288 T. JOVANOVIC ET AL.

Copyright © 2004 John Wiley & Sons, Ltd. Flavour Fragr. J. 2005; 20: 288–290

FLAVOUR AND FRAGRANCE JOURNALFlavour Fragr. J. 2005; 20: 288–290Published online 20 December 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1409

Chemical composition and antimicrobial activity of theessential oil of Acinos arvensis (Lam.) Dandy fromSerbia

Tatjana Jovanovic,1* Dusanka Kitic,* Radosav Palic,2 Gordana Stojanovic2 and Mihailo Ristic3

1 Faculty of Occupational Safety, Carnojevica 10a, 18000 Nis, Serbia–Montenegro2 Department of Chemistry, Faculty of Science, Cirila i Metodija 2, 18000 Nis, Serbia–Montenegro3 Institute of Medical Plant Research ‘Dr Josif Pancic’, Tadeusa Koscuska 1, 11000 Beograd, Serbia–Montenegro

Received 30 May 2003; Revised 21 November 2003; Accepted 1 December 2003

ABSTRACT: The hydrodistilled essential oil of Acinos arvensis (Lam.) Dandy was analysed by GC and GC/MS. Thirty

constituents representing 83.8% of the oil were identified, of which germacrene D (47.0%) was the major component.

Furthermore, b-bourbonene (7.7%), caryophyllene oxide (3.2%) and b-caryophyllene (3.0%) were noted as additional

significant constituents. The antimicrobial activity of the oil was screened against Escherichia coli, Staphylococcus aureus,

Pseudomonas aeruginosa and Klebsiella pneumoniae. All the microorganisms, except for Pseudomonas aeruginosa, were

found to be susceptible to the oil. Copyright © 2004 John Wiley & Sons, Ltd.

KEY WORDS: Acinos arvensis; essential oil; composition; germacrene D; antimicrobial activity

* Correspondence to: T. Jovanovic, Faculty of Occupational Safety,

Carnojevica 10a, 18000 Nis, Serbia–Montenegro.

E-mail: acinos2002@yahoo.com

Experimental

Plant material

The plant material was collected in Rtanj, Serbia, at the

blooming stage (July 2000). A voucher specimen is

deposited in Herbarium Moesicum Doljevac (HMD;

no. 367). Dried and pulverized aerial parts of the plant

(100 g) were hydrodistilled for 2.5 h using a Clevenger-

type apparatus.14–16

Identification procedure

The oil analysis was performed using GC and GC/MS.

Constituents of the oil were identified by comparison of

their mass spectra to those from the MS library (Wiley

275.l) using a computer search and literature.17 For the

purpose of quantitative analysis the area percentage

obtained by FID was used as the basis. The GC analysis

of the oils was carried out on a GC HP-5890 II appara-

tus, equipped with the split–splitless injector, attached to

an HP-5 column (25 m × 0.32 mm; 0.52 µm film thick-

ness) and fitted to FID. The carrier gas flow rate (H2) was

1 ml/min, the split ratio 1:30, the injector temperature

250 °C and the detector temperature 300 °C, while the

column temperature was linearly programmed from 40 to

240 °C at the rate of 4 °C/min. The same analytical con-

ditions were employed for GC/MS analysis, where an HP

G 1800C Series II GCD system was used. The transfer

line was heated to 260 °C. The mass spectra were ac-

quired in EI mode (70 eV), in m/e range 40–400 (column

HP-5MS 30 m × 0.25 mm, 0.25 µm film thickness).

Introduction

In the genus Acinos Miller (Lamiaceae), there are 10

species, which mainly inhabit alkaline soils, dry and

sunny areas in Europe, the Mediterranean, Central Asia,

N. Africa and N. America.1,2 The genus Acinos is repres-

ented by five species in the Flora of Serbia and

Montenegro.1 Acinos arvensis (Lam.) Dandy [syn. Acinos

thymoides Moench, Calamintha acinos (L.) Clairv.,

Satureja acinos (L.) Scheele] is used for medicinal

purposes as an antiseptic, stimulant, tonic and anti-

spasmotic.3 Also, it is applicable internally in melancholy,

for shortness of breath and improving digestion. A.

arvensis was once used to treat bruises, toothache,

sciatica and neuralgia.4

The chemical composition of the oils of A. arvensis,3–5

A. suaveolens,4,5,7,8 A. alpinus,9–11 A. majoranifolius,12 A.

rotundifolius4 and A. troodi13 was previously studied.

There is also one paper devoted to the oil content, the

qualitative and quantitative composition of tannins and

the qualitative analysis of the flavonoids.6 To the best of

our knowledge, there is no previous study in the literature

concerning the antimicrobial activity of the oil of A.

arvensis. In this paper, we report on the composition and

antimicrobial activity of the essential oil of A. arvensis

found in Serbia.

