1), rutin (quercetin 3-O--L-rhamnopyranosyl-(1->6)--D-glucopyranoside) (2) and quercetin (3) and of rutin de-caacetate (4) and quercetin pentaacetate (5) from acetylated methanolic extract. Their structures were elucidated by spectral methods. The bioassay-directed analysis of flavonols 1-3 indicated that rutin (2) and quercetin (3) were the most active compounds, whereas apigenin showed no significant activity. The acetylation process increased the anti-inflammatory activity but decreased the antioxidant activity. The MeOH extract and all of flavonoids tested did not show in vitro significant cytotoxic effect in J774.A1 macrophage cell line. [Editor’s note: The five compounds under discussion are denoted throughout this paper by bolded number (1, 2, etc.). This should not be confused with the referenced ancillary texts, denoted in non-bolded font and within parentheses.]

Key Word Index

Phyllanthus orbicularis leaf and steam extracts, flavonoids, anti-inflammatory activity, antioxidant activity.



Introduction

Phyllanthus orbicularis (Phyllantaceae) is an herbaceous plant growing in Cuba. Its leaves have been used as a remedy in local folk medicine for the treatment of pathological pro-cesses such as ulcers and rheumatism, and as a febrifuge (1). In previous papers, the aqueous extract has been reported to show antiviral activity against human hepatitis B virus, herpes simplex virus type 1 and 2, bovine herpes (2, 3) as well as shown anti-mutagenic and antioxidant properties (4, 5).

Flavonoids are reported to affect the inflammatory process of the mammalian system and possess anti-inflammatory activity in vitro and in vivo (6). Prostaglandin biosynthesis and nitric oxide production have been implicated in the process of inflam-mation and NO, produced by inducible and constitutive nitric oxide synthase (cNOS), is one of the inflammatory mediators that plays an important role in inflammation (6).

In vitro studies have confirmed that the flavonoid quercetin inhibits nitric oxide production in IL-1β-stimulated hepatocytes through the inhibition of iNOS expression (7). In addition to its anti-inflammatory activity, quercetin also has a gastric ulcer protective effect, by the reduction in lipid peroxidation and an increase in the activity of antioxidant enzymes (8). These properties make quercetin an anti-inflammatory agent with no gastrointestinal side effect. However, quercetin has not been used widely in therapeutic medicine because it is practically insoluble in water or oil, and many other reasons such as low of oral absorption and bioavailability (9). It is therefore important for the molecular modification to enhance the solubility and bioavailability of quercetin. Some acetic acid esters of quer-cetin were reported enhanced the anti-inflammatory activity of quercetin (10). We reported that quercetin pentaacetate

Gaitén et al.


showed stronger inhibitory activity on TPA-induced inflam-matory assay than quercetin.

In the context of our research on medicinal plants from Cuba, the present paper reports on the composition and anti-inflammatory activity of the methanolic and acetylated methanolic extracts of P. orbicularis and on the involvement of its major constituents, flavonols and flavonol glycosides, in mediating this activity.

Experimental

Apparatus: A Bruker DRX-600 spectrometer operating at 599.2 MHz for 1H and 150.9 MHz for 13C using the UXNMR software package was used for NMR measurements with CD3OD solutions. DEPT, 1H-1H DQF-COSY, 1D TOCSY and HMBC spectra were obtained by employing the conventional pulse sequences. Optical rotations were measured on a Perkin-Elmer 141 polarimeter using a sodium lamp operating at 589 nm in 1% w/v solutions in MeOH. Electrospray ionization mass spec-trometry (ESIMS) was performed using a Finnigan LCQ Deca instrument from Thermo Electron (San Jose, CA) equipped with Xcalibur software. Full mass and collision-induced dis-sociation (CID) MS/MS spectra were acquired both in positive and negative mode. HPLC separations were performed with a Waters model 6000A pump equipped with a U6K injector and a Model 401 refractive index detector.

Table I. Inhibitory effects of extracts and compounds 2−5 from P. orbicularis leaves and steams on TPA-induced inflammation

in mice

Samples (mg/ear) Edema (mg) Inhibition (%)

Indomethacin 0.36 2.88±0.73 78.76*MeOH extract 1 8.57±0.49 50.00*Ac-MeOH extract 1 6.83±2,53 59.80*rutin 1 12.93±0.57 24.51*rutin decaacetate 1 6.10±4.51 64.12*quercetin 1 5.47±0.56 33.73* quercetina pentaacetate 1 11.27±2.24 66.80*

*p < 0.05 by Student’s t-test as compared to control group; the results were analyzed by means of a test of t- Student. Different letters mean that differences exist statistically significant for 95% of confidence.

Table II. Antioxidant activity of extracts and compounds 2−5 from P. orbicularis leaves and stems

Samples (1 mg/mL) FRAP (μmol/g)a

l-ascorbic acid 404.0 ± 9.2bMeOH extract 1067.2 ± 22.7Ac-MeOH extract 103.1 ± 4.1rutin 467.4 ± 4.3rutin decaacetate 82.3 ± 3.2quercetin 1559.7 ± 120.5quercetin pentaacetate 90.3 ± 6.9

aFRAP, relative activities of the individual antioxidants to the reaction of Fe+2. For protocols used, see Experimental section. bMean ± SD of three determinations

Plant material: Phyllanthus orbicularis HBK was identi-fied and collected by Agronomist Engineer Rafael Carbonel Paneque in August 2007 during flowering, in the zone of Cajalbana, Pinar del Rio Province (Cuba). A specimen was deposited in the herbarium of the Nacional Botanical Garden of Cuba (HFC-85589-HAJA).

Extraction and isolation: The dried, powered stem and leaves were mixed (500 g) and partitioned successively (7 days) with n-hexane (1500 mL x 3), ethyl acetate (EtOAc) (1500 mL x 3) and methanol (MeOH) (1500 mL x 3) to yield three extracts. The methanolic extract was used in this work. A portion of methanolic extract was acetylated with acetic anhydride/pyridine (2 mL, 1:1) to 70°C by 6h. The methano-lic and acetylated methanolic extracts were fractionated on a column packed with silica gel 60 GF254 (Merck) and eluted with hexane, EtOAc, MeOH and mixtures of them. Fraction containing flavonoids was subjected to reversed-phase HPLC separation on Bondapak C18 column (30 cm x 7.8 mm i.d. flow rate 1.5 mL/min) with MeOH- H2O (1:1) as solvent system. This procedure gave three pure compounds identified by their observed NMR, FABMS spectra and optical rotation data in comparison with the literature values as apigenin, quercetin and rutin from MeOH extract and as rutin decaacetate and quercetin pentaacetate from acetylated methanolic extract.

Animals: Male CD1/c mice were housed in an environ-ment with controlled temperature, (21-24°C), lighting (12:12 light: darkness cycle), standard laboratory chow and drinking water ad libitum for a period of 7 days before any experimental manipulation. Their body weights ranged 20-25 g. All experi-ments were conducted according to guidelines established by the Animal Care Committee.

Assay of TPA-induced inflammation ear edema in mice: Ear edema was induced according to the method of De Young et al. (11). The right ear of each mouse received TPA (0.125 mg/mL acetone solution) as a topical application (10 mL for each side of the ear). The methanolic and acetylated methanolic extracts, rutin (2), quercetin (3), rutin decaacetate (4) and quercetin pentaacetate (5) from Phyllanthus orbicularis (dissolved in acetone), were applied topically immediately after TPA at doses of 1.0 mg/ear. The left ear, used as a control, received the vehicle. Indomethacin (0.36 mg/ear/20 μL) was used as a reference compound. Four hours after TPA administration, the animals were sacrificed and disks of 6 mm diameter were removed from each ear and their weights determined. Swelling was measured as the difference in weight between the punches from right and left ears, and the percent inhibition of edema was calculated in comparison with control animals.

Determination of reducing power: The total antioxidant potential of samples was determined using the ferric reduc-ing antioxidant power (FRAP) assay of Benzie and Strain (12). A solution of 10 mM TPTZ in 40 mM HCl and 12 mM ferric chloride was diluted in 300 mM sodium acetate buffer (pH 3.6) at a ratio of 1:1:10. Solutions of the MeOH and Ac-MeOH extracts and pure compound 2-5 (60 mL) were added to 3 mL of the FRAP solution, and the absorbance at 593 nm was determined every 10 min, for 90 min. Aqueous solutions of known Fe(II) concentration (FeSO4·7H2O) were used for calibration of the FRAP assay and antioxidant power was expressed as mmol/g FRAP. l-ascorbic acid was used as refer-

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160 fractions. The fractions 40-61 eluted with hexane:EtOAc (60:40) gave 27.5 mg of a crystalline powder cream color (4) and fractions 98-109 eluted with hexane:EtOAc (10:90) were gave 33.2 mg of a yellow crystalline product (5). 4 and 5 were identified as quercetin pentaacetate (14) and rutin decaacetate (15) respectively by comparison of their physical and spectro-scopic data with reported values.

Inhibition of TPA-induced inflammation in ear mice: TPA can act as an inducer of epidermal hyperplasia, a tumor promoter, and an activator of various biological systems. TPA-induced ear edema is an in vivo model of acute inflammation; it offers a simple and useful assay for screening the efficacy of topical anti-inflammatory capacities of plant extracts, in this case blackberry preparations. The inhibitory effects of MeOH and Ac-MeOh extracts of and their isolated compounds 2-5 were evaluated on TPA-induced inflammation in mice together with those of a commercially available anti-inflammatory drugs, indo-methacin. The results are shown in Table I. All of the extracts and compounds tested showed inhibitory effects comparable or more active than indomethacin. Rutin and quercetin were less potent compared to the extract; conversely rutin decaacetate and quercetina pentaacetate showed an increase of edema percentages inhibition (64.12% and 66.80% respectively) and were more active when compared to the acetylated extract. Apigenin present in small amounts in the extract was not tested for its anti-inflammatory activity. Quercetin pentacetate exhibited a strong inhibitory effect that was almost the same order of potency as that of indomethacin (Table I). Our results suggest that chemical transformation by means of a reaction of acetylation of the methanolic extract can improve the activity and the results are in agree with those reported by Chen et al. (16) and Gusdinar et al. (17).

These flavonoids therefore contribute to the anti-inflam-matory activity of the MeOH extract of the leaves and steams of P. orbicularis. The inhibitory effect against TPA-induced inflammation has been demonstrated to closely parallel that of the inhibition of tumor promotion in two stage carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA) and TPA, a well-known promoter, in a mouse skin model. Thus, compounds 2-5 may have anti-tumor-promoting activity in this animal model.

Antioxidant activity: The antioxidant potential of extracts and pure compounds from P. orbicularis were evaluated in the antioxidant (FRAP) assays. l-Ascorbic acid was used as the reference antioxidant. The results (Table II) showed that flavonoids 2 and 3 exhibited free-radical scavenging activity at potency levels comparative to the reference antioxidant compound, while 4 and 5 had more moderate activities.

In this case, the acetylation process decreases the antioxi-dant activity. It is well known that the free radical scavenging and antioxidant activities of phenolics are dependent upon the arrangement of functional groups about the nuclear structure. Both the number and configuration of H-donating hydroxyl groups are the main structural features influencing the anti-oxidant capacity of phenolics.

Citoxicity: As previously indicated the MeOH extract of P. orbicularis and its derived flavonoids (1-3) have been also

ence compound. All extracts and compounds were diluted and analyzed in triplicate.

Cell culture: The macrophage cell line J774.A1 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin and 100 g/mL streptomycin at 37°C under 5% CO2 humidified air.

MTT assay for cell viability: Cytotoxicity studies were performed in a 96 well-plate. J774.A1 cells were mechanically scraped and plated at a seeding density of 35.000 J774.A1 cells/well to a final volume of 150 L. After 2 h of incubation in DMEM 5% FCS, cells were treated with LPS 1 g/mL alone or in combination with P. orbicularis MeOH extract (1-100 g/mL) or derived flavonoids 1-3 (0.5-50 g/mL) dissolved in DMSO. This DMSO percentage allows the optimal solubilization of flavonoids in aqueous solution. Control and LPS wells received the same amount of DMSO. After 22 h of incubation at 37°C, 25 L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 5 mg/mL) were added in each well and 3 h later the cells were lysed with 100 L of lysis buffer (20% SDS and 50% DMF, pH 4.7). After an incubation of 18 h at 37°C, the optical densities (OD620) for the serial dilutions of the methanolic extract of P. orbicularis and its derived flavonols were compared to the OD of the control or LPS-stimulated wells to assess the cytotoxicity (6).

Statistical Analysis

Experimental results are expressed as the means ± SEM of measurements of at least six different mice. Data were assessed by the method of analysis of variance (ANOVA). If this analysis indicated significant differences among the group means, then each group was compared with those for controls by the Stu-dent’s t test, and p values of less than 0.05 were considered to be statistically significant. For antioxidant activity analysis data are reported as mean ± standard deviation (SD) of triplicate determinations. The statistical analysis was carried out using the Microsoft Excel software package (Microsoft Corp.).


Chemical studies: The TLC analysis of the MeOH extract of P. orbicularis leaves and steams revealed the presence of flavonoids as major constituents. A silica–gel column of the MeOH extract (15 g) gave a flavonoid enriched fraction C that was chromatographed by reversed-phase HPLC to yield three pure compounds 1 (0.008%), 2 (0.533%) and 3 (0.035 % of MeOH extract). They were identified as apigenin (1), rutin (2) and quercetin (3) respectively (13), by ESIMS and extensive NMR analysis. In particular the sugar moiety was determined to be 3-O--L-rhamnopyranosyl-(1->6)--D-glucopyranoside linked at C3 of the aglycone in compound 2 by a combination of 1D TOCSY, 2D DQF-COSY and HMQC 2-D NMR experi-ments. The position of the interglycosidic linkage and the site of glycosylation on the aglycone were deduced by long-range C-H correlations in the HMBC spectra. The chemical shift, multiplicity, absolute value of the proton coupling constants, as well as the resonances of C2, C3 and C5, indicated the configuration of D-glucopyranose and L-rhamnopyranose. The acetylated methanolic extract was fractionated by CC obtaining

Gaitén et al.


tested for their in vitro cytotoxic activity on J774.A1 cell line, but we did not observe any significant cytotoxic effect here (data not shown).

Conclusion

The flavonoids have been considered as the active prin-ciples of many anti-inflammatory plants. It has been speculated that anti-inflammatory properties are a consequence of their inhibitory actions on arachidonic acid metabolism as demon-strated in vitro and in vivo (6). Furthermore, flavonoids have been reported to possess free radical scavenging or antioxidant properties, which can be related with the inhibition exerted in the metabolism of arachidonic acid via lipoxygenase activity (18). On the basis of our results, we can hypothesize that the anti-inflammatory activity of the extracts and compounds of P. orbicularis may be due to the presence of a combination of flavonoids and flavonoid glycosides.

Acknowledgements

The authors give thanks to the Institute of Chemical of Nacional Autonomous University of Mexico for the provision of necessary facilities.

References

J.T. Roig, 1. Diccionario botánico de nombres vulgares cubano. pp 80, Editorial científico técnico, La Habana, Cuba, (1988).

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J. Fernández, G. Del Barrio, B. Romeo, Y. Gutiérrez, S. Valdés and F. 3. Parra, In vitro antiviral activity of Phyllanthus orbicularis extract againts herpes simples type 1. Phytother. Res., 17, 980-982 (2003).

M. Ferrer, A. Sánchez, J. Fuentes, J. Barbé and M. Llagostera, 4. Antimutagenic mechanisms of Phyllanthus orbicularis when hydrogen peroxide is tested using Salmonella assay. Mutat. Res., 517, 251-254 (2002).

A.L. Sánchez, J.L. Fuentes, G. Fonseca, N. Cápiro, M. Ferrer, A. 5. Alonzo, L. Baluja, R. Cozzi, R. De Salvia, M. Fiore and M. Llagostera, Assessment of the potential genotoxic risk of Phyllantus orbicularis HBK aqueous extract using in vitro and in vivo assays. Toxicol. Lett., 136, 87-96 (2002).

G. Autore, L. Rastrelli, M.R. Lauro, S. Marzocco, R. Sorrentino, A. 6. Pinto and R. Aquino, Inhibition of nitric oxide synthase expression by a methanolic extract of Crescentia alata and its derived flavonols. Life Science, 70, 523-534 (2001).

S. Martinez-Florez, B. Gutierrez-Fernandez, S. Sanchez-Champos, 7. J. Gonzalez-Gallego, and M.J. Tunon, Quercetin attenuates nuclear

factor-kB activation and nitric oxide production in interleukin-1b-activated rat hepatocytes. J. Nutr., 135, 1359-1365 (2005).

O. Coskun, M. Kanter, F. Armutcu, K. Cetin, B. Kaybolmaz and O. 8. Yazgan, Protective effects of quercetin, a flavonoid antioxidant, in absolute ethanol-induced acut gastric ulcer. Eur. J. Gen. Med., 1, 37-42 (2004).

Y. Peng, Z. Deng and C. Whang, Preparation and pro-drug studies 9. of quercetin pentabenzensulfonate. Yakugaku Zasshi, 128, 1845-1849 (2008).

Y.Chen, S. Shen, W. Lee, W. Hou, L. Yang and T.J.F. Lee, Inhibition 10. of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin and quercetin pentaacetate in RAW 264,7 macrophage. J. Cell. Biochem., 82, 537-548 (2001).

L.M. De Young, J.B. Kheifets, S.J. Ballaron and I.M. Young, Edema and 11. cell infiltration in the phorbol ester-treated mouse ear are temporally separate and can be differentially modulated by pharmacologic agents. Agents Actions, 26, 335–341 (1989).

I.F.F. Benzie and J.J. Strain, The ferric reducing ability of plasma 12. (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem, 239, 70–76 (1996).

L. Rastrelli, P. Saturnino, O. Schettino and A. Dini, Studies on the 13. constituents of Chenopodium pallidicaule (cañihua) seeds. Isolation and characterization of two new flavonol glycosides. J. Agr. Food Chem., 43, 2020-2024 (1995).

M. S. Rao, H. Duddeck and R. Dembiinshi, Isolation and structural 14. elucidation of 3,4’,5,7-tetraacetyl quercetin from Adina cordifolia (Karam ki Gaach). Fitoterapia, 73, 353-355 (2002).

