FLAVOUR AND FRAGRANCE JOURNALFlavour Fragr. J. 2003; 18: 21–25Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1137

Composition and variability of the essential oil ofSardinian Thymus herba-barona Loisel

Marianna Usai,1* Aldo Atzei,1 Giorgio Pintore2 and Ioseph Casanova3

1 Dipartimento di Scienze del Farmaco, Universita di Sassari, Via Muroni 23/A, 07 100 Sassari, Italy2 Dipartimento Farmaco Chimico Tossicologico, Universita di Sassari, Via Muroni 23/A, 07 100 Sassari, Italy3 Universite de Corse, Equipe Chimie et Biomasse, UMR CNRS 6134, Route des Sanguinaires, 20 000 Ajaccio, France

Received 11 October 2001Revised 12 February 2002Accepted 12 March 2002

ABSTRACT: The essential oils from nine wild populations of the Sardinian Thymus herba-barona Loisel(Labiatae) were chemically characterized to identify the different chemotypes. GC and GC–MS analysis showedthat in Sardinia only two chemotypes (thymol and carvacrol) are present. The carvacrol chemotype is predominantand this compound was found in high concentration (50.7–79.4%) in seven of the nine populations studied. Thethymol chemotype was found in only two populations (34.2–56.2%). Analyses also showed that the other maincomponents of the oils of T. herba-barona, were 3-heptanone (1.4–5.6%), 3-octanone (1.4–5.5%) and borneol(2.5–8.3%). p-Cymene is present at 1.2–5.6% but is not present in plants from the Tonara medium station, whereO-cymene (1.9%) is present instead. Copyright 2002 John Wiley & Sons, Ltd.

KEY WORDS: Thymus herba-barona Loisel; essential oil; Labiatae; carvacrol; thymol

Introduction

Thymus herba-barona Loisel is a very aromatic, pul-vinate, woody suffrutescent plant with opposite leavesand simple epidermal glands. The inflorescence is rosy,cymose; the cymes at the nodes, condensed into a falsewhorl (verticillaster), are a terminal pseudo-capitulum.The calyx is united into a two-lipped funnel-shaped tubeand stamens with anthers facing inwards; the fruits aretetra-akenes.1,2

T. herba-barona Loisel is considered an endemic Sar-dinian and Corsican plant1,3,4 although there is a debatedsignalling in Majorca.5 This species grows in moun-tainous and submountainous zones at an altitude of800–2000 m. In Sardinia it is less widespread than inCorsica and prefers schist and granite substrates, there-fore it is almost absent in the centre-west of Sardinia.1

T. herba-barona is moderately rich in essential oil(0.8%) which has expectorant and antiseptic properties;it is used in folk medicine as a diaphoretic, sedative, anti-septic and antimycotic and for the treatment of insomniaand it is also utilized in perfumery, phytocosmetics andthe manufacture of liqueurs.2

* Correspondence to: Marianna Usai, Dipartimento di Scienze delFarmaco, Universita di Sassari, Via Muroni 23/A, I, 07 100 Sassari,Italy. E-mail: dsfusai@ssmain.uniss.itContract/grant sponsor: INTERRREG II.Contract/grant sponsor: MURST.

Sardinian T. herba-barona has been investigated forits essential oil composition using chemical methods6

and GC analyses for thymol and carvacrol content7 andtheir seasonal variation.8 Recently the essential oil hasalso been investigated for antibacterial properties.9 Cor-sican T. herba-barona has also been well investigatedfor the chemical composition of its essential oil10,11 andshowed appreciable differences among the populationsanalysed.

In a previous paper9 we described the essentialoil composition of Sardinian T. herba-barona and itsantibacterial activity; moreover, in that study we obser-ved the predominance of carvacrol (73.0–75.4%) in theessential oils. In this paper we enlarge our investigationon Sardinian T. herba-barona to a greater number ofsamples to investigate the oil composition, with the aimof verifying whether these populations are more homo-geneous than those growing in Corsica.

Experimental

Plant Material

In order to study the infraspecific variability, individualplants of each population were collected from each placeat the same time as homogeneous samples of correspond-ing populations. Sardinian plant populations of T. herba-barona were collected during their full flowering periodat different stations in northern Sardinia (at every station,

Copyright 2002 John Wiley & Sons, Ltd.

22 M. USAI ET AL.

three different samples of each population): Vallicciola(1000 m height, carvacrol population); Punta Balestreri(1360 m, carvacrol population); Badde Urbara low sta-tion (850 m, carvacrol population) and Badde Urbarahigh station (1050 m, thymol population); Su Ninnieri(1400 m, carvacrol population); Badde Salighes (800 m,carvacrol population); Tonara-1 (800 m, thymol pop-ulation); Tonara-2 (850 m, carvacrol population); andTonara-3 (950 m, carvacrol population).

Professor A. Atzei identified all the populations anal-ysed. Voucher specimens have been deposited at theHerbarium SASSA of the Dipartimento di Scienze delFarmaco, University of Sassari, under a collective num-ber (1077) for all Thymus herba-barona Loisel samples.

