474 C. J. SILVA ET AL. FLAVOUR AND FRAGRANCE JOURNAL Flavour Fragr. J.

2007; 22: 474478 Published online 18 July 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ffj.1823

Comparative study of the essential oils of seven Melaleuca (Myrtaceae) species grown in Brazil
Cleber J. Silva,1,2 Luiz C. A. Barbosa,1* Clia R. A. Maltha,1 Antnio L. Pinheiro3 and Fyaz M. D. Ismail4
1 2 3 4

Chemistry Department, Federal University of Viosa, 36571-000 Viosa-MG, Brazil Plant Biology Department, Federal University of Viosa, 36571-000 Viosa-MG, Brazil Forest Engineering Department, Federal University of Viosa, 36571-000 Viosa-MG, Brazil Medicinal Chemistry Research Group, School of Pharmacy and Chemistry, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK

Received 23 March 2007; Revised 23 May 2007; Accepted 30 May 2007

ABSTRACT: The compositions of the essential oils obtained from seven species of Melaleuca grown in the municipal district of Viosa, State of Minas Gerais, Brazil, were analysed by both GC and GCMS. The various Melaleuca species showed differences within their relative essential oil compositions. Each of the oils analysed revealed a dominant component: terpinen-4-ol (53.7 0.2%) for M. alternifolia; 1,8-cineole for M. armillaris (80.2 0.0%), M. ericifolia (79.5 0.4%), M. cajuputi, subspecies cajuputi (43.7 0.5%) and M. cajuputi subspecies platyphylla (41.0 8.5%); viridiorol (71.0 0.9%) for M. quinquenervia; and methyl eugenol (96.6 0.7%) for M. leucadendra. Copyright 2007 John Wiley & Sons, Ltd. KEY WORDS: Melaleuca alternifolia; Melaleuca cajuputi subsp. cajuputi; Melaleuca cajuputi subsp. platyphylla; Melaleuca armillaris; Melaleuca leucadendra; Melaleuca quinquenervia; Melaleuca ericifolia; chemotypes

Introduction
The genus Melaleuca L. (family Myrtaceae) is currently represented by approximately 250 described species which predominate in Australia.1 It is also one of the most important essential oil-producing species. The leaves and stems of several Melaleuca species produce strongly scented essential oils, some of which have useful medicinal properties.2 For instance, recent studies on the treatment of human melanoma involving essential oils from M. alternifolia (tea tree oil) demonstrate that the mechanism of action may involve the interaction of oily components with cell membranes.3 Furthermore, its essential oils possess a broad spectrum of biological activity, which upon topical application shows some effectiveness against herpes simplex virus (HSV).4 As a consequence, it is a potentially useful and cheaper alternative to more expensive anti-retrovirals (such as acyclovir, valacyclovir or fancyclovir) does not possess their side effects, increasing patient compliance and acceptability. More importantly, it poses a lesser threat of inducing resistance than systemically applied antiviral agents.5 Besides these medicinal uses, its compounds have also been extensively employed by the cosmetic industry.6 The growing inter* Correspondence to: L. C. A. Barbosa, Universidade Federal de Viosa Departamento de Qumica, Av. PH Rolfs s/n, CEP 36570-000 Viosa-MG, Brazil. E-mail: lcab@ufv.br Contract/grant sponsor: Conselho Nacional de Desenvolvimento Cientco e Tecnolgico (CNPq), Brazil. Contract/grant sponsor: Fundao de Amparo a Pesquisa de Minas Gerais (FAPEMIG), Brazil.

est in the production of essential oil of this species has also led to the development of tissue culture techniques to study the genetic and environmental inuences upon M. alternifolia oil composition.7 In many species of aromatic plants, variations in the chemical composition of the volatile oils are used for the identication of different chemotypes.8 Modern theories have established that secondary metabolites express themselves as a result of external stimuli. According to this theory, an organism can produce completely different groups of metabolites depending on the environmental conditions, duration and intensity of stress, composition and genetic plasticity of plants.9 Melaleuca species grown in Brazil may, therefore, express chemotypes different from those found in other environments, such as Australia. There is also the possibility that some of these chemotypes can be exploited for the production of commercially valuable essential oils. As part of our screening programme identify valuable commercial aromatic and plants grown in Brazil,10 we report the chemical composition of the essential oils obtained from seven Melaleuca species currently cultivated in Brazil which may also have medicinal value.