T.J. Mabry, J. Kagan and H. Rösler, NMR spectra of trimethylsilyl 15. ethers of flavonoid glycosides. Phytochemistry 4, 177-183 (1964).

Y. Chen, S. Shen, W. Lee, W. Hou, L.Yang and T.J. Lee, Inhibition 16. of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and Cyclooxygenase-2 gene expressions by rutin, quercetin and quercetin pentaacetate in RAW 264,7 macrophage. J. Cell. Biochem. 82, 537-548 (2001).

T.R.H. Gusdinar, R.E. Kartasamita and I.K. Adnyana, Anti-inflammatory 17. and antioxidant activity of quercetin-3,3´,4´-triacetate. J. Pharmacol. Toxicol. 6, 182-188 (2011).

S.J. Duthie and V.L. Dobson, Dietary flavonoids protect human 18. colonocyte DNA from oxidative attack in vitro. Eur. J. Nutr., 38, 28–34 (1999).

P. orbicularis


Rec: Nov 2010

Acc: Mar 2011

Chemical Composition of Essential Oils from Ripe and Unripe Fruits of Piper amalago L. var. medium

(Jacq.) Yunck and Piper hispidum Sw.

Maria Lúcia Ferreira Simeone*Embrapa Milho e Sorgo, Caixa Postal 151, Sete Lagoas-MG, 35700-297, Brazil

Sandra Bos Mikich Embrapa Florestas, Caixa Postal 319, Colombo-PR, 83411-000, Brazil

Lílian Cristina Côcco Centro Politécnico, Usinas Piloto A, Universidade Federal do Paraná, Caixa Postal 19.024, Curitiba-PR, 81530-990, Brazil

Fabrício Augusto HanselEmbrapa Florestas, Caixa Postal 319, Colombo-PR, 83411-000, Brazil

Gledson Vigiano BianconiInstituto Neotropical: Pesquisa e Conservação, Caixa-Postal 19009, Curitiba-PR, 81531-980, Brazil

Abstract

The chemical composition of essential oils from unripe and ripe fruits of Piper amalago L. var. medium (Jacq.) Yunck and Piper hispidum Sw. was examined using GC/MS analysis. The analysis of oils from P. amalago revealed a predominance of oxygenated sesquiterpenes and 65 compounds were identified; their main constituents are: (E)-nerolidol (14.2% and 19.9%), germacrene-D-4-ol (10.3% and 12.7%), a-cadinol (11.1% and 8.2%) in 99.6% and 98.7% of the compounds for unripe and ripe fruits, respectively. Piper hispidum revealed a predominance of sesquiterpene hydrocarbons, from which we identified 53 compounds including: a-copaene (28.7% and 36.2%), a-pinene (13.9% and 7.1%), b-pinene (13.3% and 7.5%), and (E)-nerolidol (2.9% and 7.0%) which represented 97.8% and 98.1% of the compound constituents for unripe and ripe fruits, respectively. The essential oils of fruits of P. amalago and P. hispidum are reported for the first time.

Key Word Index

Piperaceae, Piper amalago L. var. medium (Jacq.) Yunck, Piper hispidum Sw., essential oil composition, a-pinene, b-pinene, a-copaene, (E)-nerolidol, germacrene d-4-ol, a-cadinol.



Introduction

The genus Piper (Piperaceae) has been recently revised, and includes approximately 700 species, represented by herbs, shrubs and trees (1). The genus is widely distributed in tropical and subtropical regions of both hemispheres. Several plants of this genus are widely used in folk medicine in several parts of the world and have been reported to produce compounds with diverse biological and pharmacological properties (2). Many Piper species are aromatics and as a consequence the chemi-

cal composition of the essential oils from several species has been studied in detail. These studies revealed a diverse range of oil components, including monoterpenes, sesquiterpenes, arylpropanoids, aldehydes, ketones and long chain alcohols (3-5). The oil components from fruit parts of Piperaceae species have been reported in a number of investigations and consist of variable mixtures with a predominance of monoterpenes (C10) and sesquiterpenes (C15) (6).

Previous results suggest that Piperaceae are very important

Simeone et al.


Table I. Chemical composition of unripe and ripe fruits P. amalago and P. hispidum.

Oil componentsa RIb RIc Relative Area%

Piper amalago Piper hispidum

Unripe Ripe Unripe Ripe

1 heptanal 904 899 - - <0.1 -2 tricyclene 925 926 - 0,1 - -3 a-pinene 933 939 0.7 3.6 13.9 7.14 canfene 951 953 - 0.2 0.3 0.15 sabinene 973 976 1.3 3.0 - -6 b-pinene 980 980 0.2 0.6 13.3 7.57 myrcene 988 991 1.8 2.6 0.6 0.78 n-decane 1004 999 - - 0.3 0.39 a-phellandrene 1008 1005 0.7 - - -10 para-cymene 1025 1026 0.7 2.2 0.6 0.411 limonene 1030 1031 1.0 1.5 0.9 0.912 b-phellandrene 1032 1031 8.2 7.3 - <0.113 1,8-cineole 1034 1033 0.3 0.1 - -14 cis-b-ocimene 1034 1040 - - - <0.115 trans-b-ocimene 1045 1040 0.1 <0.1 0.4 0.216 g-terpinene 1058 1062 - <0.1 - -17 cis-sabinene hydrate 1072 1068 - <0.1 - -18 para-mentha-2,4(8)-diene 1087 1086 - <0.1 - -19 linalool 1101 1098 2.0 1.4 - -20 trans-hydrate sabinene 1104 1097 - - - -21 perilene 1113 1099 - - 1.6 1.922 perilene isomer 1113 1099 0.3 - - -23 4-terpineol 1183 1177 0.3 0.2 0.2 -24 exo-fenchol 1122 1117 - - 0.6 -25 cis-pinene hydrate 1127 1121 0.06 - - -26 cis-b-terpineol 1145 1144 0.04 - 27 trans-b-terpineol 1158 1163 - 0.3 -28 borneol 1175 1165 0.1 - 1.4 -29 cryptone 1191 1185 0.1 0.9 - -30 a-terpineol 1198 1189 0.2 0.5 1.5 0.431 n-decanal 1207 1201 - - 0.2 0.132 undec-9-en-1-al 1309 1308 - - <0.1 -33 g-elemene isomer 1332 1339 0.1 - - -34 g-elemene 1335 1339 0.4 0.5 - -35 a-cubebene 1347 1351 0.2 0.1 0.2 0.136 ciclosativene 1370 1368 0.1 <0.1 - -37 isoledene 1369 1373 - - 1.3 1.338 a-copaene 1376 1376 3.0 0.7 28.7 36.239 b-bourbonene 1384 1384 0.2 0.2 - -40 b-cubebene 1388 1390 3.3 2.5 - -41 b-elemene 1390 1391 - 0.5 - -42 b-isocomene 1392 1403 - - 0.2 0.243 a-gurjunene 1407 1409 - - 0.1 <0.144 trans-caryophyllene 1420 1418 2.6 2.7 1.7 4.945 b-gurjunene 1431 1432 - 0.2 - 0.246 aromandrene 1439 1439 0.1 <0.1 2.6 0.847 geranyl acetone 1448 1453 - - 0.9 3.048 a-humulene 1456 1454 1.0 0.8 3.3 4.849 trans-b-farnesene 1453 1458 - <0.1 - -50 seichelene 1460 1460 - <0.1 0.3 0.351 g-gurjunene 1475 1473 - 0.5 0.8 0.952 g-himachalene 1472 1476 - 2.3 - -53 g-muurolene 1479 1477 2.1 - - <0.154 germacrene D 1481 1480 2.0 1,0 - -55 b-selinene 1489 1485 - - - <0.156 valencene 1491 1491 0.3 - 0.3 0.257 b-cis-guaiene 1496 1490 - 0.2 0.258 bicyclogermacrene 1496 1494 9.1 3.0 - -59 a-muurolene 1498 1499 1.5 <0.1 0.6 0.860 germacrene A 1507 1503 0.9 0.7 - -61 g-cadinene 1513 1513 0.9 0.9 0.4 0.462 cubebol 1516 1514 - - - <0.163 d-cadinene 1518 1524 6.6 2.3 3.4 2.364 cis-calamenene 1521 1521 - <0.1 1.7 2.2

P. amalago and P. hispidum


65 a-colacorene 1542 1542 - <0.1 1.1 1.366 (E)-nerolidol 1561 1564 14.2 19.9 2.9 7.067 ledol 1571 1565 - - - 0.168 germacrene d-4-ol 1578 1576 10.3 12.7 - -69 spathulenol 1578 1576 - - 4,16 3,6770 caryophyllene oxide 1583 1581 - 0.7 1.82 1.3971 globulol 1588 1583 - 0.8 0.94 1.2772 humulene epoxide II 1612 1606 - - 0.97 0.8473 1-epi-cubenol 1629 1627 - <0.1 0.30 0.4474 a-acorenol 1631 1630 1.2 2.1 - -75 b-acorenol 1635 1634 - <0.1 - -76 epi-a-cadinol 1644 1640 6.1 4.9 - 0.477 epi-a-muurolol 1646 1641 1.5 - - 0.278 a-muurolol 1649 1645 2.6 2.1 0.2 0.279 a-cadinol 1658 1653 11.1 8.2 - -80 9-methoxicalamelene 1667 d - <0.1 - -81 cadalene 1674 1674 - - 1.7 1.982 a-bisabolol 1682 1683 - <0.1 - -83 n-heptadecane 1699 1700 - - 0.9 0.784 oplopanone 1737 1733 0.1 3.9 - -85 khusinol acetate 1829 1816 - <0.1 - -Terpenoid ClassesMonoterpene hydrocarbons 14.9 21.2 30.4 17.2Oxygenated monoterpenes 3.3 3.0 5.8 2.4Sesquiterpene hydrocarbons 34.5 19.1 48.6 59.3Oxygenated sesquiterpenes 46.9 55.4 13.0 19.2Total 99.6 98.7 97.8 98.1

– Not detected.a -Compounds are listed in order of their elution from a CPSIL 8 CB column; b - RI = retention indices relative to C8 – C26 n-alkanes; c - RI = retention indices from literature (DB-5 column);10

d - mass spectrum agreed with NIST mass spectral database (tentative identification);Obs: DB-5 and CPSIL 8 CB columns are similar in dimensions and chemical composition.

Table I. Continued. Chemical composition of unripe and ripe fruits P. amalago and P. hispidum.

Oil componentsa RIb RIc Relative Area%

Piper amalago Piper hispidum

Unripe Ripe Unripe Ripe

for the fruit-eating bat genus Carollia. Fruit-eating bats are amongst the main seed dispersers for a variety of species and improving this ecological role may have application in forest restoration projects since the essential oils isolated from mature chiropterochoric fruits are able to attract frugivorous bats both inside forest remnants and in order to promote tropical forest restoration (7-8).

The objective of this study was to evaluate the oil con-stituents in both unripe and ripe fruits of two Piper species (Piper hispidum Sw. and Piper amalago L. var. medium (Jacq.) Yunck) for use in future research to attract bats, encouraging seed dispersal and subsequent forest regeneration.

Experimental

Plant material: Unripe and ripe fruits of P. amalago and P. hispidum were harvested at the Parque Estadual Vila Rica do Espírito Santo, in the town of Fênix (Paraná State, Brazil; 23°55’S, 51°57’W), in 2006. Specimens were identified by Dr. Sandro M. Silva and vouchers were deposited in the Herbarium of Universidade Federal do Paraná, Curitiba, Brazil, under code numbers UPCB 32345, 32346 for P. amalago L. var. medium (Jacq.) Yunck and UPCB 32337, 32339 for P. hispidum Sw.

Isolation of the essential oils: Unripe and ripe fruits of P. amalago and P. hispidum species (200 g) were subjected to hydrodistillation for 4 h in a Clevenger-type apparatus. The oil layers obtained were dried over anhydrous sodium sulfate after extraction with ethyl ether and, after filtration, evaporated under nitrogen flux and maintained under refrigeration (-10ºC) before analysis. Yields were calculated from the weight of fresh material. The yields were averaged over three experiments and calculated through the relation of the oil weight from the Clevenger-type equipment to the mass of fruit material used in the extraction.

Analysis of the essential oils: Oil sample analyses were performed using a Varian CP-3800 gas chromatograph equipped with an auto sampler Varian 8200 and a flame ionization de-tector using a CP-SIL 8 CB (30 m x 0.25 mm id, 0.25 μm film thickness) capillary column. The injector and detector tem-perature were maintained at 250ºC and 300ºC, respectively. The samples (1.0 μL), dissolved in ethyl acetate, were injected in split mode (1:200), using He as the carrier gas, flow rate 1 mL/min-1. The oven temperature was programmed as follows: 60ºC (1 min), heating to 240ºC at 3ºC/min-1 and holding for 5 min. Peak areas were measured by electronic integration. The relative amounts of individual components were determined

Simeone et al.


on the basis of their GC peak areas, without corrections for FID response factors. GC/MS analysis was carried out on a Varian ion trap Model CP 3800/Saturn 2000. GC/MS, EI electron impact ion source, 70 eV using a CP-SIL 8 CB fused silica capillary column (30 m x 0.25 mm i.d. x 0.25 μm film thickness); helium as the carrier gas, with a flow rate of 1 mL/min-1 and a split ratio of 1:100. The injector temperature and ion trap temperature was 250ºC and 150ºC, respectively. The oven temperature was programmed as follows: 60ºC (1 min), heating to 240ºC at 3ºC.min-1 and holding for 5 min, following the same conditions from literature (11). The transfer line and manifold temperatures were 200ºC and 100ºC, respectively. Mass range was used from 35 to 500 m/z and scan time was set in 0.4 s/scan-1.

Oil components were identified by comparison of their mass spectra with those obtained from the spectrometer data base, held at the National Institute for Standard Technology–NIST library using retention indices as a pre-selection routine (9-10).

The linear retention indices were calculated by co-injection with a standard saturated n-alkanes homologous series. The identifications were confirmed by comparison of the fragmen-tation pattern and corresponding linear retention indices with those reported in the literature (10).


Average essential oils yields were for unripe and ripe P. amalago, 0.09% and 0.03%, and for P. hispidum, 0.01% and 0.04%, respectively.

In total, there were identified 85 oil constituents in the fruits of the two Piper species (Table I). The 32 monoterpenes and 53 sesquiterpenes identified are shown in order of elution on a CPSIL 8 CB column, corresponding to essential oils from the unripe and ripe fruits of P. amalago and P. hispidum.

Although P. hispidum showed a different chemical com-position of essential oils compared with those identified from P. amalago, in both species there was a predominance of ses-quiterpene compounds in both ripe and unripe fruit.

Chemical analysis using GC/MS (Table I) of essential oil from fruits of P. amalago revealed a predominance of oxy-genated sesquiterpenes and 65 compounds were identified; the main constituents are: (E)-nerolidol (14.2% and 19.9%), germacrene-D-4-ol (10.3% and 12.7%), a-cadinol (11.1% and 8.2%) and b-phellandrene (8.2% and 7.3%) in 99.6% and 98.7% of the compounds obtained from unripe and ripe fruit oils, respectively.

In the analysis of essential oils from fruits of P. hispidum there was a predominance of sesquiterpene hydrocarbons, and were identified 53 compounds including: a-copaene (28.7% and 36.2%), a-pinene (13.9% and 7.1%), b-pinene (13.3% and 7.5%), (E)-nerolidol (2.9% and 7.0%) and trans-caryophyllene (1.7% and 4.9%) the majorities constituents. The whole indentified compounds represented 97.8% and 98.1% of the constituents of unripe and ripe fruit oils, respectively.

As expected, there are some differences in the propor-tions of the oil compounds in the unripe and ripe fruits of the two Piper species. Table I showed that sesquiterpenes hydrocarbons decreased and sesquiterpenes oxygenated in-

creased from unripe to ripe fruit oil of P. amalago. As for P. hispidum, the monoterpenes decreased and sesquiterpenes increased from unripe to ripe fruit oil. These results confirm that the formation of ripe compounds in fruit is a dynamic process, during which concentrations of constituents change both qualitatively and quantitatively, which can cause changes in the oil composition.

Mesquita et al, 2005 (11) analyzed the composition of vola-tile oil from leaves of P. amalago and P. hispidum harvested in the state of Minas Gerais (Brazil). Piper amalago had a yield of 0.6% and showed a predominance of sesquiterpenoids containing the following major constituents: caryophyllene oxide (18.0%), E-caryophyllene (17.8%), bicyclogermacrene (16.4%), germacrene D (10.9%), a-pinene (9.3%). Piper hispidum had a yield of 0.2% in which the predominant com-pound was monoterpenoid b-pinene (14.0%), followed by sesquiterpenoid spathulenol (7.0%), germacrene D (6.9%), caryophyllene oxide (6.4%).

The chemical composition of the essential oils of P. hispi-dum, from Cerrado (Brazillian savannah) was determined and compared with the composition of oils from the same species collected in the Atlantic Rain Forest. The distillation of leaves of P. hispidum (12) had a yield of 0.3% and 26 compounds were identified in the essential oil. The main constituents were: b-pinene (19.7%), a-pinene (9.0%), d-3-carene (7.4%), a-cadinol (6.9%) and spathulenol (6.2%). The identification of chemical constituents of essential oils from the roots of P. hispidum were 99.9% and 92.2%, represented by phenylpro-panoids, corresponding to three major components: dillapiole (57.5%), elemicine (24.5%) and apiole (10.2%) (13).

The essential oil from the fruits of P. tuberculatum were identified 90.4% from the major constituent compounds (E)-caryophyllene with 12.3% and caryophyllene oxide 26.6%. This finding contrasts with previous studies (14) that revealed the prevalence of monoterpenes found in fruits of the other Piper-aceae species.The sesquiterpenes are frequent in the composi-tion of the oils from the leaves, roots and fruits of these species, and are also commonly recorded in the essential oils obtained from other Piper species. This is the first report of the chemical composition of essential oils from the fruits of P. amalago and P. hispidum, and the observed chemical profile is different from those obtained from other plant parts (11-13).

