36 S. GONZÁLEZ ET AL.

Copyright © 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2004; 19: 36–39

FLAVOUR AND FRAGRANCE JOURNALFlavour Fragr. J. 2004; 19: 36–39Published online 1 October 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1270

Aromatic plants from Patagonia. Part I. Antimicrobialactivity and chemical composition of Schinuspolygamus (Cav.) Cabrera essential oil

Silvia González,1 Pedro E. Guerra,1 Hugo Bottaro,1 Soledad Molares,1 Mirta S. Demo,2 Maria M. Oliva,2

Maria P. Zunino3 and Julio A. Zygadlo3*

1 Facultad de Ciencias Naturales and Facultad de Ingeniería, Universidad Nacional de la Patagonia sede Esquel, Argentina,Sarmiento 849, 9200 Esquel, Chubut, Argentina

2 Departamento de Microbiología, Universidad Nacional de Río Cuarto, Argentina3 Instituto Multidisciplinario de Biología Vegetal, Cátedra de Química Orgánica, FCEFyN-UNC, Avenida Vélez Sarsfield 1600,

5000 Córdoba, Argentina

Received 29 July 2002

Revised 16 December 2002

Accepted 17 December 2002

ABSTRACT: The essential oil of Schinus polygamus (Cav.) Cabrera f: chubutensis (syn. S. marchandii Barkley)was analysed for the first time by means of GC and GC–MS. Of 30 identified compounds, representing 96.3% ofthe oil, α-phellandrene and limonene were the major components. The oil showed high antimicrobial activity againstB. cereus. Copyright © 2003 John Wiley & Sons, Ltd.

KEY WORDS: Schinus polygamus; Anacardiaceae; α-phellandrene; limonene; essential oil composition; anti-microbial activity

* Correspondence to: J. A. Zygadlo, Instituto Multidisciplinario de BiologíaVegetal, Cátedra de Química Orgánica, FCEFyN-UNC, Avenida VélezSarsfield 1600, 5000 Córdoba, Argentina.E-mails: jzygadlo@gtwig.efn.uncor.edu and jzygadlo@efn.uncor.eduContract/grant sponsor: SECyT, Argentina.Contract/grant sponsor: CONICET, Argentina.

of Argentina, in July 2000. Voucher specimens arekept in the Herbarium of the UNPAT sede Esquel(Bottaro 52).

Preparation of the Essential Oil

Dried leaves of S. polygamus were hydrodistilled in aClevenger-type apparatus to yield 0.2% oil. The oilobtained was dried over anhydrous sodium sulphate andstored in a refrigerator until analysis.

Gas Chromatography Analyses

GC

Analyses were performed in a Shimadzu GC-R1A (FID)gas chromatograph, fitted with 30 m × 0.25 mm i.d.,0.25 µm film thickness, fused silica capillary columnscoated with a non-polar DB-5 (phase 5% phenyl 95%dimethylpolysiloxane) or with a polar Supelcowax 10phase (polyethyleneglycol). The GC operating con-ditions were: oven temperature programmed from 40to 230 °C at 2 °C/min; injector and detector tempera-tures 240 °C; carrier gas, nitrogen at a constant flow of0.9 ml/min.

Introduction

S. polygamus, molle, is an aromatic and medicinalshrub native to Patagonia.1,2 Local people chew leaves ofmolle to clean the teeth, and drink molle juice for childdiuresis.3,4 The flavonoids and extracts of S. polygamus

have been studied previously.5,6 The composition of theessential oil of S. polygamus from Patagonia is reportedin the present paper. To the best of our knowledge, noprevious studies have been reported on the compositionof this oil.

Experimental

Plant Material

Schinus polygamus (Cav.) Cabrera f. chubutensis (syn.S. marchandii Barkley), Family Anacardiaceae, commonname ‘molle’, ‘incienso’ or ‘trementina’. The plants werecollected in Arroyo Cuche, in the Chubut province

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Copyright © 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2004; 19: 36–39

GC–MS

GC–MS analyses were performed with a Perkin-ElmerQ-700 equipped with an apolar SE-30 capillary column(30 m × 0.25 mm i.d., film thickness 0.25 µm), 100%dimethylpolysiloxane. The operating conditions were:oven temperature programmed from 40 °C to 230 °C at2 °C/min; carrier gas, helium, at a constant flow of0.9 ml/min; source at 70 eV.