Materials and Methods

Plant Material Aerial parts of M. alternifolia Cheel, M. leucadendra (L.) L, M. ericifolia Sm., M. armillaris Sm., M.

Copyright 2007 John Wiley & Sons, Ltd.

Flavour Fragr. J. 2007; 22: 474478 DOI: 10.1002/ffj

ESSENTIAL OILS OF MELALEUCA SPP. 475

quinquenervia (Cav.) S. T. Blake and M. cajuputi Roxb. subsp. cajuputi Powell, and M. cajuputi Roxb. subsp. platyphylla Barlow were collected in February 2006, from plants grown in the arboretum of the Forest Engineering Department, Dendrology Sector, at the Federal University of Viosa (UFV), state of Minas Gerais, Brazil. M. alternifolia samples were obtained from a 10 year-old plantation and samples of all the other species came from 4 year-old plantations. In all cases, the seeds originated from CSIRO, Division of Forestry and Forest Products, Canberra, ACT. The materials were identied, and a voucher specimen of each plant was deposited in the VIC Herbarium (Registration Nos 3083930845) of the Plant Biology Department, Federal University of Viosa (UFV).

(Wilwaukee, WI, USA) or Acros Organics (Morris Plains, NJ, USA) (-terpineol, 99%; terpinen-4-ol, 97%). Gas ChromatographyMass Spectrometry (GCMS) The GCMS unit (Model GCMS-QP5050A; Shimadzu, Japan) was equipped with a DB-5 fused silica column (30 m 0.22 mm i.d., lm thickness 0.25 m) and interfaced with an ion trap detector. Oven and injector temperatures were as described above; transfer line temperature, 240 C; ion trap temperature, 220 C; carrier gas, He at a ow rate of 1.8 ml/min; split ratio, 1:10; column pressure, 100 kPa; ionization energy, 70 eV; scan range, 29 450 u; scan time, 1 s. The identity of the components was assigned by comparison of their relative retention indexes (RRI) to a standard alkane series (C9 C24)12 and secondly by comparison of their mass spectra with either reference data from the equipment database (Wiley 330 000) or from the literature for additional verication.13

Extraction and Analysis of Essential Oils The leaves were collected separately from random points on the trees under investigation. Stems and leaves were extracted separately for both M. alternifolia and M. armillaris. Each sample was subdivided into three portions of 100 g each, chopped and then subjected to 3 h hydrodistillation in a Clevenger apparatus. The resulting oils were weighed and the reported yields were calculated with respect to dry material weight. All distillations were repeated three times and the oils produced in this process were stored under a nitrogen atmosphere, maintained at approximately 0 C, until they were analysed chromatographically. Leaf dry weight was calculated by drying each sample (2 g, held at 103 2 C for 24 h) according to published methods (ASAE, 2000).11 Each determination was carried out in triplicate.

Results and Discussion

Hydrodistillation of M. alternifolia, M. cajuputi subsp. cajuputi, M. armillaris and M. cajuputi subsp. platyphylla gave yields of volatile oils of 4.0 0.36%, 4.0 0.21%, 3.9 0.20% and 3.7 0.22% w/w, respectively. These values are higher than the yields obtained for M. leucadendra, M. quinquenervia and M. ericifolia (1.5 0.11%, 1.6 0.17% and 0.70 0.01% w/w, respectively). In the current study, the stems and leaves of M. alternifolia and M. armillaris were extracted and analysed separately. In these cases, the oil contents found for leaves of M. alternifolia and M. armillaris were 4.0 0.36% and 3.9 0.20% w/w, respectively. In contrast, the stems of M. alternifolia and M. armillaris were a comparatively poor source of oil (0.01 0.00% and 0.02 0.01% w/w, respectively). The compounds identied within the volatile oils from the seven Melaleuca species, along with their percentage composition, are shown in Table 1. The major components found in the volatile oils were terpinen-4-ol (53.7 0.2% in M. alternifolia), 1,8-cineole (80.2 0.0% in M. armillaris; 79.5 0.4% in M. ericifolia; 43.7 0.5% in M. cajuputi subsp. cajuputi; 41.0 8.5% in M. cajuputi subsp. platyphylla), viridiorol (71.0 0.9% in M. quinquenervia) and methyl eugenol (96.6 0.7% in M. leucadendra). The percentages of 1,8-cineole, terpinolene and terpinen-4-ol in the volatile oils were used to characterize different chemotypes of Melaleuca species. The literature reports ve chemotypes for M. alternifolia, based on the concentrations of 1,8-cineole, terpinolene and terpinen-4-ol.14,15 The International Standard Organization (ISO 4730) has established that M.