ESSENTIAL OIL OF ACINOS ARVENSIS 289

Copyright © 2004 John Wiley & Sons, Ltd. Flavour Fragr. J. 2005; 20: 288–290

Antimicrobial activity

The oil was tested in vitro using the disc diffusion

method18 with the following microorganisms: Escherichia

coli 95 Torlak, Staphylococcus aureus ATCC 6538,

Pseudomonas aeruginosa ATCC 9027 and Klebsiella

pneumoniae ATCC 10031. The substrate for the micro-

organisms was Antibiotic Medium 1 (Difco Labora-

tories Detroit, MI, USA). The ethanolic solution of the

oil (50 µl) in the dilution of 1:20 (oil:abp. ethanol)

was put on discs of Antibiotica Test Blattchen (Schleicher

and Schuell, Dassel, Germany; diameter 12.7 mm).

The discs were then put on the antibiotic medium

sown with micoorganisms and kept at 37 °C. After

18 h the activities were determined on a Fisher-Lillly

Antibiotic Zone Reader (Fisher Scientific Co., USA)

by measuring the inhibition diameter around the

discs.

Results and discussion

The oil content of the aerial dried parts of A. arvensis

was 0.05 ml (0.05%, v/w). Compared with other Acinos

species, whose oil yields were between 0.01 and

2.3%2–4,8–12,19, it seems that A. arvensis has a rather low

oil yield.

The results obtained after GC and GC-MS analysis of

the oil are given in Table 1. Thirty constituents were

identified comprising 83.8% of the oil. The major

constituent was characterized as germacrene D (47.0%).

Furthermore, β-bourbonene (7.7%), caryophyllene oxide

(3.2%) and β-caryophyllene (3.0%) contents were found

to be high.

The composition of our sample of A. arvensis

was closely related to those of the Canadian oil,3

including 51.4% of germacrene D, 7.9% of caryophyllene

and 4.8% β-bourbonene. This was only qualitatively

similar to the Turkish oil4 in which germacrene D repres-

ents only 14.3%, β-bourbonene 7.0% and caryophyl-

lene oxide 3.2%. However, oil composition was totally

different from the Greek oil, consisting of pulegone,

menthone and isomenthone;5 the occurrence of chemo-

types or, better, misidentification of the species may be

hypothesized.

Table 2. Antimicrobial activity of the oil of Acinos arvensis (Lam.) Dandy

Microorganisms Inhibition zone diameter (mm)

Oil:ethanol (1:20) Thymol (10 mg/ml)

Klebsiella pneumoniae ATCC 10031 21.5 34.0

Staphylococcus aureus ATCC 6538 16.6 33.5

Escherichia coli 95 TORLAK 13.8 30.8

Pseudomonas aeruginosa ATCC 9027 — 17.0

Table 1. Composition of the essential oil of Acinosarvensis

Component Percentage RI

1,8-Cineole 0.5 1029

Camphor t 1141

Borneol t 1163

Terpinen-4-ol 0.6 1175

p-Cymen-8-ol t 1184

α-Ylangene t 1347

α-Copaene 0.6 1372

β-Bourbonene 7.7 1380

β-Cubebene t 1387

β-Elemene 0.5 1389

β-Caryophyllene 3.0 1413

β-Gurjunene 1.1 1424

Geranyl acetone 0.8 1439

α-Humulene 0.7 1448

Allo-aromadendrene 1.2 1455

9-Epi-β-caryophyllene 0.7 1457

1-Dodecanol 0.7 1458

Germacrene D 47.0 1461

Bicyclogermacrene 1.5 1492

Germacrene A 2.1 1499

(E,E)-α-Farnesene 0.5 1500

δ-Cadinene 0.6 1520

11-Nor bourbonan-1-one 0.7 1550

Germacrene D-4-ol 0.6 1560

Spathulenol 2.5 1571

Caryophyllene oxide 3.2 1576

8-Cedren-13-ol 1.5 1680

Phytol 2.3 1845

Pentadecanoic acid 1.9 1916

Hexadecanoic acid 1.3 1959

t, less than 0.1.

The results of the antimicrobial activity of the oil of A.

arvensis, which was determined by measuring the inhibi-

tion zone diameter of investigated microorganisms, are

given in Table 2. It was found that all the microorgan-

isms, except for Pseudomonas aeruginosa, were suscep-

tible to the oil. The susceptibility decreased in the

following order: Klebsiella pneumoniae, Staphylococcus

aureus and Escherichia coli. Pseudomonas aeruginosa

was resistant to the oil.

Acknowledgements—We thank Mr Bojan Zlatkovic, Institute of NatureProtection of Republic of Serbia, Nis, for the botanical identification ofthe plant species. We also thank the Ministry of Science, Technologiesand Development of the Republic of Serbia for the financial support ofthis study.

290 T. JOVANOVIC ET AL.

Copyright © 2004 John Wiley & Sons, Ltd. Flavour Fragr. J. 2005; 20: 288–290

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