Oil Distillation and Yield

The fresh plant material was hydrodistilled for 4 h usinga Clevenger-type apparatus.12 The oils were dried overanhydrous sodium sulphate and stored at �20 °C untilthey were analysed.

Oil Analyses

GC

Four replicates of each sample were analysed, usinga Hewlett-Packard Model 5890A GC equipped witha flame ionization detector (FID) and fitted with a60 m ð 0.25 mm, film thickness 0.25 µm, AT-5 fusedsilica capillary column (Alltech). Injection port anddetector temperatures were 280 °C. The column temper-ature was programmed from 50 °C to 135 °C at 5 °C/ min(1 min), rising to 225 °C (5 min) and then to 260 °C andheld for 10 min. The samples (0.2 µl each), generallyanalysed without dilution (using 2,6-dimethylphenol asinternal standard), were injected using a split/splitlessautomatic injector HP 7673, with helium as the carriergas. Measurements of peak areas were performed with aHP workstation; the threshold was set at 0, peak width at0.02. The data reported in Table 2 are the average of fourGC injections. The quantitation of each compound wasexpressed as a relative percentage, using internal stan-dard and response factors. The detector response factors�RFs� were determined for key components relative to2,6-dimethylphenol and assigned to other components onthe basis of functional group and/or structural similarity,

since oxygenated compounds have a lower detectabilityby FID than hydrocarbons.13 The standards were >95%pure and actual purity was checked by GC. Severalresponse factor solutions were prepared that consisted ofonly four or five components, plus 2,6-dimethylphenol,to prevent interference from trace impurities.

GC–MS

GC/MS analyses were carried out with a Hewlett-Packard G1800B-GCD System using the same condi-tions and column as described above. The column wasconnected with the ion source of the mass spectrometer.Mass units were monitored from 10 to 450 at 70 eV.The identification of compounds was based on a com-parison of their retention times with those of authenticsamples and/or by comparison of their mass spectra withthose of published data and HP Libraries14–16 or on theinterpretation of the EI-fragmentation of the molecules.

13C-NMR

To confirm all the constituents and to discriminatesecurely, carvacrol and thymol NMR spectra were recor-ded on a Bruker AC 200 Fourier transform NMR spec-trometer operating at 50.323 MHz for 13C, equippedwith a 10 mm (or 5 mm) probe, in deuterated chloro-form, with all shifts referred to tetramethylsilane (TMS).13C spectra were recorded with the following param-eters: pulse width (PW), 5 µs; acquisition time, 1.3 for32 K data table with a spectral width (SW) of 12 500 Hz(250 ppm); CPD mode decoupling; digital resolution,0.763 Hz/pt.

Results and Discussion

In order to study the chemical differences in T. herba-barona, nine populations, represented by 27 differentgroups of plants (three for each station) widely dis-tributed in Sardinia, were collected at full flowering time.The essential oil yields obtained from the different pop-ulations were in the range 0.2–0.8% (Table 1).

Considering all the analysed stations, 14–35 con-stituents can be identified, accounting for 98.5–99.8% ofthe total components of T. herba-barona Loisel essen-tial oils. Table 2 reports the variation of percentages ofevery component in the studied stations of wild T. herba-barona.

Table 1. Yield of the oil of the nine wild Sardinian Thymus herba-barona Loisel populations

Valliciola(1000 m)

BaddeUrbara(850 m)

BaddeUrbara

(1050 m)

PuntaBalestreri(1360 m)

Su Ninnieri(1400 m)

Tonara-1(800 m)

Tonara-2(850 m)

Tonara-3(950 m)

BaddeSalighes(800 m)

0.4% 0.4% 0.8% 0.8% 0.4% 0.2% 0.8% 0.7% 0.7%

Copyright 2002 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 21–25

ESSENTIAL OIL OF THYMUS HERBA-BARONA 23Ta

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Copyright 2002 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 21–25

24 M. USAI ET AL.

This study shows differences when compared withprevious studies on T. herba-barona collected inCorsica,11 where eight different chemotypes (thymol,carvacrol, linalool, geraniol, ˛-terpenyl acetate, terpinen-4-ol, carvone and cis-dihydrocarvone) can be found.

Analyses of the other data reported in Table 2 showthat among the 35 compounds identified, including thetwo major constituents carvacrol and thymol, only 15reached 1%.

Analysing the major alcohols, we found that borneol,linalool and terpinen-4-ol were always present in everystation, but the borneol content was always >2.5%. Itis noteworthy that this compound reached a content of8.3% in Tonara 1 and 6.5% in Badde Urbara-b stations;the lowest concentration of borneol was found in PuntaBalestreri (2.5%).

3-octanol reached its maximum in Punta Balestreriand Tonara-1 (2.8–2.6%) whereas it was not detectedin Badde Urbara-b and in Badde Salighes stations. Thepresence of 3% of 1,8-cineole was noted because in theother station this compound varies between 0% (less thanthe lower sensitivity threshold of the instrument) and0.5%.