Gas Chromatography GC analysis were accomplished using a GC-17A Series instrument (Shimadzu, Japan) equipped with a ame ionization detector (FID). Chromatographic conditions were: fused silica capillary column (30 m 0.22 mm i.d.) with a DB-5 bonded phase (0.25 m lm thickness); carrier gas, nitrogen at a ow rate of 1.8 ml/min; injector temperature, 220 C; detector temperature, 240 C; column temperature was programmed to start at 55 C (isothermal for 2 min), then rising at 3 C/min to 240 C, then held isothermal at 240 C for 15 min; injection, 1.0 l (1% w/v in CH2Cl2); split ratio, 1:10; column pressure, 115 kPa. Quantitative analyses were carried out in triplicate by external standardization for major constituents within each oil sample. Reference samples and analytical chemicals (i.e. 1,8-cineole, 99%; -terpinene, 97%; citronellol, 98%; methyl eugenol, 98%) were purchased from Aldrich

Copyright 2007 John Wiley & Sons, Ltd.

Flavour Fragr. J. 2007; 22: 474478 DOI: 10.1002/ffj

Table 1. Percentage composition of the essential oils from the leaves of seven species of Melaleuca MCJ MER SD MLD SD MPT SD MQQ SD MAL* MAR*
SD 7.2 1.1 3.2 0.0 1.9 1.0 1.2 0.3 0.1 0.5 0.6 2.6 7.0 3.0 5.7 3.1 0.2 1.2 1.6 0.5 41.0 0.8 0.3 0.2 0.7 7.0 2.0 7.0 13.0 0.9 1.9 1.5 3.5 1.5 0.3 1.0 0.3 0.7 0.8 0.3 1.3 0.9 0.8 1.1 95.8 8.1/80.5 2.1 0.4 0.3 0.1 8.5 0.2 0.1 0.03 0.1 0.2 0.06 1.5 3.1 0.2 0.4 0.2 0.04 0.3 0.2 0.7 0.2 0.4 0.1 0.2 0.3 0.06 0.09 0.5 4.7 7.2 2.2 3.5 71.0 88.6 4.7/9.4 2.0 1.6 94.2 33.2/57.4 3.9/3.3 96.6 0.0/74.5 23.7/25.9 5.3 0.0/3.6 19.5/42.1 1.3 SD 0.7 0.06 0.1 0.0 0.1 0.6 leaves 7.6 3.7 18.9 3.0 53.7 3.7 SD 0.2 0.5 0.2 0.03 0.2 0.1 0.1 0.1 0.6 0.7 0.03 0.02 0.1 0.1 0.09 Stem 14.2 2.4 15 1.8 1.3 13.6 2.8 1.4 2.3 1.7 1.7 5.0 17.6 10.8 7.0 98.6 1.8/33.9 0.1 0.3 0.5 0.1 0.1 0.3 0.0 0.1 0.3 0.3 0.1 5.9 79.5 0.3 8.0 93.7 87.5/6.2 0.4 0.4 0.0 0.2 96.6 0.3 0.3 0.2 0.8 0.5 98.7 0.0/0.0 0.5/1.6 0.0/0.0 2.8 0.9 43.7 0.5 1.0 22.6 0.8 0.6 7.6 13.4 0.6 94.5 66.3/5.2 22.2/0.8 0.5 0.6 0.2 0.02 0.9 Stem 24.2 2.2 5.3 10.2 3.6 5.4 4.5 2.2 2.0 14.2 7.5 81.3 0.0/26.4 SD 1.75 0.1 0.0 0.9 0.4 0.2 1.0 0.2 0.02 0.1 0.3 leaves 4.8 1.6 2.5 80.2 1.09 9.2 99.4 8.9/90.4