In summary, the study of essential oil components of Piper species collected in Paraná state showed a predominance of oxygenated sesquiterpenes in P. amalago and sesquiterpene hydrocarbons were mostly detected in P. hispidum. The es-sential oils of these chiropterochoric fruits can be used to improve autoecological studies of fruit-eating bats and to promote tropical forest restoration through the attraction of frugivorous bats to degraded areas.

Acknowledgements

This work was funded by the Conselho Nacional de Desenvolvi-mento Científico e Tecnológico - CNPq (Process 476253/2007-1). The authors are grateful to Dr. Carlos Itsuo Yamamoto (Laboratório de Análise de Combustíveis Automotivos – LACAUT – UFPR) for record-ing the GC and GC/MS spectra. S.B. Mikich is grateful to CNPq for the Productivity Fellowship (Process 308419/2008-1).

P. amalago and P. hispidum


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V.A. Facundo, A.R. Polili, R.V. Rodrigues, J.S.L.T. Militão, R.G. Stabelli 13. and C.T. Cardoso, Constituintes químicos fixos e voláteis dos talos e frutos de Piper tuberculatum Jacq. e das raízes de P. hispidum H. B. K. Acta Amazon., 38, 733-742 (2008).

H.D. Navickiene, A.A. Morandim, A.C. Alécio, L.O. Regasini, 14. D.C.B. Bergamo, M. Telascrea, A.J. Cavalheiro, M.N. Lopes, V.S. Bolzani, M. Furlan, M.O. Marques, M.C.M. Young and M.J. Kato, Composition and antifungal activity of essential oils from Piper aduncum, Piper arboreum and Piper tuberculatum. Quim. Nova, 29, 467-470 (2006).

Simeone et al.


Rec: Nov 2010

Acc: June 2011

Chemical Composition and Larvicidal Effects of Essential Oil from Bauhinia acuruana (Moric) against

Aedes aegypti

Roberto W. da Silva Gois, Leôncio M. de Sousa, Telma L. G. Lemos, Angela M. C. Arriaga and Manoel Andrade-Neto

Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CEP 60451-970, Fortaleza, CE, Brazil

Gilvandete M. P. Santiago* and Yana S. FerreiraDepartamento de Farmácia, Universidade Federal do Ceará, Rua Capitão Francisco Pedro 1210, CEP 60430-370,

Fortaleza, CE, Brazil

Péricles B. Alves and Hugo C. R. de JesusDepartamento de Química, Universidade Federal de Sergipe, CEP 49100-00, São Cristovão, SE, Brazil

Abstract

The essential oil from leaves of Bauhinia acuruana Moric was obtained by hydrodistillation and analyzed by gas chromatography (GC-FID) and gas chromatography/mass spectrometry (GC/MS). In total, thirty compounds compris-ing 91.4% of the total peak area were identified. The main constituents of the essential oil were the sesquiterpenes spathulenol (23.4 ± 0.08%), epi-a-cadinol (20.7 ± 0.12%) and caryophyllene oxide (16.4 ± 0.04%). The essential oil was tested against Aedes aegypti larvae and showed LC50 value of 56.2 ± 0.4 mg/mL.

Key Word Index

Bauhinia acuruana, Caesalpinioideae, essential oil composition, sesquiterpenes, epi-a-cadinol, spathulenol, Aedes aegypti.



Introduction

Bauhinia (family: Leguminosae, subfamily: Caesalpin-ioideae) is a genus of shrubs or trees, very rarely climbers, distributed throughout the tropical regions of the world (1). This genus comprises 300 species and is popularly known in the northeast Brazil as “pata-de-vaca” due to their leaf format (2). Species of this genus have been frequently used in folk medicine to treat diabetes (3-5).

There are reports in the literature on the chemical compo-sition of some species of the genus Bauhinia. Thus, different classes of compounds such as lactones, flavonoids. terpenoids, steroids, tannins and quinones were isolated and identified from these species (3). No previous work on the chemical composi-tion of Bauhinia acuruana has been reported.

Mosquitoes play a predominant role in the transmission of several diseases which are today among the greatest health problems in the world (6). Aedes aegypti is the principal vector for the arboviruses responsible for yellow fever and dengue, including the hemorrhagic form. The incidence of dengue has grown significantly around the world in recent years. Today,

dengue is the most important mosquito-borne viral disease affecting humans (7). Due to the lack of vaccines for dengue, vector control is the main strategy to prevent or contain disease outbreaks. Many synthetic insecticides such as organochlorides, organophosphates and carbamates have been used to control A. aegypti, but the toxicity problem, together with the growing incidence of insect resistance (8,9), has called attention to the necessity for novel insecticides.

The demand for new natural larvicides has increased and plant essentials oils are a source of potential larvicides (6,10-16) because they are, in some cases, highly active, economically viable, and biodegradable (16).

As part of our program to evaluate essential oils from northeastern Brazilian flora, this work reports the composition and larvicidal activity of the essential oil from the leaves of B. acuruana against Aedes aegypti.

Experimental

Plant material: Leaves of B. acuruana were collected in May 2008 in Tianguá County, State of Ceará, northeast Brazil.

B. acuruana


A voucher specimem (#EAC42405) has been deposited at the Herbário Prisco Bezerra, Departamento de Biologia, Univer-sidade Federal do Ceará, Ceará, Brazil.

The fresh leaves of B. acuruana were subjected to hydro-distillation in a Clevenger-type apparatus for 2 h to afford a pale yellow oil. The isolated oil, after drying over anhydrous sodium sulfate and filtration, was stored in sealed glass vials and maintained under refrigeration until further analysis. The yield (w/w) was calculated based on the fresh weight of the leaves.

Analysis of the essential oils: The essential oil was analyzed by GC/MS on a Shimadzu QP5050A (Shimadzu Corporation, Kyoto, Japan) system equipped with a AOC-20i autosampler

Table I. Chemical composition (%) of essential oil from the leaves of Bauhinia acuruana.

Peak Compound RI1 RI Lit (18) Percentage Identification

1 d-Elemene 1334 1338 0.3 ± 0.01 RI, MS2 a-Copaene 1375 1376 0.4 ± 0.01 RI, MS3 b-Elemene 1388 1390 2.3 ± 0.03 RI, MS4 cis-a-Bergamotene 1412 1412 0.3 ± 0.01 RI, MS5 b-Caryophyllene 1418 1419 1.5 ± 0.01 RI, MS6 b-Copaene 1429 1432 0.2 ± 0.01 RI, MS7 trans-a-Bergamotene 1432 1434 0.8 ± 0.01 RI, MS8 (E)-b-Farnesene 1452 1456 0.4 ± 0.03 RI, MS9 a-Humulene 1454 1454 0.6 ± 0.00 RI, MS10 allo-Aromadendrene 1458 1460 0.7 ± 0.00 RI, MS11 Germacrene D 1480 1485 0.7 ± 0.02 RI, MS12 b-Selinene 1487 1490 0.4 ± 0.05 RI, MS14 Bicyclogermacrene 1493 1500 0.7 ± 0.01 RI, MS15 g-Cadinene 1511 1513 0.8 ± 0.00 RI, MS16 b-Sesquiphellandrene 1523 1522 1.8 ± 0.02 RI, MS17 Elemol 1547 1549 0.1 ± 0.01 RI, MS18 Germacrene B 1557 1561 0.7 ± 0.01 RI, MS20 (E)-Nerolidol 1560 1563 2.4 ± 0.03 RI, MS22 Spathulenol 1576 1578 23.4 ± 0.08 RI, MS24 Caryophyllene oxide 1581 1583 16.4 ± 0.04 RI, MS26 Globulol 1585 1590 2.4 ± 0.02 RI, MS27 Viridiflorol 1593 1592 1.6 ± 0.01 RI, MS29 M 220 1603 - 1.0 ± 0.01 -31 Humulene epoxide II 1609 1608 2.2 ± 0.02 RI, MS32 M 220 1627 - 1.2 ± 0.00 -33 g-Eudesmol 1631 1632 1.0 ± 0.01 RI, MS34 epi-a-Cadinol 1641 1640 20.7 ± 0.12 RI, MS35 a-Muurolol 1646 1646 0.4 ± 0.04 RI, MS37 Valerianol 1654 1658 5.7 ± 0.09 RI, MS38 M 220 1658 - 1.4 ± 0.07 -39 trans-Calamenen-10-ol 1664 1669 0.5 ± 0.03 RI, MS40 14-Hydroxy-9-epi(E)-caryophyllene 1668 1668 0.6 ± 0.00 RI, MS41 M 222 1674 - 0.4 ± 0.01 -42 2,3-Dihydrofarnesol 1686 1689 1.4 ± 0.01 RI, MS43 Unidentified 1739 - 0.4 ± 0.08 -44 M 222 1749 - 1.1 ± 0.21 -45 Unidentified 1844 - 0.5 ± 0.02 -46 Unidentified 2057 - 1.2 ± 0.03 - Sesquiterpene hydrocarbons - 12.6 Oxygenated sesquiterpenes - 78.8

Total - 91.4

1RI: Relative retention index calculated against n-alkanes applying the Van den Dool & Kratz (1963) equation. RI (17). m/z (rel. int.): RI=1603, 220[M+] (5), 204(28), 189(24), 177(12), 161(49), 147(40), 133(36), 119(42), 107(76), 105(85), 91(74), 79(49), 69(41), 55(52), 41(100); RI =1627, m/z (rel. int.): 220[M+] (1), 202(36), 187(25), 183(20), 159(100), 145(40), 131(56), 119(49), 117(34), 105(46), 91(43), 41(60); RI=1658, m/z (rel. int.): 220[M+] (7), 204(44), 189(60), 175(16), 159(65), 147(35), 133(46), 119(40), 105(66), 93(62), 81(66), 67(42), 55(61), 41(100); RI=1674, m/z (rel. int.): 222[M+] (10), 202(24), 187(26), 159(50), 145(38), 131(32), 125(43), 119(37), 107(49), 105(49), 93(43), 91(55), 79(41), 67(40), 55(41), 41(100); RI=1749, m/z (rel. int.): 222[M+] (5), 202(17), 200(18), 185(13), 177(24), 159(76), 143(27), 131(26), 117(22), 105(23), 91(47), 79(17), 65(13), 55(18), 43(100).

under the following conditions: J&W Scientific DB-5MS fused silica capillary column (30 m x 0.25 mm i.d., x 0.25 mm film thickness, composed of 5% phenyl/95% methylpolysiloxane) operating in EI mode at 70 eV. Helium (99.999%) was used as the carrier gas at a constant flow of 1.2 mL/min. The injection volume was 0.5 μL (split ratio of 1:100), the injector tempera-ture 250oC and the ion-source temperature 280oC. The oven temperature was programmed from 50oC (isothermal for 1.5 min), with an increase of 4oC/min to 200oC, then 10oC/min to 300oC, ending with a 10 min isothermal period at 300oC. Mass spectra were taken at 70 eV with a scan interval of 0.5 s and fragments from 40 to 500 Da.

da Silva Gois et al.


91.4% of the essential oil. Its chemical composition, includ-ing retention index (RI) values listed in order of elution from the DB-5MS column, and the percentage relative to each constituent, are presented in Table I. The GC chromatogram with peak identification of essential oil from the leaves of B. acuruana is presented in Figure 1.

The major components of this essential oil were spathulenol (23.4 ± 0.08%), epi-a-cadinol (20.7 ± 0.12%) and caryophyllene oxide (16.4 ± 0.04%). Other minor constituents were found to be valerianol (5.7 ± 0.09%), (E)-nerolidol (2.4 ± 0.03%), globulol (2.4 ± 0.02%), b-elemene (2.3 ± 0.03%) and humulene epoxide II (2.2 ± 0.02%).

The large abundance of sesquiterpenoid compounds in the essential oil from leaves of B. acuruana is in accordance with findings about the chemical composition of the essential oils from leaves of B. aculeata (20), B. brevipes (20), B. longifolia (20), B. pentandra (20), B. rufa (20), B. variegata (20), B. forficata (20, 21) and B. ungulata (22). Thus, it seems that the occurrence of sesquiterpenes as the predominant constituents in the essential oil from leaves of Bauhinia species is a chemical characteristic of this genus (20).

Furthermore, the larvicidal potential of the essential was evaluated against A. aegypti larvae and exhibited an LC50 value

of 56.2 ± 0.4 mg/mL. Several studies have shown that sesquiter-penoid compounds possess significant larvicidal activities (15, 23, 24), and that essential oils containing sphathulenol exhibit properties against the larvae of Aedes aegypti (15, 25).

This is the first report on the chemical composition and larvicidal activity against A. aegypti of essential oil from B.

Figure 1. GC chromatogram of essential oil from the leaves of Bauhinia acuruana.

Quantitative analysis of the chemical constituents was per-formed by flame ionization gas chromatography (FID), using a Shimadzu GC-17A (Shimadzu Corporation, Kyoto, Japan) instrument, under the following operational conditions: capillary ZB-5M5 column (5% phenyl-arylene/95% methylpolysiloxane fused silica capillary column 30 m x 0.25 mm i.d. x 0.25 mm film thickness), under the same conditions reported for the GC/MS. Quantification of each constituent was estimated by area normalization (%). Compound concentrations were calculated from the GC peak areas and they were arranged in order of GC elution. Three independent analyses were carried out, and data on average value and standard deviation were calculated.

Identification of individual components of the essential oils was performed by computerized matching of the acquired MS with those stored in NIST21 and NIST22 mass spectral library of the GC/MS data system. Retention indices (RI) for all compounds were determined according to literature (17) for each constituent, as previously described (18).

Larvicidal bioassay: Aliquots of the essential oils tested (12.5-500 mg/mL) were placed in a beaker (50 mL) and dis-solved in DMSO/H2O 1.5% (20 mL). Fifty instar III larvae of Aedes aegypti were delivered to each beaker. After 24 h at room temperature, the number of dead larvae was counted and the lethal percentage calculated. A control using DMSO/H2O 1.5% was carried out in parallel. For each sample, three independent experiments were run (19).


The essential oil yield was 0.01%. A total of 30 compounds were identified, all of which were sesquiterpenes, representing

B. acuruana


acuruana and the findings of the present study indicate this essential oil as a potential natural larvicide, effective in the control of the A. aegypti.

Acknowledgements

The authors thank the Brazilian agencies CNPq, CAPES, FUNCAP, PRONEX for fellowships and financial support, and Laboratório de Entomologia, Núcleo de Endemias da Secretaria de Saúde do Estado do Ceará, Brazil, where the bioassays were performed.

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V.S.S. Dharmagadda, S.N. Naik, P.K. Mittal and P. Vasudevan, 9. Larvicidal activity of Tagetes patula essential oil against three mosquito species. Biores. Technol., 96, 1235-1240 (2005).

E.C.C. Araújo, E.R. Silveira, M.A.S. Lima, M. Andrade-Neto, I.L. 10. de Andrade, M.A.A. Lima, G.M.P. Santiago and A.L.M. Mesquita, Insecticidal activity and chemical composition of volatile oils from Hyptis martiusii Benth. J. Agric. Food Chem., 51, 3760-3762 (2003).

M.R.J.R. Albuquerque, E.R. Silveira, D.E.A. Uchoa, T.L.G. Lemos, E.B. 11. Souza, G.M.P. Santiago and O.D.L. Pessoa, Chemical composition and larvicidal activity of the essential oils from Eupatorium betonicaeforme (D.C.) Baker (Asteraceae). J. Agric. Food Chem., 52, 6708-6711 (2004).

G.M.P. Santiago, T.L.G. Lemos, O.D.L. Pessoa, A.M.C. Arriaga, F.J.A. 12. Matos, M.A.S. Lima, H.S. Santos, M.C.L. Lima, F.G. Barbosa, J.H.S. Luciano, E.R. Silveira and G.H.A. de Menezes, Larvicidal activity against Aedes aegypti L. (Diptera : Culicidae) of essential oils of Lippia species from Brazil. Nat. Prod. Commun., 1, 573-576 (2006).

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da Silva Gois et al.


Rec: Dec 2010

Acc: Mar 2011

Chemical Composition and Biological Properties of the Leaf Essential Oil of Tagetes lucida Cav. from

Cuba

Erik L. Regalado and Miguel D. FernándezCenter of Marine Bioproducts, Loma y 37, Havana, C.P. 10400, Cuba

Jorge A. Pino*Food Industry Research Institute, Carretera al Guatao km 3½, Havana, C.P. 19200, Cuba

Judith Mendiola Institute of Tropical Medicine “Pedro Kourí”, P.O. Box 601, Havana, Cuba

Olga A. Echemendia Institute Finlay. Ave 27 319805, Havana, C.P.11600, Cuba

Abstract

The leaf essential oil of Tagetes lucida Cav. (Asteraceae) from Cuba has been obtained by hydrodistillation and analyzed by GC-FID and GC/MS. Forty volatile compounds were identified, of which estragole (96.8%) was the major constituent. The antioxidant capacity of this essential oil was measured by two different in vitro assays (DPPH and TBARS) and significant activities were evidenced. The preliminary screening of its antiplasmodial, antibacterial, antifungal and antiviral activities was carried out against Plasmodium berghei, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, Acinetobacter lwoffi, Enterobacter aerogenes and against strains HHV 1 and HHV 2. The results showed a moderate activity against P. berghei and E. coli.

Key Word Index

Tagetes lucida, Asteraceae, essential oil composition, estragole, antioxidant capacity, antiplasmodial activity, an-timicrobial activity, antiviral activity.



Introduction

Tagetes lucida Cav. (syn. T. florida Sweet, T. schiedeana Less.), commonly called Pericón, hierbanís, anís, santa María, Mexican mint marigold, Mexican tarragon, Spanish tarragon, or Texas tarragon, is a perennial herb that grows in dry rocky slopes and woods native to Central America and South America and naturalized elsewhere in the tropics and subtropics (1, 2). It is cultivated commercially in Costa Rica as a spice herb; it contains an essential oil having an anise-like odor, and the fresh aerial parts of this plant are sold in the supermarket as a substitute of tarragon (3), which have been very used as spice and to preserve meat (4). This species has been cultured in Cuba in gardens due to the beauty of its foliage, but it is not common its exploitation for medicinal aims.