Among the terpene hydrocarbons, camphene was notdetected at Badde Salighes station and reached its max-imum at Badde Urbara-b. ˇ-Caryophyllene was presentat only five stations and its maximum was only 1.1% inPunta Balestreri. �-Terpinene was always higher than1%, reaching its maximum at Punta Balestreri andTonara-1. p-Cymene was the most represented terpene inT. herba-barona essential oil. It reached a maximum of5.6% at Tonara-1 but was not present at Tonara-2, whereit was substituted by the presence of O-cymene, which

was found at only one other station (Tonara-3) where,however, it was present together with p-cymene in highpercentage (3.9%) similar to that found in Vallicciola.

3-Heptanone and 3-octanone were always present inhigh percentages; 3-heptanone had a minimum at BaddeSalighes (1.3%) and a maximum of 5.6% at BaddeUrbara-b. A high percentage was also found at Tonara-3and Tonara-1, while in the other investigated stations thepercentage was ca. 2%.

Every station was characterized by a different dis-tribution of compounds in the analysed essential oils.Tonara 1 station was characterized, besides high value ofborneol, by the high values of 3-heptanone, 3-octanone,p-cymene, �-terpinene and linalool, which were alwayshigher than 4.5%. Comparing the other Tonara stations,it was possible to distinguish the oil’s owing to somesubstantial differences. In fact, Tonara-2 was character-ized by the lowest content of 3-heptanone, 3-octanone,�-terpinene, linalool and borneol and by the presenceof O-cymene instead of p-cymene. This last differencepermitted this station to also be recognized among allthe others studied; moreover, it was a carvacrol chemo-type, with only 4.5% of thymol. Tonara-3, by contrastshowed the presence of phellandrene and iso-thymol andalso may be considered a carvacrol chemotype, having11.4% of thymol.

A compound that may be discriminating to recognizePunta Balestreri oil among all those of the investigatedstations is 1-8-cineole. In Punta Balestreri oil it reached3%, while in the other samples it was absent or presentin very low concentrations (0.2–0.3%). It was alsocharacterized by the highest concentration of �-terpinene(5.3%).

Figure 1. Multivariate statistical analysis at the stations analysed

Copyright 2002 John Wiley & Sons, Ltd. Flavour Fragr. J. 2003; 18: 21–25

ESSENTIAL OIL OF THYMUS HERBA-BARONA 25

Table 3. Ratio between [A] C [B]/[A] � [B] A D carvacrol percentage, B D thymol percentage

Valliciola1000 m

BaddeUrbara850 m

BaddeUrbara1050 m

PuntaBalestreri1360 m

Su Ninnieri1400 m

Tonara800 m

Tonara850 m

Tonara950 m

BaddeSalighes800 m

1.02 1.03 1.36 1.04 1.04 2.69 1.12 1.58 3.16

The two stations Su Ninnieri and Badde Urbara-ahad a very similar composition of the oils; also, atBadde Urbara-a 3-heptanone, 3-octanone and borneolwere slightly higher than at Su Ninnieri (Table 2). BaddeUrbara-b differed from Badde Urbara-a because this wasa thymol chemotype with only 8.7% of carvacrol.

Vallicciola station was characterized by the pres-ence of thymol-methyl ether (2%). This compound waspresent in the same amount only at Tonara-1, while inthe other samples it is present only in traces or not at all.Badde Salighes is recognizable as a carvacrol chemotypewith a very high content of thymol (28%) and for 1.1%of trans-carveol.

To better compare all the stations investigated, wesubmitted the collected data to a multivariate analysis(Figure 1) by the principal components method.17,18 Theplotting of the results using eigenvectors 1 and 2 showeda good separation among the analysed samples.

Five groups were located in the plot. In particular,Tonara-1 and Badde Salighes single shot. In fact, ifwe analyse the following ratio: [A] C [B]/[A] � [B],where A is the carvacrol percentage and B is the thymolpercentage, the two above-mentioned stations showedstrong differences in respect to all the other stationsanalysed (Table 3). The Punta Balestreri and Su Ninniericonstituents seem to be very similar and are also thehighest stations above sea level (ca. 1400 m). The othertwo groups are constituted by Vallicciola and BaddeUrbara LS and HS and by the remaining two Tonarastations.

In conclusion, it is possible to say that among thenine stations it was possible to observe that only twochemotypes were present, carvacrol and thymol, and nopopulation showed any other chemotype, such as thatreported for Corsica;11 moreover, plant at seven of thestations were carvacrol chemotype and only two werethymol chemotype.

Acknowledgements—The work was financially supported byINTERRREG II and ex 60% Consiglio Nazionole delle

Ricerel (CNR) Istituto Aplicazioui Tecniche ChimicheAveuzede ei Problemi Agzobiologici (IATCAPA), Min-istero Universita Ricerca Scientifica e Tecnologica(MURST). The authors gratefully acknowledge CNR(IATCAPA) for permitting the use of GC–MS instru-mentation and Miss M. Stella Foddai and Mr GianuarioManca for technical assistance.

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