Identification

The constituents of the essential oils were identifiedon the basis of their GC retention indices (RI) withreference to a homologous series of n-alkanes (C12–C25), by comparison of their retention times with thoseof pure authentic samples from the Sigma, Fluka andPalma Companies, peak enrichment on co-injectionwith authentic standards wherever possible, by GC–MS library search (Wiley and Nist) and using visualinspection of the mass spectra from literature, forconfirmation.

Testing for Antimicrobial Activity

Antimicrobial activity was assayed against 10 micro-organisms (four Gram-positive, five Gram-negative anda yeast): Micrococcus luteus (ATCC 9341); Bacillus

cereus; Staphylococcus aureus (ATCC 25512); Staphylo-

coccus epidermidis; Escherichia coli; Proteus mirabilis;Enterococcus faecalis (ATCC 29212); Pseudomonas

aeruginosa; Klebsiella sp.; and Candida albicans. Strainsobtained from water, food and clinical samples weretypified in the Microbiology and Immunology Depart-ment Laboratory of the National University of Rio Cuarto(Argentina). All the strains tested were maintained at4 °C in tryptone-soya agar and were subcultured everymonth. The fungus was stored at 4 °C in Sabouraudagar and subcultured every month. The paper disc dif-fusion method was used for testing antimicrobial acti-vity. It was performed using an 18 h culture, growth at37 °C and adjusted to approximately 106 colony-formingunits (cfu)/ml; 200 microliters of the inoculum werespread over plates containing Mueller–Hinton agar anda paper filter disc (diameter 6 mm) impregnated with10 µl essential oil was placed on the surface of themedia. The plates were left for 30 min at room temper-ature to allow the diffusion of the oil, then they wereincubated at 37 °C for 24 h. After this time, the inhibi-tion zone around the disc was measured using a verniercaliper.

The minimum inhibitory concentration (MIC) wasprobed only with microorganisms that showed inhibitoryzones with a diameter >10 mm, following as criterion

that an inhibitory area of essential oil of 10 mm is appro-ximately 50% of the inhibitory area of gentamicin.The MIC was made in Mueller—Hinton agar and theinoculum density was to 106 microorganisms/ml.

MIC was determined by the dilution of essential oilin dimethyl sulphoxide, absorbing 10 µl of each dilutionon a filter paper disc. Dilutions of the emulsions of essen-tial oils were made over the concentration range of10– 45 000 µg/ml. MIC was defined as the lowest con-centration that inhibited visible growth.7

Two controls were included in the test. The first wasa filter paper disc to which 10 µl dimethyl sulphoxidewas applied. The second involved the antibiotic gentamicin(10 µg) for bacteria and amphotericin B (20 µg/ml) forC. albicans, in order to check the sensitivity of the testorganisms.

Antifungal experiments were made in the same wayusing malt-extract broth for the culture and Sabouraudagar for the plates.

Sensorial Test

This was done in two human groups, one from Patagonia,where the plant grows and is used in medicine andanother from the central area of Argentina where theplant is unknown. The preferential test was used8 assensorial test.

Results and Discussion

The percentage composition of the oil of S. polygamus isshown in Table 1. The concentration of each componentwas calculated from GC peak areas without correctionfactors. Components are listed in order of elution froma DB-5 column. Thirty compounds were identified, re-presenting 96.3% of the total oil. The volatile oil ofS. polygamus was characterized by a high monoter-pene content 58.3%, where α-phellandrene (12.9%) andlimonene (22.6%) were the major components. Amongthe sesquiterpenes, the oxygenated sesquiterpenes repre-sented 20.4%, with α-cadinol (7.1%) and cubenol (5.6%)being the main compounds.

In accordance with the identified components of theessential oil of S. polygamus, the following statements onthe above-mentioned applications of this essential oilcan be given. The folk medicinal application as a teethcleaner can be partly correlated to flavour and antimicro-bial activity although the essential oil of S. polygamus

essential oil has not shown strong antibacterial activity,except against B. cereus (Table 2). The sensorial testshowed that the essential oil of S. polygamus wasaccepted by the Patagonian people as a pleasant flavour,while it was not accepted by inhabitants from central areaof Argentina (Figure 1).