Tagetes lucida has been referred in Mexican traditional medicine for different therapeutic applications. The infusion of leaves and flowers is drunk to combat diarrhea, rheumatism, asthma, and cold (5, 6). The decoction of the aerial parts is

employed in the treatment of amoebic dysentery, giardiasis, ascaridiasis and other infections caused by helminthes (6). Moreover, a bibliographic survey of plants for malaria in Latin America (2) reported the use of the dried powdered plant or the plant decoction for the treatment of malaria in Mexico.

Essential oils from aromatic and medicinal plants have been known to possess important biological properties, nota-bly antibacterial, antifungal and antioxidant activities. Their biological potential depends on their chemical composition determined by genotype and influenced by environmental and agronomic conditions (7). The chemical composition of T. lucida volatile oil has been the subject of previous studies. The major constituents of this volatile oil were determined to be methyl eugenol (80%) and estragole (12%) in México (8); estragole (45%) and methyl eugenol (20%) in Hungary (9); anethole (23.8%), eugenol (24.3%) and estragole (33.9%) in Guatemala (10) or estragole (95-97%) in Costa Rica (3). T. lucida extracts have reported to act on bacteria and phytopathogenic fungi (5) and also possesses antidepressant-like properties in rats (11).

T. lucida


Moreover, considering both chemical composition and biomass yields, T. lucida appeared to be a promising species, with high potential for use as biocidal crops for the implementation of pest control practices (12).

In spite of the worldwide use of T. lucida in the folk medicine, the biological properties of its essential oil based on experimental models have remained largely unexplored. In this context, the present work describes a detail chemical composition and examines the antiplasmodial, antibacterial, antifungal and antiviral activities of the essential oil isolated from the leaves of Tagetes lucida Cav. from Cuba.

Experimental

Plant material: Leaves of T. lucida were collected in February 2010, in the medicinal plants field of the Food Industry Research Institute in Havana, Cuba. The plant was identified by Dr. Pedro Herrera of the Institute of Ecology and Systematic (IES) and a voucher specimen was deposited at the Herbarium of IES (HAC 44100). Leaves (200 g) were submitted to hydrodistillation in a Clevenger-type apparatus for 2 h. At the end of each distillation the oils were collected, dried with anhydrous Na2SO4, measured, and transferred to glass flasks that were filled to the top and kept at a temperature of −18°C for further analysis. Analyses were made by duplicate. Yields were calculated according to the weights of oils and plant material before distillation.

Analysis of the essential oils: Oil sample analyses were performed on A Konik 4000A instrument (Barcelona, Spain) equipped with a HP-5ms fused silica column (25 m x 0.25 mm i.d., film thickness 0.25 μm), split injection 1:10, and flame ionization detection. Injector and detector temperatures were at 220ºC and 250ºC. The oven temperature was held at 70ºC for 2 min and then raised to 250ºC at 4ºC/min and held for 10 min. The carrier gas was H2 at 1 mL/min. Samples were injected by splitting and the split ratio was 1:20. The lineal retention indices (RI) were obtained from GC by logarithmic interpolation between bracketing a homologous series of n-alkanes used as standards. Peak areas were measured by electronic integration using the EZChrom Chromatography Data System 6.07 program (Scientific Software, Inc., FL). The relative amount of the individual components was based on the peak areas.

GC/MS analysis was performed on a Shimadzu 17A (Tokyo, Japan) gas chromatograph coupled to a Shimadzu QP-5000 high performance quadrupole mass selective detector was used. The GC was fitted with a HP-5ms fused silica column (25 m x 0.25 mm i.d., film thickness 0.25 μm). The GC operating conditions were identical with those described above except that He was used as carrier gas. The MS operating conditions were: ionization potential 70 eV with scan mass range of 35-400 m/z and ion source temperature at 250ºC. Compounds were identified by computer search using digital libraries of mass spectral data (NIST 02, Wiley 275, Adams 2001, Palisade 600, and Flavorlib homemade library) and by comparison of their retention indices of either reference substances or literature values (13), relative to C8-C32 n-alkane series in a temperature-programmed run.

2,2-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scav-

enging Assay: The antioxidant activity of the essential oil was measured in terms of free-radical scavenging ability according to DPPH reported method (14) with minor modifications. Basically, a 60-μM methanolic solution of DPPH (980 μL) [Sigma-Aldrich Co. (St. Louis, MO)], prepared daily, was placed in a spectrophotometer cuvette, and eight concentrations of the essential oil of 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0 and 3.0 mg/mL or ascorbic acid (standard) (0.16, 0.26, 0.6, 1.0 and 1.30 mg/mL) in methanol (v/v) solution (20 μL) were added. The decrease in absorbance at 515 nm was determined in a UV-1201 spectrophotometer, until the reaction plateau step was reached. Methanol was used to zero the spectrophotometer. EC50 values were determined from the plotted graph of scav-enging activity against sample concentrations, which is defined as the total antioxidant necessary to decrease the initial DPPH radical concentration by 50%. Triplicate measurements were carried out, and their scavenging effect was calculated based on the percentage of DPPH scavenged.

TBARS (thiobarbituric acid reactive species) as-say: The lipid peroxidation assay as TBARS was carried out according to a modified method (15). The reaction mixture containing, in a final volume of 1.1 mL, 100 mL of cerebral tissue (whole brain) and 1 mL (0.05M) of KH2PO4/K2HPO4 buffer, pH 7.4 in NaCl (0.9%), and seven concentrations of the essential oil (20, 50, 100, 150, 200, 250 and 500 μg/mL) was incubated at 37°C for 1 h. Then, 1 ml of thiobarbituric acid (0.5%) and 1 mL of acetic acid (20%) were added to the test tubes and were incubated at 100°C for 60 min. After cooling, absorbance was measured at 532 nm against control and buffer, BHT being used as reference compound. All the experiments were performed in triplicate, and the results were averaged. The inhibition percentage was determined by comparison of the results between the samples and control.

Antimalarial assay: In vitro drug susceptibility was de-termined in the standard short-term cultures of Plasmodium berghei ANKA blood stages as described before (16). Briefly, erythrocytes infected with parasites of P. berghei ring forms/young trophozoites are incubated at 2% parasitemia at a final cell concentration of 1% in complete culture medium (RPMI 1640 with 20% Fetal Calf Serum, Sigma, St. Louis, MO) containing serial dilutions of essential oil maximal concentration tested, from 200 μg/mL to 12.5 μg/mL, each in duplicate wells of 96-well culture plates. These plates are incubated for a period of 24 h at 37°C under standardized in vitro culture conditions. The antimalarial activity was expressed as inhibitory concentration 50 (IC50), defined as the concentration of the volatile oil that induces 50% reduction of production of schizonts, which was calculated according to reported methodologies (17). IC50 was expressed as mean ± standard deviation of tests performed in two different assays. Chloroquine diphosphate and artemisinin (Sigma, St. Louis, MO) were used as references.

Antibacterial activity: The antibacterial activity was car-ried out by the diffusion method according to the National Com-mittee for Clinical Laboratory Standard Guideliness (NCCLS) (18) and evaluated against several bacterial reference strains: Staphylococcus aureus (ATCC 6538), Pseudomona aeruginosa (ATCC 9027), Escherichia coli (10576), and Candida albicans (ATCC10231) and strains from clinical samples: Pseudomona aeruginosa, E. coli, Acinetobacter lwoffi, and Enterobacter

Regalado et al.


Figure 1. Total ion chromatogram of Tagetes lucida leaf essential oil.

aerogenes. All the suspensions of microorganism were adjusted 0.5 MacFarland and six concentrations of the essential oil were tested. The plates were incubated to 37ºC in a humidified atmosphere, containing 5% CO2 for 24 h.

Cytotoxicity assay: Vero line was grown (37°C, 5% CO2) in 96-well culture plates, in 199 medium, supplemented with 10% fetal calf serum. Confluent monolayers were incubated 3 days with each dilution of the essential oil. All experiments were performed in triplicate. The effect of samples on cell viability was measured using the Naftol Blue Black (NBB) method (19).

Antiviral activity: The antiviral activity was evaluated against strains HHV 1 and HHV 2 and performed in 96-well flat bottom tissue culture plates. Different dilutions of the essential oil were added to confluent monolayer of Vero cell. After 1 hour of incubation at 37°C in a 5% CO2, the virus suspensions were added. All plates were further incubated at 37°C in a 5% CO2 atmosphere for 3 days.


A yield of 0.79% (v/m) of leaf essential oil was obtained by hydrodistillation of the fresh leaves of T. lucida.

A gas chromatogram of the volatile compounds of the leaf oil is shown in Figure 1, indicating the presence of 40 components, all of them were identified by comparing and matching the mass spectra and GC retention index of the unknown compounds with those of reference.

Table I lists the volatile components identified in the leaf oil. Sixteen compounds are reported for the first time. Estragole (96.8%) dominated the leaf oil composition. The pleasant sweet odor of T. lucida leaves is assumed to be caused by the anise-like smelling estragole. Among all molecules identified, the other quantified components included myrcene, germacrene D, (E)-b-ocimene, linalool, ß-caryophyllene and (E,E)-a-farnesene.

Previous studies on leaf oils classified T. lucida into different chemotypes (3, 9, 10) on the basis of the main constituents. The leaf oil of T. lucida from Cuba could therefore be classified as the estragole high content type, similar to the species reported in Costa Rica (3). A minor difference with the Costa Rican leaf oil is that other phenylpropanoids such as (E)-anethole and eugenol were found in trace in the Cuban leaf oil and they were not detected in Costa Rican leaf oil.

To establish the antioxidant activity of this essential oil, we used two well-established in vitro assays. The first is based on the free-radical-scavenging capacity of the stable DPPH radi-cal and the second concerns the spectrophotometric detection of TBARS, namely being malonaldehyde (MDA), one of the secondary lipid peroxidation products, whose quantification gives a measure of the extent of lipid degradation.

For the first assay, solutions with eight volatile-oil concen-trations of 0.1–3 mg/mL, and different doses of ascorbic acid (positive control) were prepared to evaluate the DPPH radical-scavenging capacity. The respective scavenging capacities ranged from 10.2 ± 0.3% to 85.4 ± 1.1% with an EC50 value of 1.4 ± 0.3 mg/mL for the essential oil and 0.025 ± 0.004 mg/mL for ascorbic acid. For the second test, seven different concentration of volatile oil (20–500 μg/mL) and BHT as positive control, also showed antioxidant activities in a dose dependent manner and had 14.04 ± 0.09% to 96.26 ± 0.05% inhibition on lipid peroxidation. The IC50 value was found to be 0.23 ± 0.03 mg/mL for the essential oil and 0.18 ± 0.04 μg/mL for BHT.

These results were in agreement with published data, which demonstrated that estragole is a weak DPPH radical scavenger (IC50 > 400 μM) (20). However, several investigations have demonstrated that some essential oils (estragole chemotype) exhibit antioxidant potential (15, 16). At the same time, the other significant component of this volatile oil, myrcene (2.3%) and a minor one (linalool, 0.1%) have been previously found to possess substantial protective effect against oxidant induced genotoxicity, which is predominately mediated by their radi-

T. lucida


Table I. Chemical composition of Tagetes lucida leaf essential oil

Peak Nr. Compound RIE1 RIR %

1 ethyl 2-methylbutanoate 851 852 tr2 (Z)-3-hexenol 859 860 tr3 myrcene 991 992 2.3 ± 0.044 (Z)-3-hexenyl acetate 1005 1007 tr5 (Z)-b-ocimene 1037 1038 tr6 (E)-b-ocimene 1050 1048 0.2 ± 0.017 linalool 1097 1097 0.1 ± 0.018 estragole 1194 1196 96.8 ± 1.19 carvone* 1243 1243 tr10 chavicol 1250 1252 tr11 (E)-anethole 1285 1287 tr

12 a-cubebene 1350 1351 tr13 eugenol* 1359 1359 tr

14 b-bourbonene* 1385 1388 tr

15 b-elemene 1391 1393 tr

16 b-caryophyllene 1419 1419 0.1 ± 0.01

17 b-copaene* 1432 1432 tr18 trans-a-bergamotene 1435 1435 tr19 aromadendrene* 1441 1441 tr

20 (E)-b-farnesene 1456 1457 tr21 germacrene D 1481 1485 0.3 ± 0.02

22 (Z,E)-a-farnesene* 1490 1491 tr23 bicyclogermacrene 1500 1499 tr24 a-muurolene 1502 1500 tr

25 (E,E)-a-farnesene 1505 1505 0.1 ± 0.0126 d-cadinene 1523 1526 tr27 elemol 1550 1550 tr28 1,10-di-epi-cubenol* 1619 1619 tr29 epi-a-muurolol* 1642 1642 tr

30 a-muurolol* 1646 1646 tr

31 a-cadinol 1654 1656 tr32 14-hydroxy-9-epi-(E)-caryophyllene* 1670 1670 tr33 n-octadecane* 1800 1800 tr34 hexahydrofarnesylacetone* 1840 1844 tr35 n-nonadecane* 1900 1900 tr36 n-eicosane* 2000 2000 tr37 n-heneicosane 2100 2100 tr38 phytol 2112 2115 tr39 n-docosane* 2200 2200 tr

40 n-tricosane* 2300 2300 tr

*Reported for the first time in this essential oil; 1RIE and RIR: Experimental and reference retention index; Trace constituent (< 0.1%)

cal scavenging activity (21). In this context, the weak (DPPH) and moderate (TBARS) antioxidant effects of T. lucida es-sential oil could be attributed in a great part to these volatile metabolites.

T. lucida essential oil exhibited a moderate antimalarial activity, with IC50 value equal to 72 ± 3.62 μg/mL, which was evaluated following recommended endpoint criteria for natural complex mixtures (22). IC50 values for chloroquine (30.92 ng/mL) and artemisinin (18.3 ± 4.48 ng/mL) were significantly lower than this obtained for the oil. In addition, they closely corresponded with previously reported IC50 values of P. berghei ANKA (23). Previous studies have underlined the potential biological activities of various essential oils against malaria parasites. At earliest research, eight essential oils were tested in vitro against P. falciparum and displayed IC50 values ranging

149-1000 μg/mL (24), while more recently, other five essential oils (Xilopia phloiodora, Pachypodanthium confine, Antidesma laciniatum, Xylopia aethiopica, Hexalobus crispiflorus) (25) with IC50 values ranging 2-30 μg/mL were considered very active. Essential oils from fresh leaves of Cymbopogon citratus and Ocimum gratissimum growing in Cameroon showed significant antimalarial activities in the four-day suppressive in vivo test in mice infected with Plasmodium berghei, at concentrations of 200, 300 and 500 mg/kg of mouse weight per day (26). In these reports, no clear conclusions can be derived from the chemical composition of the tested essential oils in association with their antimalarial activity.

The antioxidant activity of T. lucida essential oil is an inter-esting biological property. Endothelial cell injury by adherent parasitized red blood cells is ameliorated by superoxide dis-

Regalado et al.


mutase, ascorbic acid, or tocopherol in vitro, highlighting the potential therapeutic benefit of antioxidants for severe malaria. At present, it is difficult to predict what will be their effect on malaria disease, but future strategies might specifically target antioxidants to the endothelium while keeping or enhancing oxidative stress in infected RBC (27). It will depend on the distribution of the essential oil in the cells (28).

To the best of our knowledge, only extracts of flowers of T. erecta in ethanol/water and methanol/water solvent mixtures were evaluated in the in vitro antimalarial screening and against P. berghei in mice, which gave negative results (29), so the moderate antimalarial activity exhibited by T. lucida essential oil should be further explored.

Additionally, T. lucida essential oil only showed a moderate antibacterial activity against E. coli (10576) using the diffusion method (18). This essential oil was not cytotoxic from 103 dilutions and did not show antiviral activity with strains HHV 1 and HHV 2. Estragol-rich essential oils are reputed for their antifungal properties (30), but have also exhibited significant activity against bacterial strains (31), including against E. coli (32). The antibacterial properties of T. lucida extracts have been previously demonstrated where some isolated coumarins were the most effective compounds against Gram-negative and Gram-positive bacteria (5). However, we report for the first time the activity of its essential oil against E. coli.

Acknowledgements

The authors thank Pedro Herrera for the identification of the species.

References

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W. Milliken,2. Plants for malaria. Plants for fever. Medicinal species in Latin America- a bibliographic survey. The Royal Botanic Gardens, Kew; United Kingdom (1997).

J. F. Cicció, 3. A source of almost pure methyl chavicol: volatile oil from the aerial parts of Tagetes lucida (Asteraceae) cultivated in Costa Rica. Rev. Biol. Trop. (Int. J. Trop. Biol.), 52, 853-857 (2004).

L. Kershaw, 4. Edible & Medicinal Plants of the Rockies. Lone Pine, Edmonton, Canada (2000).

C. L. Céspedes, J. G. Avila, A. Martínez, B. Serrato, J. A. Calderón-5. Mugica and R. Salgado-Gaeciglia, Antifungal and antibacterial activities of Mexican tarragon (Tagetes lucida), J. Agric. Food Chem., 54, 3521-3527 (2006).

C. Márquez, O. F. Lara, R. B. Esquivel and E. R. Mata, 6. Plantas Medicinales de México II. Composición, Usos y Actividad Biológica. Universidad Nacional Autónoma de México, México, 123 (1999).

F. Bakkali, S. Averbeck, D. Averbeck and M. Idaomar, 7. Biological effects of essential oils – A review. Food and Chemical Toxicology, 46, 446–475 (2008).

A. Guzmán and A. Manjarrez, 8. Estudio del aceite esencial de Tagetes florida. Bol. Inst. Quím. Univ. Nac. Autón. Méx., 14, 48-54 (1962).

E. Hethelyi, B. Danos, P. Tetenyi and G. Juhasz, 9. Phytochemical studies on Tagetes species; infraspecific differences of the essential oil in T. minuta and T. tenuifolia. Herba Hungarica, 26, 145-158 (1987).

C. Bicchi, M. Fresia, P. Rubiolo, D. Monti, C. Franz and I. Goehler, 10. Constituents of Tagetes lucida Cav. ssp. lucida Essential Oil. Flavour Fragr. J., 12, 47-52 (1997).