476

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Constituent

RIIa SDc

RIb

C. J. SILVA ET AL.

-Thujene -Pinene Sabinene -Pinene Myrcene -Careno Terpinene p-Cymene 1,8-Cineoled -Terpinened -Terpinolene Linalool Neo-isopulegol Citronellal Terpinen-4-old -Terpineold Citronellold Neral Geraniol Geranial -Terpinyl acetate Citronellyl acetate Geranyl acetate Tetradecane Methyl eugenold (E)-Cariophyllene -(Z)-Farnesene -(E)-Farnesene Aromadendrene -Humulene Germacrene D (Z)--Guaiene Viridiorene Geranyl-isobutyrate -Cadinene Ledol Spathulenol Caryophyllene oxide Globulol Viridiorol epi--Cadinol -Muurolol Identied (%) Monoterpene hydrocarbons/oxygenated Sesquiterpene hydrocarbons/oxygenated Others

932 935 976 976 990 1014 1019 1023 1032 1060 1089 1097 1144 1154 1176 1189 1234 1240 1254 1270 1349 1353 1382 1399 1406 1417 1438 1457 1437 1451 1478 1490 1493 1511 1521 1564 1575 1581 1583 1589 1640 1651

931 939 976 980 991 1011 1018 1026 1033 1062 1088 1098 1145 1153 1177 1189 1228 1240 1255 1270 1350 1354 1383 1399 1401 1418 1443 1458 1439 1454 1480 1490 1493 1514 1524 1565 1576 1581 1583 1589 1640 1645

Flavour Fragr. J. 2007; 22: 474478 DOI: 10.1002/ffj

RRI, relative retention indices relative to C9C24 n-alkanes on a DB5 column. RI, literature retention indices from ref. 13. SD, standard deviation. d Retention time identical to authentic compounds and quantitative results based on external standardization analyses. * For these two species, both the leaves and stems were analysed. MCJ, M. cajuputi subsp. cajuputi; MER, M. ericifolia; MLD, M. leucadendra; MPT, M. cajuputi subsp. platyphylla; MQQ, M. quinquenervia; MAL, M. alternifolia; MAR, M. armillaris.