38 S. GONZÁLEZ ET AL.

Copyright © 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2004; 19: 36–39

Table 1. Essential oil composition of S. polygamus

Compounds % Retention Index Methods of

DB5 Supelcowax 10identification

Sabinene 3.7 976 1128 MS–COMyrcene 3.3 992 1161 MS–COααααα-Phellandrene 12.9 1005 1167 MS–COα-Terpinene 6.2 1016 1172 MS–COLimonene 22.6 1031 1190 MS–COZ-β-Ocimene 3.0 1039 MSE-β-Ocimene 1.0 1050 1254 MScis-Linalool oxide (furanoid) 2.0 1062 MSMenth-2-en-1-ol (cis-p) 2.0 1126 MSMenth-2-en-1-ol (trans-p) 2.1 1140 MSCamphor 0.6 1143 MS–COCitronellyl acetate 0.9 1314 MS–COβ-Elemene 0.8 1394 1588 MSUnknown 0.2α-Gurjunene 0.8 1403 1525 MSLongifolene 2.0 1404 MSβ-Caryophyllene 0.1 1419 1598 MS–COUnknown 0.1 1420α-Humulene 0.8 1454 1663 MSFarnesene (E,β) 0.8 1458 1672 MSGermacrene D 3.3 1480 1707 MSα-Muurolene 0.6 1499 1695 MSγ-Cadinene 0.1 1506 1824 MSδ-Cadinene 3.2 1524 1744 MSGermacrene B 2.8 1557 1811 MSSpathulenol 3.7 1576 2110 MS–COCaryophyllene oxide 2.5 1581 2032 MSCubenol 5.6 1632 2097 MSβ-Eudesmol 1.5 1649 2283 MSα-Cadinol 7.1 1653 2212 MS

Total 96.3

Compounds are listed in order of elution from a DB-5 column. MS, compounds identified by mass spectra and CO compounds thathave also been identified by co-injection of standards.

On other hand, the high content of limonene andα-cadinol could explain the anti-inflammatory pro-perties9,10 of S. polygamus. Moreover, α-cadinol hasshown selectivity for the human colon tumour cell line(HT-29).11

Acknowledgement—We are grateful to SECyT-UNC and CONICET forfinancial support.

References

1. Cabrera, AL. Rev. Museo la Plata, Botán., 1938; 6: 3–64.2. Correa, M. Flora Patagonica, vol V. Colección Científica del

INTA, 1988.3. San Martín J. Econ. Bot., 1983; 37: 216–227.4. Martínez Crovetto R. Breve Panorama de las Plantas Utilizadas

por los Indios de Patagonia y Tierra del Fuego—Suplemento

Antropológico. Asunción, Paraguay, 1982; 17: 20–24.

Table 2. Antimicrobial activity of essential oil of S. polygamus (inhibition zone diameter measured in mm, thedisc diameter of 6 mm being included). All values are average of three replicates

Microorganism Inhibition zone (mm) MIC (µg/ml) Gentamicin Amphotericin-Bof essential oil of essential oil (10 µg/ml) (20 µg/ml)

B. cereus 17.5 56.2 20.5 NTS. aureus 9.0 NT 26 NTS. epidermidis 8 NT 26 NTM. luteus 9.5 NT 20.5 NTE. faecalis 9.5 NT 18.5 NTE. coli 8 NT 18 NTKlebsiella sp. 7 NT 19 NTP. mirabilis NA NT 20 NTP. aeruginosa NA NT 20 NTC. albicans 10 237 NT 20

NT, not tested; NA, not active.

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Copyright © 2003 John Wiley & Sons, Ltd. Flavour Fragr. J. 2004; 19: 36–39

5. Mandich LM, Barros C, Silva MJ. Bol. Soc. Chil. Quim., 1982;27: 175–177.

6. Mandich LM, Bittner M, Silva M, Barros C. Rev. Latinoam.

Quim., 1984; 15: 80–82.7. Demo, MS, Oliva MM, Ramos B, Zygadlo JA. Higiene

Alimentar., 2001; 15: 87–90.8. Anzaldua Morales A. La Evaluación Sensorial de los Alimentos

en la Teoría y la Práctica. Acribia. Zaragoza, Spain, 1994.9. Lu M, Battinelli L, Daniele C et al. Planta Med., 2002; 68: 213–

216.10. Andersson M, Bergendorff O, Shan R et al. Planta Med., 1997;

63: 251–254.11. He K, Zeng L, Shi G. J. Nat. Prod., 1997; 60: 38– 40.

Figure 1. Preferential test of essential oils of S.polygamus, p < 0.01 (n = 33)