G. Guadarrama-Cruz, F. J. Alarcon-Aguilar, R. Lezama-Velasco, G. 11. Vazquez-Palacios and H. Bonilla-Jaime, Antidepressant-like effects of Tagetes lucida Cav. in the forced swimming test. J. Ethnopharmacol., 120, 277-281 (2008).

I. Marotti, M. Marotti, R. Piccaglia, A. Nastri, S. Grandi and G. Dinelli, 12. Thiophene occurrence in different Tagetes species: agricultural biomasses as sources of biocidal substances. J. Sci. Food Agric., 90, 1210-1217 (2009).


W. Brand-Williams, M. E. Cuvelier and C. Berset, 14. Use of a free radical method to evaluate antioxidant activity. Food Sci. Technol., 28, 25-30 (1995).

H. Ohkawa, N. Ohishi and K. Yagi, 15. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem., 95, 351-358 (1979).

C. J. Janse and A. P. Waters, Plasmodium berghei:16. the application of cultivation and purification techniques to molecular studies of malaria parasites. Parasitol. Today, 11, 138-143 (1995).

M. Schlichtherle, M. Wahlgren, H. Perlmann and A. Scherf, 17. Methods in malaria research, Malaria Research and Reference Reagent Resource Center, Virginia (2000).

P.A. Wayne, National Committee for Clinical Laboratory Standards. 18. Performance standards for antimicrobial disk susceptibility test. Approved Standards MZ-8, Vol 25, NCCLS (2004).

E. A. Gould and J. C. S. Clegg, 19. Growth, titration and purification of alphaviruses and flaviviruses. In: Virology a practical approach, Fifth edition. Edit. B. W. J. Mahy, IRL Press-Oxford University, London, 76 (1985).

H. Tominaga, Y. Kobayashi, T. Goto, K. Kasemura and M. Nomura, 20. DPPH radical-scavenging effect of several phenylpropanoid compounds and their glycoside derivatives. Yakugaku Zasshi 125 (4), 371-375 (2005).

D. Mitić-ćulafić, B. Žegura, B. Nikolić, B. Vuković-Gaćić, J. Knežević-21. Vukćević and M. Filipić, Protective effect of linalool, myrcene and eucalyptol against t-butyl hydroperoxide induced genotoxicity in bacteria and cultured human cells. Food Chem. Toxicol, 47, 260-266 (2009).

P. Cos, A. J. Vlietinck, D. Vanden Berghe and L. Maes, 22. Anti-infective potential of natural products: How to develop a stronger in vitro “proof-of-concept”. J. Ethnopharmacol. 2006, 106, 290-302 (2006).

B. Franke-Fayard, D. Djokovic, M. W. Dooren, J. Ramesar, A. P. 23. Waters, M. O. Falade, M. Kranendonk, A. Martinelli, P. Cravo and C. J. Janse, Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites. Int. J. Parasitol., 38, 1651-1662 (2008).

G. Milhau, A. Valentin, F. Benoit, M. Mallie, J. Bastide, Y. Pelissier 24. and J. Bessiere, In vitro antimicrobial activity of eight essential oils. J. Essent. Oil Res., 9, 329-333 (1997).

F. F. Boyom, V. Ngouana, P. H. A. Zollob, C. Menut, J. M. Bessiere, J. 25. Gut and P. J. Rosenthal, Composition and anti-plasmodial activities of essential oils from some Cameroonian medicinal plants. Phytochem., 64, 1269-1275 (2003).

F. Tchoumbougnang, P. H. Amvam Zollo, E. Dagne and Y. Mekonnen, 26. In vivo antimalarial activity of essential oils from Cymbopogon citratus and Ocimum gratissimum on mice infected with Plasmodium berghei. Planta Med., 71, 20-23 (2005).

H. C. Ackerman, S. D. Beaudry and R. M. Fairhurst, 27. Antioxidant therapy: Reducing malaria severity? Crit. Care Med., 37, 758-760 (2009).

F. Bakkali, S. Averbeck, D. Averbeck and M. Idaomar, 28. Biological effects of essential oils – A review. Food Chem. Toxicol., 46, 446-475 (2008).

M. Misra, R. E. Rodriguez, S. L. North and K. S. Kasprzak, 29. Nickel-induced renal lipid peroxidation in different strains of mice: concurrence with nickel effect on antioxidant defense systems. Toxicol. Lett., 58, 121-133 (1991).

S. Shin and C. A. Kang,30. Antifungal activity of the essential oil of Agastache rugosa Kuntze and its synergism with ketoconazole. Lett. Appl. Microbiol., 36, 111-115 (2003).

K. Carović-Stanko, S. Orlić, O. Politeo, F. Strikić, I. Kolak, M. Milos 31. and Z. Satovic, Composition and antibacterial activities of essential oils of seven Ocimum taxa. Food Chem., 119, 196-201 (2010).

A. Guerrini, G. Sacchetti, M. Muzzoli, G. Moreno Rueda, A. Medici, 32. E. Besco and R. Bruni, Composition of the volatile fraction of Ocotea bofo Kunth (Lauraceae) Calyces by GC-MS and NMR fingerprinting and its antimicrobial and antioxidant activity. J. Agric. Food Chem., 54, 7778-7788 (2006).

T. lucida


Rec: Dec 2010

Acc: Jan 2011

Analytical Characterization of Industrial Essential Oils from Fruits and Leaves of C. aurantifolia Tan.

and C. latifolia Swing.

Ivana Bonaccorsi*, Paola Dugo, Luigi Mondello, Danilo Sciarrone and Giovanni DugoDipartimento Farmaco-chimico, University of Messina, V.le annunziata -98168-Messina, Italy

Luis Haro-GuzmanJosè Vasconcelos, 105, Col. Jard. Vista Hermosa, 28010 Colima Col., Mexico

Abstract

The physicochemical indices, the composition of the volatile fraction, the enantiomeric ratios of some volatile components and the oxygen heterocyclic fraction of cold-pressed Key lime oils (types A and B), Persian lime oils, and petitgrain lime oils are reported. The volatile fraction of cold-pressed Persian lime oil is characterized by a higher content of limonene, g-terpinene and esters and a lower content of b-pinene, sesquiterpene hydrocarbons, alcohols and aldehydes than cold-pressed Key lime oils. In petitgrain oils the oxygenated compounds are present at levels higher than the peel oils. Oxypeucedanin, probably due to the extraction technology, was almost absent in cold-pressed Key lime type A, while it is present in cold-pressed Key lime type B and in Persian lime oil. The en-separation was performed by direct enantioselective GC (esGC) and by multidimensional GC (MDGC) to obtain the most appropriate antiomeric separation of all the components analyzed. The enantiomeric excess of S-(-)-a-pinene, 1S,4R-(-)-camphene, S-(-)-b-pinene, S-(-)-sabinene, and R-(-)-b-phellandrene are lower in cold-pressed Persian lime oil than in Key lime oils.

Key Word Index

C. aurantifolia Tan., C. latifolia Swing., peel and leaf lime oils, volatiles, non-volatiles, enantiomeric ratios, esGC, GC/MS, MDGC, HPLC.



Introduction

Lime essential oil is known as one of the most complex citrus essential oils, difficult to characterize for a series of variables (variety, extraction method, geographic origin) and it is extremely appreciated in the food and beverage market. Usually Key lime (C. aurantifolia Tanaka), also known as acid lime or Mexican lime, is the most appreciated, although Persian lime (C. latifolia Swingle) is commonly used in transformation industry.

From the Key lime tree it is possible to obtain the following products: Key lime oil type A (cold-pressed by screw press from the whole fruits, then separated by centrifuge); distilled lime oil (obtained by steam distillation of the juice/oil emulsion); Key lime type B (cold-extracted by common extractor/roller machines which rasp the peel to release the oil. The oil is never in contact with the acid juice); Key lime petitgrain (obtained from the young green parts of the tree by distillation).

According to the “world map showing production centers of essential oils” created by Treat PLC, attached to the very

recent volume edited by Baser and Buckbauer (1) the main producer countries of lime oils are Mexico, Perù, Brazil, Haiti, Cuba, Guatemala, Ivory Coast, Ghana, Gambia, Madagascar, India and Indonesia.

This study reports results on the analytical characteriza-tion of the different types of lime oils, of different geographic origin as described in Table I to complete and update the knowledge on these products which is often obsolete and scant in recent literature.

The composition of the volatile fraction of Key lime oils type A and B, and of Persian lime oil was reviewed in 1979 by Shaw (2) and successively, for the articles published until 1999, by Dugo et al. (3). These authors also published in the same year a review on the composition of petitgrain lime oils (4). Also in 2002 Haro-Guzman (5) revised the composition of distilled lime oils and Mondello et al. (6) reported on the enantiomeric distribution of some volatile components of these oils. The composition of the oxygen heterocyclic compounds was extensively revised by Dugo and McHale (7). Information on the composition of these oils can be found in periodical

Bonaccorsi et al.


reviews by Lawrence (8). In the above mentioned reviews are included samples obtained by industrial processes of secure origin, and results relative to commercial samples and oils extracted in laboratory by different techniques.

Few are the articles published on industrial genuine oils after those revised in the above mentioned reviews in 2002. Those relative to the volatile fraction of cold-pressed lime oils found in literature in the new millennium are quite scant (9-15). These are unfortunately relative to a single sample or focused on particular aspects of the composition of the oils.

Table I. Analyzed samples of lime essential oils and their physicochemical indices.

Key lime oils Persian lime oils

Cold-pressed Petigrain Cold-pressed Commercial

Type A Type B Crude Colorlessc

(1)a (2)a (3)a (4)a (5)a (6)a (7)b (8)a (9)a (10) (11)

Refractive index 1.484 1.484 1.486 1.484 1.483 1.480 1.484 1.482 1.482 1.479 n.d.

CD 5.05 6.76 5.55 7.00 5.50 0 0 7.06 7.06 3.47 0

a) Mexican; b) Egyptian; c) obtained by distillation of sample 10

The results relative to the oxygen heterocyclic fraction of lime oils are even fewer (16-19). More data is available in literature relative to commercial oils of uncertain origin, some probably polluted.

Recent literature is unavailable concerning the composi-tion of lime petitgrain, and the enantiomeric distribution of any lime oil industrially produced.

More information on oils extracted in laboratory, published in the last decade, is available. The results relative to the vola-tile fraction of peel oils (20-29) were published between 1998

Figure 1. GC-FID chromatogram of cold pressed Key lime Type B. Peak identification: 1 tricyclene; 2 a-thujene; 3 a-pinene; 4 a-fenchene; 5 camphene; 6 thuja-2,4(10)-diene; 7 sabinene; 8 b-pinene; 9 6-methyl-5-hepten-2-one; 10 myrcene; 11 decane; 12 octanal; 13 a-phellandrene; 14 d-3-carene; 15 a-terpinene; 16 p-cymene; 17 limonene; 18 (Z)-b-ocimene; 19 (E)-b-ocimene; 20 g-terpinene; 21 cis-sabinene hydrate; 22 terpinolene; 23 p-cymenene; 24 linalool; 25 trans-sabinene hydrate + nonanal; 26 fenchol*; 27 trans-p-mentha-2,8-dien-1-ol; 28 cis-p-menth-2-en-1-ol; 29 allocimene; 30 cis-limonene oxide; 31 trans-limonene oxide; 32 trans-pinocarveol; 33 citronellal; 34 (Z)-isocitral; 35 borneol; 36 isogeranial + terpinen-4-ol; 37 p-cymen-8-ol; 38 a-terpineol; 39 decanal; 40 citronellol; 41 nerol; 42 neral; 43 carvone; 44 geraniol; 45 geranial; 46 perilla aldehyde; 47 bornyl acetate; 48 trans-pinocarvyl acetate; 49 tridecane; 50 undecanal; 51 methyl geranate; 52 d-elemene; 53 citronellyl acetate; 54 neryl acetate; 55 geranyl acetate; 56 b-elemene; 57 dodecanal; 58 cis-a-bergamotene; 59 b-caryophyllene; 60 g-elemene; 61 trans-a-bergamotene; 62 (E)-b-farnesene; 63 a-humulene; 64 b-santalene; 65 g-curcumene; 66 germacrene D; 67 trans-b-bergamotene; 68 b-selinene; 69 a-selinene; 70 (Z)-a-bisabolene; 71 (E,E)-a-farnesene; 72 b-bisabolene; 73 (E)-g-bisabolene; 74 (E)-a-bisabolene; 75 germacrene B; 76 caryophyllene oxide*; 77 dodecyl acetate; 78 tetradecanal; 79 a-bisabolol; 80 (E,E)-farnesal.

C. aurantifolia and C. latifolia


Figure 2. GC-FID chromatogram of Persian lime oil. Peak identification: 1 tricyclene; 2 a-thujene; 3 a-pinene; 4 camphene; 5 sabinene; 6 b-pinene; 7 6-methyl-5-hepten-2-one; 8 myrcene; 9 octanal; 10 a-phellandrene; 11 d-3-carene; 12 a-terpinene; 13 p-cymene; 14 limonene + 1,8-cineole + (Z)-b-ocimene; 15 (E)- b-ocimene; 16 g-terpinene; 17 cis-sabinene hydrate; 18 terpinolene; 19 linalool; 20 nonanal; 21 cis-limonene oxide; 22 trans-limonene oxide; 23 citronellal; 24 borneol; 25 terpinen-4-ol; 26 a-terpineol; 27 decanal; 28 nerol + citronellol; 29 neral; 30 geraniol; 31 geranial; 32 perilla aldehyde; 33 bornyl acetate; 34 undecanal; 35 d-elemene; 36 citronellyl acetate; 37 neryl acetate; 38 geranyl acetate; 39 cis- b-elemene; 40 dodecanal; 41 cis-a-bergamotene; 42 b-caryophyllene; 43 g-elemene; 44 trans-a-bergamotene; 45 (Z)- b-farnesene; 46 (E)- b-farnesene; 47 a-humulene; 48 b-santalene; 49 g-curcumene; 50 b-selinene; 51 (Z)-a-bisabolene; 52 (E,E)-a farnesene; 53 b-bisabolene; 54 (Z)-g-bisabolene; 55 (E)-g-bisabolene; 56 E)-a-bisabolene; 57 germacrene B; 58 caryophyllene oxide*; 59 tetradecanal; 60 2,3-dimethyl-3-(4-methyl-3-penten-1-yl)-2-norbornanol; 61 campherenol; 62 a-bisabolol

and 2008. Five articles are relative to laboratory extracted leaf oils (30-34). The enantiomeric distribution of some volatile components were the object of two articles (35,36).

Experimental

Plant material: The present study was carried out on eleven samples of industrially produced oils described in Table I.

Physicochemical indices: The analyses of CD were carried out on a Hitachi U-2000 spectrophotometer, UV absorbance was measured in the range of 200-400 nm. The CD values were determined in accordance with the method of Sale (37), 25 mg of lime essential oil accurately weighed was diluted in 100 mL of ethanol. The analyses of refractive index were car-ried out on an Optec refractometer at 21°C with a Carlo Erba Kryo-Thermo stat WK5.

GC-Flame ionization detector (GC-FID): A Shimadzu GC2010 gas chromatograph, equipped with an AOC-20i series autoinjector, was used in all applications (Shimadzu, Kyoto, Japan).

All the samples were injected and analyzed in triplicates under the following conditions: column, SLB-5MS (silphenylene polymer, virtually equivalent in polarity to 5% diphenyl/95% methylpolysiloxane) 30 m x 0.25 mm i.d. x 0.25 mm df (Supelco, Milan, Italy); temperature program: 50°C to 250°C at 3.0°C

min; split/splitless injector (250°C); injection mode: split, ra-tio:1:100; injection volume: 1.0 mL; inlet pressure: 99.5 kPa; carrier gas: He; constant gas linear velocity: 30.0 cm/s. Data handling by means of GCsolution software.

GC/Mass spectroscopy (GC/MS): Samples were analyzed by GC/MS (EI) on a GC/MS-QP2010 system coupled to FF-NSC ver. 1.3 database (38), created in the authors’ laboratory now commercially available (Shimadzu, Japan, Wiley, USA) and to Adams library (39); GC conditions: fused silica capil-lary column SLB-5MS 30 m x 0.25 mm i.d., 0.25 mm df film thickness; column temperature, 50-250°C (10 min) at 3°C/min; carrier gas, He delivered at a constant pressure of 30.6 KPa (30.1 cm/s); 1.0 mL of solution (1/10, v/v, essential oil/hexane) injected on a split-splitless injector; injector temperature, 250°C; injection mode, split; ratio: 1:50. MS scan conditions: source temperature, 200°C; interface temperature, 250°C; E energy 70eV; mass scan range, 40-400 amu. Data were handled through the use of GC/MSsolution software.

Direct enantioselective GC (esGC): All the samples were analyzed in triplicate under the following conditions: column, Megadex DETTBS-b (diethyl-tert-butil-silyl b-cyclodextrin) 25 m x 0.25 mm i.d. x 0.25 mm df (Mega, Legnano, Italy); tem-perature program: 50°C to 200°C at 2.0°C min; split/splitless injector (220°C); injection mode: split, ratio: 1:10; injection volume: 1.0 mL (1/100, v/v, essential oil/hexane); inlet pressure:

Bonaccorsi et al.