ESSENTIAL OILS OF MELALEUCA SPP. 477

alternifolia commercial oil should have a minimal of 30.0% composition of terpinen-4-ol and maximum level of 15.0% content of 1,8-cineole.16 These recommendations reect the concentrations required to maintain the antiseptic property attributed to the volatile oil of M. alternifolia, which is primarily dependent on the concentration of oxygen-containing monoterpene terpinen4-ol. The chemical analysis of M. alternifolia essential oils from plants cultivated in Viosa showed that the concentrations of the principal constituents comply with the ISO 4730 (2004)16 specications, and constitutes a chemotype possessing 1,8-cineole concentrations below 8.0%, adequate for commercialization. Investigations on both the chemistry and essential oils chemical composition of M. armillaris are rare. The leaf oil of M. armillaris growing in Egypt contains 33.7 33.9% of 1,8-cineole, whereas the terpinen-4-ol content is 18.724.8%.17,18 In the present study, 1,8-cineole (80.2 0.0%) was identied as the major component of the volatile oils from the leaves of this species, followed by -terpineol (9.2 0.6%). Oil obtained from the stems principally consisted of -terpineol (15.0 3.2%), 1,8cineole (14.2 7.2%), globulol (17.6 7.0%) and E-caryophyllene (13.6 1.0%) (Table 1). The literature reported the existence of six chemotypes of M. quinquenervia: four occur in Madagascar, consisting of one chemotype with 37.0% 1,8-cineole within the volatile oil; a second containing 20.0% viridiorol and 5.0% terpinolene; a third rich in viridiorol (48.0%); and a fourth having (E)-nerolidol (87.0%) as the major volatile oil component)19. Other two chemotypes are from Australia and New Guinea: one is comprised of Enerolidol (7495%) and linalool (1430%), whilst another contains 1,8-cineole (1075%), viridiorol (1366%), -terpineol (0.514%) and -caryophyllene (0.528%), in varying proportions and in order of dominance within the oils.20 Other data, however, described the existence of three chemotypes for M. quinquenervia populations occurring in New Caledonia, whose compositions were dominated by either (a) 1,8-cineole (up to 80.0%); (b) terpinene derivatives; or (c) -pinene and viridiorol.21 Studies accomplished with this species in India revealed the presence of 1,8-cineole (48%) as the major component, followed by -terpineol (13%), nerolidol (9%), -pinene (56%) and viridiorol (5%).22 The results found in the present study showed that the plants grown in Viosa, Brazil, correspond to a chemotype with high viridiorol concentration (71.0 0.9%), followed by 1,8cineole (7.2 0.6%) (Table 1). For M. leucadendra, four essential oil chemotypes have been reported: one with 1,8-cineole (64.3%) as the major component.18 Two chemotypes characterized by very high contents of the phenylpropanoids, viz. methyleugenol and (E)-methylisoeugenol (up to 99% and 88%, respectively);23 and one containing signicant quantities of both viridiorol (28.2%) and 1,8-cineol

(21.3%).24 In contrast, analysis of the species grown in Viosa demonstrate high concentrations of methyl eugenol (96.6 0.7%). The economic potential of this plant arises from its eugenol-like scent (imparted by methyl eugenol), which could be exploited either in perfumery or as a food aroma.25 The volatile oils obtained from M. ericifolia had 1,8cineole (79.5% 0.4) as the principal component. Chemical analyses of the essential oils of M. ericifolia, occurring in Australia, also lead to the identication of populations with high contents of either 1,8-cineole or linalool as the oil major component,26 as opposed to methyl eugenol (96.8%) from species grown in Egypt.17 Three chemotypes of M. cajuputi subsp. cajuputi were reported in studies carried out in Japan, with leaf samples and commercial essential oils acquired from Indonesia (four samples) and Japan (two samples). The results indicated either high (5070%), low (31%) or zero levels of 1,8-cineole.27 M. cajuputi subsp. cajuputi grown in Viosa can be identied as a chemotype having moderately high concentrations of 1,8-cineole (43.7 0.5%) in the volatile oils. A comparative study of the oil chemical composition of M. cajuputi subsp. cajuputi and subsp. platyphylla was then carried out. The monoterpene 1,8cineole was the major component, followed by oxygencontaining monoterpene -terpineol (22.6 0.3%) in subsp. cajuputi and citronellol (13.0 3.1%) in subsp. platyphylla. The volatile oils from subsp. cajuputi also contained viridiorol (13.4 0.3%) and -pinene (2.8 0.1%) (Table 1). Selected data presented in Table 1 possessed a high standard deviation (SD) which could be attributed to either hydrodistillation-induced artifact formation or, more likely, biological sample variation. To our knowledge, the observations reported here on the essential oil chemical composition of these subspecies grown in Brazil are novel. Chemotypes of M. alternifolia, M. quinquenervia and M. leucadendra have their volatile oils rich in terpinen-4-ol, viridiorol and methyl eugenol, respectively. In contrast, the present study conrmed that plants of M. armillaris, M. ericifolia, M. cajuputi subsp. cajuputi, and M. cajuputi subsp. platyphylla growing in Viosa, Brazil, have 1,8-cineole-enriched volatile oils.
AcknowledgementsThe authors thank the Brazillian agencies Conselho Nacional de Desenvolvimento Cientco e Tecnolgico (CNPq) for research fellowships (to L.C.A.B. and C.J.S.) and Fundao de Amparo a Pesquisa de Minas Gerais (FAPEMIG) for nancial support.

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Flavour Fragr. J. 2007; 22: 474478 DOI: 10.1002/ffj