Figure 3. GC chromatogram of the essential oil of Petitgrain key lime from Egypt. Peak identification: i.s.: internal standard; 1 a-thujene; 2 a-pinene; 3 camphene; 4 sabinene; 5 b-pinene; 6 6-methyl-5-hepten-2-one; 7 myrcene; 8 octanal; 9 d-3-carene; 10 p-cymene; 11 limonene; 12 (Z)-b-ocimene; 13 (E)-b-ocimene; 14 bergamal; 15 cis-linalool oxide; 16 trans-linalool oxide; 17 a-pinene oxide; 18 linalool; 19 6-methyl-3,5-heptadien-2-one; 20 4,8-dimethyl-1,3,7-nonatriene (3E)-; 21 trans-p-mentha-2,8-dien-1-ol; 22 cis-limonene oxide; 23 trans-limonene oxide; 24 isopulegol; 25 citronellal; 26 (Z)-isocitral; 27 rose furan oxide; 28 nonanol; 29 isogeranial; 30 terpinen-4-ol; 31 trans-isocarveol; 32 a-terpineol; 33 g-terpineol; 34 decanal; 35 octyl acetate; 36 nerol; 37 citronellol; 38 cis-p-mentha-1(7),8-dien-2-ol; 39 neral; 40 carvone; 41 linalyl acetate; 42 geraniol; 43 geranial; 44 neryl formate; 45 limonen-10-ol; 46 geranyl formate; 47 undecanal; 48 methyl geranoate; 49 d-elemene; 50 citronellyl acetate; 51 neryl acetate; 52 geranyl acetate; 53 b-bourbonene; 54 b-elemene; 55 tetradecane; 56 dodecanal; 57 cis-a-bergamotene; 58 b-caryophyllene; 59 trans-a-bergamotene; 60 aromadendrene; 61 geranyl acetone; 62 a-humulene; 63 neryl isobutyrate; 64 g-muurolene; 65 b-selinene; 66 a-selinene; 67 (E,E)-a-farnesene; 68 b-bisabolene; 69 g-cadinene; 70 7-epi-a-selinene; 71 (Z)-nerolidol; 72 cis-sesquisabinene hydrate; 73 germacrene B; 74 caryophyllene oxide; 75 humulene epoxide II; 76 spathulenol; 77 a-cadinol; 78 selin-11-en-4-a-ol; 79 a-bisabolol.

96.6 kPa; carrier gas: He; constant gas linear velocity: 35.0 cm/s. Detector: FID (220°C); H2: 50.0 mL/min; air: 400 mL/min; makeup (N2): 40.0 mL/min; sampling rate: 80 msec. Data were collected by the GCSolution software (Shimadzu).

Multidimensional enantio-GC (MDGC): The MDGC system consisted of two GC2010 (defined as GC1 and GC2) gas chromatographs, equipped with a Deans’ type switch transfer device, an MS-QP2010 quadrupole mass spectrometer, and an AOC-20i autosampler (Shimadzu). GC1 was equipped with a split/splitless injector and a flame ionization detector (FID1). The MDGC switching element, located inside the oven, was connected to an advanced pressure control (APC) system which supplied carrier gas (He) at constant pressure. GC1 column was an SLB-5MS 30 m x 0.25 mm i.d. x 0.25 mm df . All samples were analyzed in triplicate. The operational conditions were as follows: constant inlet pressure 220 kPa (300°C), split mode 1:20 (gas carrier, He); injected volume, 1.5 mL; initial linear velocity, 30 cm/sec. Temperature program: 50-280°C at 3°C/min. The FID (300°C) was connected, via a stainless steel retention gap, to the transfer system; sampling rate: 80 ms. APC constant pressure: 130 kPa. GC2 was equipped with a split/splitless injector and a flame ionization detector (both not used in the present research). Transfer line between GC1 and GC2: 180°C. The chiral column was a Megadex DETTBS-b

(diethyl-tert-butil-silyl b-cyclodextrin) 25 m x 0.25 mm i.d. x 0.25 mm df (Mega, Legnano, Italy). Temperature program: 40-100°C (20 min) at 1°C/min, to 160°C at 3°C/min.

Reversed phase liquid chromatography (RP-HPLC/PDA): Samples were analyzed in triplicates by HPLC using a Shimadzu instrument equipped with two LC 10 AD Vp pumps, an SPD-M10 Avp UV detector, a SCL-10 Avp controller, a CTO-20AC column oven at 40°C and a DGU-14A degasser. Sample: ca. 100 mg of oil accurately weighed were diluted in 6 mL of ethanol. Before HPLC analysis, 50 mL of I.S. coumarin (53.1 mg in 50 mL of ethanol) were added to 1.0 mL of this solution. The injection volume was 2.0 mL. The column used was a partially porous Ascentis Express C18, 150 x 4.6 mm i.d. with particle size of 2.7 mm (Supelco, Bellefonte, PA). Two mobile phases were used: eluent A (Water:Methanol:THF, 85:10:5) and eluent B (Methanol:THF, 95:5). The HPLC analyses of lime oil were performed according to the following program: 0-5 min, 0% B, 5-25 min, 0-40% B, 25-45 min, 40-90% B, 45-55 min, 90% B, 55-60 min, 0% B. The flow-rate was 1.0 mL/min, the pressure was 225 bar. Detection was performed by a photodiode array (PDA) detector in the range 190-370 nm and the chromatograms were extracted at 315 nm. Time constant was 0.64 s and sample frequency 1.5625 Hz. Data was handled by Shimadzu LCsolution software Ver. 3.3.




Physicochemical indices: In Table I for each of the samples analyzed are reported the physicochemical indices. CD values are in agreement with those previously reported (40) for cold-extracted peel oils of secure origin; the sample of crude commercial Persian oil has a low CD value.

Volatile fraction: Table II reports the composition of the volatile fraction in single components (average values determined out of triplicates) and in class of substances of the samples analyzed. In the same table are reported the LRI experimentally determined compared to those reported

in literature. The coefficient of variation determined for the peak area % of each component out of triplicates was never above 5.9%. In general, the repeatability of the method was excellent.

GC/MS identified 137 components. The composition is expressed as relative percent of peak area determined by GC-FID without considering the response factors nor the non-volatile residue. The gas chromatograms in Figures 1-3, respectively show Key lime oil cold-pressed, Persian lime oil cold-pressed and Key lime petitgrain oil. The components identified (Table II) in peel oils represent about 98-99% of

Figure 4. A) TIC chromatogram obtained by MDGC of a cold-pressed Key lime Type A. B) enlargement of the direct esGC separation showing the separation of the enantiomers of a-pinene and of camphene; C) enlargement of the direct esGC separation of (-)/(+) a-terpineol separation. Peak identification: 1 S-(+)-a-thujene; 2 R-(-)-a-thujene; 3 R-(+)-a-pinene; 4 S-(-)-a-pinene; 5 1S,4R-(-)-camphene; 6 1R,4S-(+)-camphene; 7 R-(+)-b-pinene; 8 S-(-)-b-pinene; 9 R-(+)-sabinene; 10 S-(-)-sabinene; 11 R-(-)-a-phellandrene; 12 S-(+)-a-phellandrene; 13 R-(-)-b-phellandrene; 14 S-(-)-limonene; 15 S-(+)-b-phellandrene; 16 R-(+)-limonene; 17 R-(-)-linalool; 18 S-(-)-citronellal; 19 R-(+)-citronellal; 20 S-(+)-linalool; 21 S-(+)-terpinen-4-ol; 22 R-(-)-terpinen-4-ol; 23 S-(-)-a-terpineol; 24 R-(+)-a-terpineol.

Bonaccorsi et al.


Table II. Composition of the volatile fraction (relative percentage of uncorrected peak areas).

Key Lime oils Persian Lime oils

Cold-pressed Petitgrain Cold-pressed Commercial

Type A Type B

Compound LRIexp LRIlit (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11)

Tricyclene 923 923 0.03 0.01 0.01 0.01 0.01 - - 0.03 0.01 0.01 0.03a-Thujene 925 927 0.33 0.27 0.31 0.29 0.31 0.05 0.01 0.48 0.61 0.57 0.51a-Pinene 933 933 2.09 1.96 1.92 1.91 2.03 0.30 0.11 1.99 2.24 2.25 2.05a-Fenchene 947 948 tr - 0.01 - 0.01 - - tr - tr trCamphene 953 953 0.07 0.08 0.09 0.07 0.09 tr 0.01 0.04 0.06 0.06 0.06Thuja-2,4(10)-diene 954 953 tr - tr - tr - - - - - -Sabinene 972 972 2.45 2.43 2.31 3.02 2.40 0.89 0.14 1.70 1.95 2.10 1.92b-Pinene 978 978 20.17 21.16 18.25 20.10 18.01 0.37 0.32 10.52 11.39 12.16 11.826-Methyl-5-hepten-2-one 983 986 0.03 tr 0.03 0.10 0.03 0.87 0.34 tr 0.01 0.02 0.01Myrcene 988 991 1.01 1.05 1.07 1.04 1.13 0.78 0.45 1.26 1.46 1.38 1.306-Methyl-5-hepten-2-ol 994 995 tr - - - - - - - - - -Decane 1000 1000 tr - - - 0.01 - - - - - -Octanal 1004 1006 0.03 0.05 0.04 0.03 0.04 0.07 0.01 0.01 0.01 0.01 0.01a-Phellandrene 1006 1007 0.03 0.03 0.05 0.03 0.05 - - 0.04 0.05 0.04 0.04d-3-Carene 1010 1009 tr - tr - 0.01 0.04 0.02 tr 0.01 0.01 tra-Terpinene 1017 1007 0.20 0.19 0.23 0.13 0.22 0.03 - 0.23 0.28 0.12 0.20p-Cymene 1024 1025 0.24 0.11 0.33 0.10 0.31 0.25 0.14 0.08 0.18 1.07 0.30Limonene* 1030 1030 51.14 50.29 50.96 49.60 49.25 45.22 45.29 58.15 54.77 53.61 54.201,8-Cineole 1033 1032 - - - - - 0.14 - (Z)-b-Ocimene 1035 1026 0.09 0.12 0.13 0.12 0.13 0.45 0.23 0.18 0.22 (E)-b-Ocimene 1045 1046 0.22 0.29 0.30 0.28 0.30 2.33 0.50 0.08 0.08 0.12 0.11Bergamal 1053 1053 - - - - - - 0.03 - - - -g-Terpinene 1058 1058 9.29 7.66 9.79 8.68 9.87 1.09 - 14.61 13.90 12.38 12.84cis-Sabinene hydrate 1068 1069 0.04 - 0.05 - 0.05 - - 0.04 0.05 0.04 0.04Octanol 1070 1073 - - tr 0.06 - 0.03 - - - - -cis-Linalool oxide 1069 1072 - - - - - - 0.01 - - - -Terpinolene 1086 1086 0.38 0.33 0.52 0.33 0.53 0.10 - 0.49 0.63 0.45 0.51p-Cymenene 1095 1093 tr - 0.01 - 0.01 - - - - - -a-Pinene oxide 1097 1099 - - - - - - 0.04 - - - -Linalool 1099 1101 0.15 0.17 0.24 0.22 0.24 1.20 0.61 0.13 0.18 0.18 0.14trans-Sabinene hydrate 1100 1099 0.03 - tr - 0.02 - - 0.05 0.07 0.05 -6-Methyl-3,5-heptadien-

2-one 1102 1103 - - - - - - 0.04 - - - -n-Nonanal 1105 1107 0.02 0.03 0.03 0.03 0.04 - 0.01 - 0.01 0.014,8-Dimethyl-1,3,7-

nonatriene, (3E)- 1113 1115 tr - - - - - 0.02 - - - -1,3,8-p-Menthatriene 1110 1108 - - 0.01 - 0.01 - - - tr tr -Fenchyl alcohol 1122 1123 tr - 0.04 - 0.05 - - - - 0.01 -trans-p-Mentha-2,8-dien-

1-ol 1122 1125 tr - 0.01 - 0.01 0.01 0.05 - - tr -cis-p-Menth-2-en-1-ol 1125 1124 tr - tr - 0.01 - - - - 0.01 -(4E,6Z)-Allocimene 1128 1132 tr - - - 0.01 0.02 - - - - -cis-p-Mentha-2,8-dien-1-ol 1132 1133 tr - - - - - - - - - -cis-Limonene oxide 1134 1137 - tr 0.01 - 0.01 - 0.37 0.01 0.04 0.01trans-Limonene oxide 1138 1140 - tr tr tr 0.01 0.03 0.25 tr 0.03 0.01trans-Pinocarveol 1140 1141 tr - - - 0.01 - - - - - -Isopulegol 1142 1145 - - - - tr - 0.04 - - - -Citronellal 1152 1152 0.01 tr 0.03 0.03 0.03 0.74 1.64 0.03 0.04 0.06 0.06(Z)-Isocitral 1161 1160 - - 0.02 - 0.02 0.24 0.04 - 0.01 tr -Pinocarvone 1159 1164 tr - - - - - - - - - -Rose furan oxide 1169 1170 - - - - - - 0.10 - - - -Borneol 1170 1173 tr - 0.04 - 0.02 - - 0.01 0.02 0.02 0.01cis-Pinocamphone 1173 1176 0.01 tr 0.02 tr - - - - tr tr -Nonanol 1176 1178 - - - - - - 0.01 - - - -Isogeranial 1180 1179 0.30 0.41 0.33 - 0.32 0.47 0.16 0.01 0.02 0.04 -Terpinen-4-ol 1182 1180 0.06 0.01 0.07 0.08 0.05 0.05p-Cymen-8-ol 1188 1189 - - 0.02 - 0.02 - - - tr 0.03 -trans-Isocarveol 1189 1190 - - - - - - 0.04 - - - -a-Terpineol 1197 1195 0.27 0.22 0.80 0.31 0.80 0.16 0.18 0.25 0.32 0.26 0.21g-Terpineol 1200 1200 - - - - - - 0.08 - - - -Decanal 1206 1208 0.17 0.24 0.18 0.22 0.17 0.28 0.18 0.05 0.06 0.06 0.06trans-Piperitol 1208 1209 tr - - - - - - - - - -Octyl acetate 1214 1210 - - tr tr - - 0.02 - - - -



Nerol 1225 1229 0.04 - 0.01 - 0.04 - 1.12 0.16 0.19 0.08 0.09Citronellol 1227 1232 0.01 0.01 - - 0.01 3.27 0.59 0.03 0.06 -cis-p-Mentha-1(7),8-dien-2-ol 1230 1233 - - - - - - 0.06 - - - -Neral 1239 1238 1.12 1.36 1.61 1.82 1.65 10.38 7.45 1.09 1.25 1.46 1.35Carvone 1246 1244 - - tr - tr - 0.10 - - 0.01 -Linalyl acetate 1249 1243 - - - - - - 0.12 - 0.01 0.01 -Geraniol 1251 1255 0.07 tr 0.07 tr 0.09 3.91 1.82 0.04 0.04 0.03 0.03Piperitone 1263 1267 tr - tr - - - - - 0.01 0.01 -Geranial 1269 1268 1.90 2.29 2.63 2.88 2.67 13.96 10.75 1.79 2.02 2.42 2.20Decanol 1276 1278 0.01 - - - - - - - - - -Perillaldehyde 1276 1278 0.02 0.01 0.02 0.02 0.02 - - 0.02 0.03 0.04 0.03Neryl formate 1276 1280 - - - - - 0.03 0.05 - - - -Bornyl acetate 1283 1285 tr - - - 0.01 - - - - - -Isobornyl acetate 1284 1287 - - 0.01 - - - - - 0.01 0.02 -Geranyl formate 1298 1298 - - - - - 0.05 0.11 - - - -2,3-Benzopyrrole 1299 1297 - - 0.01 - - - - - - - -trans-Pinocarvyl acetate 1300 1296 - - 0.01 - 0.01 - - - - - -Tridecane 1300 1300 0.01 - 0.01 - tr - - - - - -Undecanal 1308 1309 0.02 0.02 0.02 0.02 0.01 0.05 0.27 0.01 0.01 0.03 0.02Geranic acid methyl ester 1321 1320 - - tr tr tr 0.01 0.17 - - - -d-Elemene 1337 1335 0.02 0.56 0.39 0.46 0.38 0.57 0.58 0.03 0.06 0.04 0.07a-Terpinyl acetate 1347 1349 - tr - - - - - - - - -Citronellyl acetate 1349 1353 0.01 tr 0.01 - 0.01 0.06 0.53 0.01 0.01 0.03 0.01Neryl acetate 1358 1361 0.16 0.07 0.14 0.12 0.13 0.45 2.11 1.11 1.21 1.31 1.08Geranyl acetate 1377 1380 0.18 0.19 0.25 0.19 0.24 1.31 6.22 0.14 0.19 0.28 0.23b-Bourbonene 1382 1381 - - - - - 0.01 0.09 - - - -

b-Elemene 1391 1391 0.16 0.29 0.19 0.27 0.18 0.81 0.67 0.04 0.05 0.07 0.06Tetradecane 1400 1400 - - - - - - 0.08 - tr 0.01 -Dodecanal 1409 1410 0.10 - 0.09 - 0.09 0.11 0.15 0.01 0.04 0.04 0.04Decyl acetate 1410 1412 - 0.13 tr 0.12 - - - - - - -cis-a-Bergamotene 1415 1416 0.07 - 0.06 - 0.05 0.02 0.04 0.08 0.08 0.08 0.06b-Caryophyllene 1423 1424 0.79 0.96 0.73 0.97 0.72 2.72 2.63 0.29 0.59 0.68 0.61a-Santalene 1416 1418 - - - - - - - - - -g-Elemene 1432 1431 0.03 - 0.06 - 0.06 0.20 - 0.01 0.01 0.01 0.01trans-a-Bergamotene 1435 1434 1.12 1.14 0.86 0.93 0.86 0.28 0.25 0.67 1.11 1.18 0.98Aromadendrene 1438 1439 - - - - 0.03 0.01 0.05 - - - -(Z)-b-Farnesene 1140 1439 - - 0.03 0.09 - - - - 0.05 0.05 0.01b-Sesquisabinene 1450 1455 - - 0.01 - - - - - 0.01 0.01 -(E)-b-Farnesene 1451 1452 0.10 0.11 0.06 - 0.08 - - 0.10 0.12 0.12 0.10Geranyl acetone 1450 1449 - - - - - - 0.34 - - - -a-Humulene 1459 1454 0.09 0.11 0.10 0.11 0.09 0.39 0.02 0.03 0.05 0.06 0.05b-Santalene 1460 1459 0.04 0.04 0.03 0.03 0.04 - - 0.01 0.04 0.05 0.04g-Curcumene 1479 1482 0.03 - 0.02 - 0.02 - - 0.01 0.03 0.02 0.02Germacrene D 1481 1480 0.26 0.07 0.27 0.06 0.26 - - 0.05 0.06 0.06 -trans-b-Bergamotene 1484 1483 0.07 - - - 0.06 - - 0.05 0.07 0.08 -Valencene 1489 1492 - 0.07 0.01 0.06 - - - - - - -Neryl isobutyrate 1485 1482 - - - - - - 0.09 - - - -g-Muurolene 1485 1485 - - - - - 0.29 0.11 - - - -b-Selinene 1492 1490 0.04 - 0.02 0.03 - 0.15 0.02 0.01 0.02 0.02Bicyclogermacrene 1499 1497 - 0.15 tr 0.12 - - - - - - -a-Selinene 1501 1500 0.06 - 0.07 - 0.07 0.06 0.16 0.02 0.03 0.04 -(Z)-a-Bisabolene 1502 1503 0.14 - 0.12 - 0.11 - - 0.18 0.15 0.15 0.13(E,E)-a-Farnesene 1504 1501 1.00 1.27 1.06 1.20 1.09 0.99 0.48 0.21 0.27 0.25 0.23b-Bisabolene 1509 1508 1.85 1.83 1.35 1.50 1.34 0.58 0.48 1.67 1.70 1.79 1.53(Z)-g-Bisabolene 1510 1511 - - tr - 0.01 - - - 0.02 0.02 0.02b-Sesquiphellandrene 1521 1523 - 0.01 - - - - - - - - -(E)-g-Bisabolene 1530 1528 - 0.04 0.02 0.03 tr - - - 0.01 0.01 0.01(E)-a-Bisabolene 1539 1540 0.03 - 0.03 - 0.03 - - - 0.04 0.04 0.03g-Cadinene 1512 1510 - - - - - tr tr - - - -7-epi-a-Selinene 1520 1520 - - 0.01 - 0.03 - 0.03 - - 0.01 -(Z)-Nerolidol 1531 1530 - - - - - - 0.29 - - - -cis-Sesquisabinene hydrate 1544 1545 0.01 - 0.01 tr 0.01 - 0.03 tr 0.01 0.02 tr

Table II. Continued



Type A Type B


Bonaccorsi et al.


the volatile fraction; those determined in the two samples of petitgrain represent 93% in the Egyptian sample and 98% in the Mexican one; in the sample of distilled colorless Persian lime the components identified represent about 96%. Accord-ing to what previously determined (40), the main components determined in Key lime oil type A and B, are in decreasing amount order limonene (49-51%), b-pinene (18-21%) and g-terpinene (8-10%); in Persian lime oils the main components are the same, but limonene riches the value of 54-58% while, with an opposite trend compared to Key lime oils, g-terpinene is higher (~14%) than b-pinene (~12%). The total content of sesquiterpene hydrocarbons is about 6-7% in Key lime oils, while in Persian lime decreases to about 2/3: this is due to the behavior of many single sesquiterpene hydrocarbons such as b- and d-elemene, b-caryophyllene, germacrenes B and D, and mainly (E,E)-a-farnesene; this behavior was also determined by Dugo et al. (40) with the exception of d-elemene in Key lime

Table II. Continued



Type A Type B


Germacrene B 1564 1557 0.54 0.55 0.52 0.45 0.51 1.33 0.31 0.09 0.12 0.11 0.12trans-Sesquisabinene hydrate 1573 1577 - 0.01 tr 0.02 tr - - - - - -Spathulenol 1576 1577 - - - - - - 0.58 - - - -Caryophyllene oxide 1587 1587 tr 0.01 0.01 - 0.01 0.15 2.09 - tr 0.05 0.01Dodecyl acetate 1609 1610 - - 0.01 - 0.01 - - - tr 0.01 -Humulene epoxide II 1612 1613 - - - - - - 0.28 - - - -Tetradecanal 1617 1614 0.04 - 0.03 - 0.03 - - - 0.02 0.03 0.02a-Cadinol 1652 1652 - - - - - - 0.09 - - - -Selin-11-en-4-a-ol 1655 1658 - - - - - - 0.17 - - - -Norbornanol** 1662 - - 0.04 tr 0.04 - - - - - - 0.05cis-Nerolidyl acetate 1669 1665 - - 0.01 - - - - 0.01 0.01 -Campherenol 1675 - - 0.02 tr 0.02 - - - - - - 0.05a-Bisabolol 1688 1685 0.08 0.07 0.06 0.06 0.06 - 0.09 - 0.09 0.10 0.08(E,E)-Farnesal 1739 1737 0.01 - 0.02 tr 0.01 - - - 0.02 0.02 -HYDROCARBONS 94.19 93.18 92.33 91.99 90.75 60.18 53.29 93.26 92.46 91.52 89.99Monoterpene 87.74 85.98 86.30 85.71 84.69 51.92 47.22 89.70 87.78 86.56 85.89Sesquiterpene 6.44 7.20 6.02 6.28 6.05 8.26 6.05 3.56 4.67 4.95 4.10Aliphatic 0.01 - 0.01 - 0.01 - 0.02 - 0.01 0.01 -ALDEHYDES 3.74 4.41 4.72 5.05 5.06 25.87 20.68 3.03 3.53 4.23 3.80Monoterpene 3.35 4.07 4.31 4.75 4.71 25.32 20.07 2.94 3.37 4.03 3.64Sesquiterpene 0.01 - 0.02 - 0.01 - - - 0.02 0.02 -Aliphatic 0.38 0.34 0.39 0.30 0.34 0.55 0.61 0.09 0.14 0.18 0.16KETONES 0.04 - 0.05 0.01 0.03 0.87 0.82 - 0.02 0.04 0.01Monoterpene 0.01 - 0.02 - - - 0.44 - 0.01 0.02 -Aliphatic 0.03 - 0.03 0.10 0.03 0.87 0.38 - 0.01 0.02 0.01ALCOHOLS 0.71 0.54 1.68 0.79 1.44 9.05 5.86 0.75 1.09 0.95 0.75Monoterpene 0.61 0.40 1.61 0.59 1.37 9.02 4.6 0.75 0.98 0.83 0.57Sesquiterpene 0.09 0.14 0.07 0.14 0.07 - 1.25 - 0.11 0.12 0.18Aliphatic 0.01 - - 0.06 - 0.03 0.01 - - - -ESTERS 0.35 0.39 0.44 0.43 0.41 1.91 9.42 1.26 1.45 1.68 1.32Monoterpene 0.35 0.26 0.42 0.31 0.40 1.91 9.40 1.26 1.44 1.66 1.32Sesquiterpene - - 0.01 - - - - - 0.01 0.02 -Aliphatic - 0.13 0.01 0.12 0.01 - 0.02 - - - -OXIDES and ETHERS - 0.01 0.02 - 0.03 0.32 2.86 - 0.01 0.12 0.03OTHERS 0.07 - 0.01 - 0.21 - 0.28 - - - -ALL 99.11 98.53 99.25 98.36 97.93 98.20 93.21 98.30 98.56 98.54 95.90

* coeluted with ß-phellandrene; **2,3-dimethyl-3-(4-methyl-3-pentenyl)-2-norbornanol; a: crude; b: colorless; LRIexp : LRI measured on SLB-5MS column; LRIlit : FFNSC 1.3 GC/MS library, Wiley, USA, 2008; Adams RP, Identification of essential oil components by gas chromatography/massspectrometry, 4th Edn,: Allured Pub Corp; 2007; Hochmuth, D.H., Joulain, D., König, W.A., 2002, MassFinder Software and Data Bank, University of Hamburg.

oil type B, which presented, in the single sample analyzed in 1997, a value significantly lower than what presently determined (0.07% vs. 0.4%). Total oxygenated components are higher in Key lime type B (~7%) compared to type A (~5%); in Persian lime the content is about the same as Key lime type A. This is probably due to the behavior of total aldehydes and alcohols. The values determined for these two classes of substances slightly differ from what previously determined (40), mainly for Key lime type B and Persian lime. These differences are probably due to the conditions used for the extraction of the oil.

In the two samples of petitgrain, as it happens for all the citrus leaf oils, hydrocarbons are present at lower levels and oxygenated compounds at higher levels, if compared to peel oils. In the two oils, among oxygenated compounds, monot-erpene aldehydes are predominant. Among the two samples some important quantitative differences are however noticed. These can be only explained by the different geographic



Figure 5. A) RP-HPLC Chromatotram of Key lime essential oil type A. B) RP-HPLC Chromatotram of key lime essential oil type B. C) RP-HPLC Chromatotram of Persian lime essential oil. For peak identification see Table IV.

Bonaccorsi et al.


Table III. Enantiomeric distribution of some volatile components of the oils analyzed.


Type A Type B Petigrain Cold-pressed Commercial

(1)a (2)a (3)a (4)a (5)a (6)a (7)b (8)a (9)a (10) (11)

a-thujene S-(+) 1.00 1.06 0.62 1.15 0.48 12.62 12.45 0.50 0.74 1.28 1.56 R-(-) 99.00 98.94 99.38 98.85 99.52 87.38 87.55 99.50 99.26 98.72 98.44a-pinene* R-(+) 21.83 22.55 22.27 22.72 23.34 78.08 77.80 29.96 28.57 28.61 32.56 S-(-) 78.17 77.45 77.73 77.28 76.66 21.92 22.20 70.04 71.43 71.39 67.44camphene 1S,4R-(-) 92.80 92.89 93.85 94.60 91.78 28.52 27.81 89.27 88.37 88.96 87.80 1R,4S-(+) 7.20 7.11 6.15 5.40 8.22 71.48 72.19 11.73 11.63 11.04 12.20b-pinene R-(+) 3.58 4.47 3.71 4.45 4.24 31.05 5.89 10.34 10.10 9.20 9.50 S-(-) 96.42 95.53 96.29 95.55 95.76 68.95 94.11 89.66 89.90 90.80 90.50sabinene R-(+) 15.23 15.44 15.37 14.83 15.18 85.68 31.32 18.60 19.28 18.61 20.35 S-(-) 84.77 84.56 84.63 85.17 84.82 14.32 68.68 81.40 80.72 81.39 79.65a-phellandrene R-(-) 50.86 53.30 52.19 54.53 58.35 16.66 15.45 55.16 55.82 56.15 49.06 S-(+) 49.14 46.70 47.81 45.47 41.65 83.34 84.55 44.84 44.18 43.85 50.94b-phellandrene R-(-) 64.94 65.59 73.70 68.54 69.09 30.05 26.67 55.08 54.48 30.45 27.89 S-(+) 35.06 34.41 26.30 31.46 30.91 69.95 70.33 44.92 45.52 69.55 72.11limonene S-(-) 2.82 2.93 2.91 2.86 2.91 2.43 0.75 2.66 2.69 2.59 4.07 R-(+) 97.18 97.07 97.09 97.14 97.09 97.57 99.25 97.34 97.31 97.41 95.93linalool R-(-) 72.70 68.69 65.23 66.11 66.01 58.80 70.71 63.45 63.94 68.01 52.34 S-(+) 27.30 31.31 34.77 33.89 33.99 41.20 29.29 36.55 36.06 31.99 47.66citronellal S-(-) - 77.04 81.04 73.41 72.79 53.43 89.25 78.68 80.50 80.49 - R-(+) - 22.96 18.96 26.59 27.21 46.57 10.75 21.32 19.50 19.51 -terpinen-4-ol S-(+) 29.22 29.15 20.78 29.13 29.14 95.87 88.29 19.34 20.41 23.64 29.52 R-(-) 70.78 70.85 79.22 70.87 70.86 4.13 11.71 80.66 79.59 76.36 70.48a-terpineol* S-(-) 84.08 85.37 79.89 78.49 83.77 77.62 80.40 77.36 78.04 79.79 77.26

R-(+) 15.92 14.63 20.11 21.51 16.23 22.38 19.60 22.64 21.96 20.21 22.74

* values determined by direct esGC.

Table IV. Composition of the oxygen heterocyclic fraction of the analyzed oils (ppm).


N° Group Compound Cold-pressed Petigrain Cold-pressed Comm.

Type A Type B

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

1 CUM Herniarin 1460 2970 3880 4670 3350 120 170 7860 4132 22202 PSO Oxypeucedanin hydrate 780 1160 1690 1710 1620 - - 500 174 1903 CUM Citropten 7350 11740 10950 9230 5940 40 80 8860 6415 16504 PSO Isopimpinellin 5670 10210 7300 6540 3010 60 60 3830 2014 11405 PSO Bergapten 2000 3450 3000 3920 2160 140 120 3620 1977 5606 PSO Byacangelicol 90 - 1020 460 80 - - 220 49 707 PSO Oxypeucedanin 260 - 10720 6660 7560 - 140 10540 5543 40908 PSO Isoimperatorin 370 390 70 210 410 2010 1160 40 58 3109 PSO Imperatorin 830 900 380 430 660 2780 1720 120 153 51010 CUM 5-Isopentenyloxy-7-methoxy-coumarin 4170 4830 2790 2670 2100 - - 580 322 37011 PSO 5-Isopentenyloxy-8-(2’,3’- epoxyisopentenyloxy)-psoralen n.a.* n.a.* n.a.* n.a.* n.a.* - - - n.a.* n.a.*12 PSO Cnidicin 340 90 250 110 70 - - - 24 4013 PSO 8-Geranyloxy-psoralen 6520 8100 4470 4540 3800 - - 1880 1164 99014 PSO 5-Geranyloxy-8-methoxy-psoralen n.a.* n.a.* n.a.* n.a.* n.a.* - - - n.a.* n.a.*15 PSO Bergamottin 37300 56130 36400 41590 25320 460 760 48830 33012 2643016 CUM 5-Geranyloxy-7-methoxy-coumarin 41550 63320 43140 45350 27770 400 240 53040 30049 8120 Coumarins 54530 82860 60750 61920 39160 560 500 70340 40918 12360 Psoralens 54160 80410 65300 66160 44700 5450 3960 69580 44168 34320

ALL 108690 163270 126050 128080 83860 6010 4460 139920 85086 46680

*n.a.: standard was not available in sufficient amount for quantitative calculationCUM = coumarinPSO = psoralen



origin, since one sample is from Egypt, the other from Mexico. In fact, further comments cannot be given, due to the lack of information available in literature on industrial lime petitgrain oils. The data available on laboratory petitgrain oils, relative to samples of different geographic origin, obtained by different techniques, provide a very large range of variability of their composition. In all these oils, as confirmed in this study, are present high amounts of neral and geranial.

Enantiomeric ratios (MDGC): Table III reports the enantiomeric ratios of the volatiles determined in all samples analyzed by MDGC and, when necessary, by direct esGC. The CV% values determined from triplicates ranged from 0 to 9.78. The highest values were determined for (+)-camphene and (-)-a-thujene (9.78 and 5.52 respectively). The elution order of the a-thujene enantiomers was established based on Casabianca et al. (41). The enatiomeric excesses determined by the two techniques were in excellent agreement. Figure 4 shows the TIC chiral chromatogram obtained by MDGC of a cold-pressed Key lime oil type A. In the same figure are reported two enlargements of the enantio-selective separation obtained by direct GC. In the first enlargement (B) it can be observed the separation achieved for (+)/(-)-a-pinene. The enatio separation of the two a-pinene enantiomers cannot be accomplished by MDGC, since the diethyl-tert-butil-silyl b-cyclodextrin column shows poor selectivity for these two enantiomers, providing sufficient resolution by direct esGC, while under the conditions applied in MDGC the resolution of this critical pair is completely lost (42). For this reason the peak of a-pinene is not transferred during MDGC analysis. The enlargement (C) relative to a-terpineol shows the excel-lent separation by direct esGC, used for quantitative analysis of the two enantiomers. It must be however underlined that these enantiomers are usually well determined by MDGC for all citrus oils (43), but in lime essential oil the separation is compromised due to a probable coelution of the (+) isomer with an unknown compound.

The MDGC system allowed to determine the enantiomeric distribution of camphene, a- and b-phellandrene in lime essential for the first time. The results determined in the five samples of Key lime oils are in good agreement among each other; the only exception is given by a-thujene determined in sample 4, with a percentage of the (+)-a-thujene about double than what determined in the other tow samples of Key lime Type B. The results determined in Persian lime are also in good agreement. Some differences are noticed between Key and Persian cold-pressed lime oils: the enantiomeric excess of S-(-)-a-pinene and of S-(-)-b-pinene is slightly lower in Persian lime oils; the enantiomeric excess of R-(-)-b-phellandrene is higher in Key lime. The enantiomeric distribution of b-phellandrene in the two commercial samples (10,11) differs from that determined in the cold-pressed oils of secure origin. As predicted, a slight tendency to racemization is noticed for many components analyzed in the colorless sample (sample 11) obtained by distillation from the cold-pressed oil (sample 10).

The values here determined for the samples of cold-pressed lime oils are in very good agreement with those previously determined by Mondello et al. and by Dugo et al., at least for the components investigated in these studies (42,44).

The enantiomeric ratios determined in the Egyptian and

Mexican petitgrain oils are similar for a-thujene, a-pinene, camphene, a- and b-phellandrene, and a-terpineol; those determined for all the other components analyzed differ more or less evidently, and for sabinene it can be observed the inversion of the enantiomeric excess. It is impossible to provide further comments on these two oils, due to the lack of information in literature.

Oxygen heterocyclic components (RP-HPLC/PDA): To determine this fraction an Ascentis Express column was employed, which is packed with partially porous particles of 2.7 mm based on Fused-Core technology. This consists of 1.7 mm solid core and a 0.5 mm porous shell, with the advantage of a small diffusion path, compared to a totally porous particle, which reduces axial dispersion of solutes and minimized peak broadening allowing for higher resolving power (19). Table IV reports the qualitative and quantitative composition (expressed as mg/100 g of oil) of the oxygen heterocyclic components. The quantitative analysis was tested for repeatability with an average CV% value of 6.63%. Figure 5A-5C shows the HPLC separations of the three samples of cold-pressed lime oils. The total content of this class of components ranges from 8% to 16% in Key lime oils; in the sample of Persian lime of secure origin the amount of these fractions ranges from 8% to 14%; in commercial Persian lime the oxygen heterocyclic components are less than 5% as confirmed by the CD values. Thus, there are rising doubts on the genuineness of this oil, which could have been diluted by the addition of distilled prod-ucts, although this hypothesis cannot be proven by the other analytical results. Sixteen components were identified in this fraction, 4 coumarins and 12 psoralens. The main components, as reported in literature, are bergamottin and 5-geranyloxy-7-methoxycoumarin. In Key lime type A oxypeucedanin is absent or present at levels lower than in Key lime type B and in the Persian lime oil of secure origin. In fact, as previously observed by Radford and Olansky (45), and by Dugo et al (40,46) re-spectively in lemon, bitter orange and lime oils, oxypeucedanin is destroyed by contact with the water and juice phase during extraction. When these conditions occur, the epoxy ring is opened by hydrolysis, forming oxypeucedanin hydrate, which is completely water-soluble.

Although some qualitative and quantitative differences are noticed, the results here obtained are in agreement with those previously determined (19, 40). The qualitative differences are due to the presence of imperatonin and byacangelicol, not identified in the article published in 1997 by Dugo et al. The quantitative differences are mainly relative to the values of herniarin, citropten and bergapten. It is possible to assume, from the present results, that the sample used by Dugo et al. (19), of unknown origin, could have been Key lime type A, due to its content of oxypeucedanin and oxypeucedanin hydrate.

As predictable, oxygen heterocyclic components are absent in Persian lime oil obtained by distillation (sample 11). The CD value for this oil was almost zero.

The two samples of petitgrain (samples 6 and 7) have very low amounts of oxygen heterocyclic compounds, among which imperatorin and isoimperatorin are the most abundant.

Acknowledgements

The authors thank Shimadzu for its continuous support.

Bonaccorsi et al.


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Manuscripts should be e-mailed to: Prof. Luigi Mondello at [email protected].

In order to speed up processing times, authors are strongly encouraged to prepare their manuscript using the JEOR Word Template.

Authors are encouraged to supply the names, addresses, and email addresses of 3-4 potential referees. Authors may also mention persons who they would prefer not to review their paper.

Submission ChecklistLetter including statement of justification for publication •A single Word file containing text, tables, and graphic materials •Names of preferred referees •Supporting materials as appropriate •

Completed • Copyright Transfer Form

Appendix

Preparation of ManuscriptManuscripts must be written in English using American spelling. Manuscripts written in ambiguous English or otherwise incomprehensible, in the opinion of the Editor, will be returned to the authors with the request to resubmit once the language has been improved.

In order to achieve uniform presentation and to avoid unnecessary delays because of further inquiries, all authors are invited to observe these guidelines.

JEOR Word TemplateThe use of the JEOR Word Template allows authors to view their paper in a style close to the final printed form. All manuscripts will be fully typeset from the author’s electronic files. It should be noted that due to defined typesetting standards and the complex requirements of electronic publishing, the Publisher will not always be able to exactly match the layout the author has submitted. The use of journal templates is preferable and its adoption will speed the publication process.

The elements of the template are:

Title

The Title should be concise and specific, describing the nature of the paper. If the paper has been previously reported in whole or in part at a scientific meeting, this should be stated as a footnote on the introductory page.

Authors’ Names

The Authors’ Names should have forename in full and middle initial(s) (e.g., Hubert H. Smith). Give full addresses for each author, and connect each author with an address. Authors with the same address must be given together. Do not list all of the authors and then all of their affiliations separately. Indicate with an asterisk the author to whom all correspondence concerning the manuscript should be sent, and indicate email address for this author. Specify very clearly the affiliation of each author.

Abstract

The Abstract is an important summary of the work because it is often the only portion of the paper read by abstracting journals. As a consequence, the abstract should be written so that it can be used verbatim in an abstracting journal. It should be a concise summary giving essential information, data, etc., and be intelligible without reference to the paper itself. No references to cited publications are permitted in the Abstract, nor should there be any numerical reference to a structure found in the text.

Key Word Index

The Key Word Index should provide relevant key words for both indexing and abstracting. For a specific plant, list species name and plant family name, and also common name if well known. For reports on the analysis of an essential oil, list “essential oil composition” and all the components found in amounts greater than 10%.

Introduction

The Introduction should present the object or reason for the investigation, and clearly states what is new in the paper submitted. A summary of the pertinent literature should be included; however, only the relevant work should be described in a brief, concise manner.

Experimental

The Experimental section should describe clearly and in sufficient detail the materials and methods used so that the experiment could be reproduced by others. Only new techniques need to be described fully, while known methods must have adequate references.


The Results should be presented in a clear, concise manner using tables and illustrations for clarity. Do not list tabular data in the text. Do not list significant data figures for which the level of experimentation error is unacceptable; e.g., for capillary GC data obtained from electronic integration, 1.4%, not 1.35 or 1.349% unless

this is a mean of a large number of analyses. Following presentation of the results they should be discussed and interpreted where possible. The results of other studies of a similar or related nature should be compared with only the most pertinent data and can be listed in tabular form for comparative purposes.

References

The References must be numbered consecutively in the text (one reference per number) and should be typed in order on a separate page. Within the text, the reference should appear as follows: “…as described by Lincoln et al. (5).” A maximum of 30 references is suggested unless the manuscript is a review article. Recent review articles can be used as a substitute for all but the most pertinent original articles. All references must be typed in full, including all editors’ names for book citations, using the following style:

B. M. Lawrence, 1. Essential Oils 1988–1991. Allured Publ. Corp., Carol Stream, IL (1993).

B.M. Lawrence, 2. A Study of the Monoterpene Interrelationships in the Genus Mentha with Special Reference to the Origin of Pulegone and Menthofuran. Ph.D. Thesis, Rijksuniversiteit, Groningen (1978).

B.M. Lawrence and J.K. Morton, 3. Cytological and Chemical Variation in Mentha. Paper No. AG/b-01, Vth International Essential Oil Congress, Sao Paulo, Brazil (1971).

B.M. Lawrence, 4. A further examination of the variation of Ocimum basilicum L. In: Flavors and Fragrances: A World Perspective. Edits., B.M. Lawrence, B.D. Mookerjee and B.J. Willis, pp. 161–170, Elsevier Sci. Publ. B.V., Amsterdam (1988).

D.E. Lincoln, M.J. Murray and B.M. Lawrence,5. Chemical composition and genetic basis for the isopinocamphone chemotype of Mentha citrata hybrids. Phytochemistry, 8, 1857–1863 (1986).

L. Mondello, R. Shellie, A. Casilli, P.Q. Tranchida, P. Marriott, G. Dugo, Ultra-fast essential oil characterization by capillary GC on 50 μm ID column6. . J. Sep. Sci., 27, 699-702 (2004).

The International Fragrance Association Website. 7. http://www.ifraorg.org/ (Feb 10, 2010)

Acknowledgments

The number of acknowledgments should be kept to a bare minimum.

Footnotes

Footnotes should be kept to a minimum. They should be indicated by a superscript number.

GC DataAll reported GC analyses must contain a description of the analytical procedures used including the make and model number of the equipment, the column type and dimensions (e.g. OV-101 30 m x 0.22 mm fused silica capillary column, film thickness 0.25 μm; or 20 ft. x 3/4 inch stainless steel packed column, coated with 10% Carbowax 20 m on 80/100 mesh Chromosorb W NAW). The temperature programming conditions used along with carrier gas flow rate must be described. It is no longer sufficient to refer to a previous publication for a description of analytical conditions. A statement as to how the quantitative data was obtained should be included.

In summary:

Column: dimensions (length, internal diameter), manufacturer and location, type of column (packed, capillary, etc.), support material, film thickness.

Carrier gas: type, purity, flow-rate/linear velocity, inlet pressure and/or pressure programmes.

Temperatures: temperatures of injector, detector, oven (and temperature programmes)

e.g.: GC-FID analyses were carried out on a Shimadzu GC-2010 gas chromatograph operated with a split/splitless injector and a Shimadzu AOC-20i autoinjector (Shimadzu, Kyoto, Japan). Column: SLB-5MS (silphenylene polymer) 30 m x 0.25 mm x 0.25 μm film thickness (Supelco, Bellefonte, IL, USA). Temperature program: from 50–250°C (10 min) at 3°C/min. Injection temperature: 250°C. Injection volume: 1.0 μL. Inlet pressure: 100 kPa. Carrier gas: He, linear velocity (u): 30 cm/sec. Injection mode: split (50:1). FID (250°C): H2 flow: 50 mL/min; air flow: 400 mL/min; make up flow (N2/Air): 50 mL/min. Sampling rate: 40 msec. Data handling was carried out by means of GCsolution 2.3 (Shimadzu).

GC/MS DataIn addition to compliance with the above GC instructions, add EI mode operating at 70 eV, scan time and acquisition mass range. Add modifier if operating in CI mode. Specify type of mass analyzer, Scan mode.

Detection conditions, ions monitored in SIM and dwell time.

e.g.: GC/MS analyses were performed with a Shimadzu GCMS-QP2010 model gas chromatograph-mass spectrometer equipped with an AOC-20i autoinjector. Column: SLB-5MS, 30 m x 0.25 mm ID x 0.25 μm film thickness. Temperature program: from 50°C (2 min) to 250°C (10 min.) at 3°C/min. Injection temperature: 250°C. Injection volume: 1.0 μL. Inlet pressure: 37.1 kPa. Carrier gas: He, linear velocity (u): 32.4 cm/sec. Injection mode: split (10:1). MS interface temp.: 250°C; MS mode: EI; detector voltage: 0.9 kV; mass range: 40-400 u; scan speed: 769 u/s; interval: 0.50 s (2 Hz). Data handling was made through GCMSsolution 2.5 (Shimadzu).

HPLC DataAll reported HPLC analyses must contain a description of the analytical procedures used including the make and model number of the equipment, the column type and dimension (e.g. RP-18 150 x 2.1 mm HPLC column packed with particles of 5 μm). The solvents used, column temperature, flow rate and gradient program/isocratic conditions must be described. Type of detector used, and specific conditions used. It is no longer sufficient to refer to a previous publication for a description of analytical conditions.

e.g.: HPLC separation was carried out on a Shimadzu system equipped with two LC 10 AD Vp pumps, an SPD-M10 Avp UV detector, a SCL-10-Avp controller, a CTO-20AC column oven thermostated at 30°C and a degasser DGU-14A, data were acquired and processed by LC-solution ver 3.3 software. The column used was an Ascentis Express C18 150 mm x 4.6 mm i.d. with particle size of 2.7 μm (Supelco, Bellefonte, PA). The injection volume was 2 μl, mobile phase consisted of Water, Acetonitrile, THF (85:10:5) (solvent A) and Acetonitrile, Methanol, THF (65:30:5) (solvent B), the linear gradient profile was as follow: 0-5 min, 0% B, 5-25 min, 0-40% B, 25-45 min, 40-90% B, 45-55 min, 90% B, 55-60 min, 0% B. Flow-rate was 1.0 mL/min, data were acquired using a photodiode array detector in the range 190-370 nm and the chromatograms were extracted at 315 nm. Time constant was 0.64 s and sample frequency 1.5625 Hz. Data acquisition was performed by Shimadzu LC solution software ver 3.3.

HPLC/MS DataIn addition to compliance with the above HPLC instructions, specify inlet system, source (vaporizer and capillary temperature, nebulising, auxiliary or ionizing gases, source voltage, CID voltage), mass analyzer (scan mode, resolution and mass range), detection.

SpectraThe inclusion of MS, IR or NMR spectra of uncommon or newly characterized compounds is encouraged. If no adequate MS or IR spectrum of a compound can be found in the readily accessible literature, then its inclusion is also encouraged.

ChromatogramsThe inclusion of a chromatogram adds substantial value to the paper, the incorporation of chromatographic profiles is strongly encouraged.

TablesTables should be double-space typed in the same form as the manuscript and should be numbered using Roman numerals; however, they should not be formatted within the text but should be included as an attachment, at the end of the article. Each table should be on a separate page. The inclusion of retention indices in tables of components identified is encouraged whereas the inclusion of retention times is not. Unless the quantitative data is an average of more than 5 analyses, only one decimal place is acceptable. Averages of more than 5 analyses can be presented in two decimal places. Tables should be typed as word documents only, using tabs, and not the space bar. Tables should be typed using Microsoft Word program only. Do not use programs like Excel to create tables, because eventually when you send the article to us on disk, we are not able to read anything that is created with cells.

Keep the number of columns in a table as few as possible and keep the titles or headings concise. Essential details in the title can be added as a footnote to the table. The compounds must be listed in the table in elution order from the GC column. Compounds that can exist in isomeric form that have not been fully characterized

should have an asterisk and footnote stating “correct isomeric form not identified.” Unknowns, tentatively identified compounds or those partially characterized as, for example, “sesquiterpene hydrocarbon,” can only be included in the table if the MS data is included as a footnote to the table.

FiguresFigures can be high quality photographic prints or camera-ready original diagrams, graphs or drawings. Spectra or chromatograms should be presented as high quality photographic prints or camera-ready computer drawn representations. All figures should be accompanied by a descriptive phrase or sentence typed on a separate page. All figure captions can be listed on the same page. The size of figures submitted should not exceed 6 x 10 inches.

Chemical NomenclatureUse generally accepted chemical nomenclature. For example, the use of trivial terpene names is recommended: a-pinene, spathulenol, b-bourbonene, etc. For IUPAC names, the author is requested to determine whether there is a trivial name for the compound; if there is, it should be used.

Species NamesAll experimental plants listed must be given their correct taxonomic classification, including the author citation; e.g., Micromeria teneriffae Benth. Once cited in the text, the following reference to the species can be written as M. teneriffae. Depositing a voucher specimen of each plant species in the herbarium of a reputable university or institution is mandatory for all plants collected from the wild state. Also, if other Micromeria species are mentioned after the genus has been introduced, then they may be cited as follows: M. benthamii: Webb. & Berthel., M. biflora Benth., etc. When a sentence commences with a species name, the species should be written in full, i.e. Micromeria biflora, not M. biflora.

Structural FormulaeAll structural formulae should be drawn with the aid of a graphics software package, dry transfers, a template, or some accurate structural design facsimile. Under each structure should be a boldface number. This is the same number that should appear in the text in bold (within square brackets) after the compound has been named; e.g., 4-ketoisophorone [8]. The inclusion of structural formulae of known compounds is discouraged unless they are considered to be essential for a better understanding of the text.

NMR DataNMR Data must be specified as either 1H-NMR or 13C-NMR. It is necessary to state the frequency of the instrument, the solvent used, and the internal standard. Chemical shifts should be noted in d (ppm) values relative to TMS. The type of signal should also be noted; e.g., singlet s, doublet d, triplet t, multiplet m, etc. Two examples of NMR data presentation are:

1. 1H-NMR (250 CDCl3/TMS): d 0.87(d, CH3), d 0.89(d, CH3), d 1.28(s, CH3OH), d 1.93(bm, CH2OH), d 5.63(bs, HC=CH).

2. 13C-NMR (25.15 MHz CDC13): d 67.9(C-1), d 134.0(C-2), d 133.7(C-3), d 42.6(C-4), d 22.7(C-5), d 37.7(C-6), d 32.3(C-7), d 32.1(C-8), d 20.1(C-9), d 19.1(C-10)—a decoupled experiment. If coupled experiments are performed, then the type of signal should be included.

MS DataPresentation of data should indicate the method used; e.g., MS (this is for EIMS), CIMS, GC/MS, and the ionizing energy. An example of data presentation can be seen as follows: MS, 70 eV, 210°C, m/z(rel. int.): 154[M]+(6), 139(28), 136(20), 121(22), 111(27), 93(100), 84(30), 79(50), 77(48), 71(45), 69(35), 55(22), 43(37).

Qualitative Analysis/Identification of CompoundsCompound identification must be supported by at least two methods of analysis. They can be mass spectrometry and gas chromatography with calculation of retention indices. Retention Indices should be included in the tabular data and compared to literature data obtained on the same stationary phase.

Some available databases of LRI are suggested here:http://www.alluredbooks.com/idofesoilbyg.htm• l http://www.odour.org.uk/index.htm• l http://www.shimadzu.com/products/lab/ms/oh80jt00000059kf.htm• l

http://www.wiley.com/WileyCDA/WileyTitle/productCd-0470425210.htm• l

Quantitative Analysis/Composition of the SampleAuthors may report simple GC percentages obtained using FID or TCD as detector, assuming response factors equal to unity for all the components. This procedure is not possible if mass spectrometer and NPD are used as detectors. Authors are strongly encouraged to provide a true quantification at least of major components (greater than 10% of the whole oil) of the analyzed sample. If standards are not commercially available, for the measurement of response factors, compounds can be grouped into chemical classes (hydrocarbons, aldehydes, etc.) and subclasses (monoterpenes, sesquiterpenes, etc.) and semi-quantification can be carried out using one (or more) pure standard for each class of similar components. Refer to “Results and Discussion” paragraph for an appropriate number of significant figures to be reported.

AbbreviationsStandard abbreviations should be used throughout the manuscript, particularly in the experimental section. Some examples of common abbreviations are:

°C, IR, GC, GC/MS, HPLC, TLC, FTIR, 1H-NMR, 13C-NMR, CD, l max (solvent), [a]D Temp, nm, cm-1, μL, °C/min, μm, kg/ha, mL, L, mg, min, eV, ppm, FID, TC, EC, etc. (Note: No periods are needed).

CopyrightThe copyright of all papers remains the property of the author unless transferred to JEOR, but the Journal has the sole right of publication for a period of six months from the date of publication. Papers appearing in JEOR may be published elsewhere after the six-month grace period has elapsed, provided that acknowledgment of the original publication is given.

Manuscript ChecklistManuscript prepared according to the journal guidelines •Experimental part includes the description of all the techniques and conditions used, so that methods can •be reproduced by others. Correct taxonomic classification of all plant material has been given •A voucher specimen of each plant specie collected from the wild state has been deposited in the •herbarium of a reputable university or institution Identification of components has been carried out using at least two methods (e.g. MS and GC data •correlated with LRI)

LRI compared with those reported in referred literature or reputable databases •

ProofsPrior to publication, galley proofs will be sent to the contact author for checking. Corrections should be restricted to typographical or similar errors. Modifications to the original text should be avoided at all costs, otherwise the publication of the article will be seriously delayed. Galley proofs should be returned to the publisher within three days of receipt. Please provide e-mail address to receive proof.

ReprintsThe contact author will be e-mailed a PDF file of the paper to use for a limited number of reprints. To order additional reprints of the article, please check with the publisher regarding the prices.

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