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Phytochemical analysis of the essentialoil of Thymus serpyllum L. growingwild in EstoniaUrve Paaver a , Anne Orav b , Elmar Arak a , Uno Mäeorg c & AinRaal aa Institute of Pharmacy, University of Tartu , Nooruse 1, 50411Tartu, Estoniab Institute of Chemistry, Tallinn University of Technology ,Akadeemia tee 15, 12618 Tallinn, Estoniac Institute of Organic and Bioorganic Chemistry, University ofTartu , Jakobi 2, 51014 Tartu, EstoniaPublished online: 27 Jul 2010.

To cite this article: Urve Paaver , Anne Orav , Elmar Arak , Uno Mäeorg & Ain Raal (2008)Phytochemical analysis of the essential oil of Thymus serpyllum L. growing wild inEstonia, Natural Product Research: Formerly Natural Product Letters, 22:2, 108-115, DOI:10.1080/14786410601035118

To link to this article: http://dx.doi.org/10.1080/14786410601035118

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Natural Product Research, Vol. 22, No. 2, 20 January 2008, 108–115

Phytochemical analysis of the essential oil

of Thymus serpyllum L. growing wild in Estonia

URVE PAAVERy, ANNE ORAVz, ELMAR ARAKy,UNO MAEORGx and AIN RAAL*y

yInstitute of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, EstoniazInstitute of Chemistry, Tallinn University of Technology,

Akadeemia tee 15, 12618 Tallinn, EstoniaxInstitute of Organic and Bioorganic Chemistry, University of Tartu,

Jakobi 2, 51014 Tartu, Estonia

(Received 20 April 2006; in final form 22 September 2006)

Variations in the essential oil composition of Thymus serpyllum L., growing wild in Estonia(33 samples) and in some other countries (Russia, Latvia and Armenia, seven samples)were determined. The oil were obtained from Estonia (46 samples) in yields 0.6–4.4 and1.9–8.2mLkg–1 in other countries. The T. serpyllum herb grown in Estonia usually did notconfirmed to the EP standard in the aspect of the essential oil contents (3.0mLkg–1). Variationsin the essential oil composition of wild thyme were studied using capillary gas chromatographicmethods. A total of 94 components were identified. Thymol and carvacrol, mentionedin literature as principial components, are not the main components of the essential oil of wildthyme growing in Estonia. (E)-nerolidol, caryophyllene oxide, myrcene and borneolchemotypes of wild thyme drug are distinguishable. The chemical composition of samplesfrom Russia, Latvia and Armenia is very variable.

Keywords: Thymus serpyllum L.; Estonia; Essential oil; Terpenes; Chemotypes; EuropeanPharmacopoeia

1. Introduction

Wild thyme (Thymus serpyllum L.) is regarded as a source of essential oil containingaromatic terpenes. Since 2003, Serpylli herba belongs to the European Pharmacopoeia(EP). The EP standard to the essential oil content is not less than 3mLkg�1 [3].The content of the essential oil in the wild thyme drug varies in great extent dependingon the origin of the plant; it is between 0.1 and 0.6% [21].

The essential oil of T. serpyllum growing in different countries of the world has beenstudied by several researches, 34–95 compounds are identified [7–11,14–17,19,20].

*Corresponding author. Tel.: þ372-7-375281. Fax: þ372-7-375289. Email: ain.raal@ut.ee

Natural Product Research

ISSN 1478-6419 print/ISSN 1029-2349 online � 2008 Taylor & Francis

http://www.tandf.co.uk/journals

DOI: 10.1080/14786410601035118

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The main components of the essential oil of the wild thyme are considered to be:thymol, carvacrol, p-cymol, linalool, �-pinene and other terpenes, particularlythymol (up to 30% in essential oil) and carvacrol (up to 20%) are mentioned [21].However, the content of �-terpinene is higher than 22% and p-cymene is higher than20% in the essential oil of wild thyme. The authors also mention thymol as mainconstituents of essential oil of S. herba (>18%). In contrast, the content of carvacrolin the essential oil was insignificant (�1%) [13].

The main constituents of essential oils of wild thyme growing wild in Lithuaniawere 1,8-cineole, caryophyllene oxide, borneol, �-caryopyllene, germacrene D,camphor, camphene and myrcene. The 1,8-cineole-caryophyllene oxide chemotypewas determined in Lithuania [10]. Also 1,8-cineole, E-carvyl acetate, E-�-ocimene,caryophyllene oxide, �-caryopyllene and cis-p-menth-2-en-1-ol chemotypes are detectedin earlier paper from Lithuania [8].

While studying S. herba originating from different natural habitats in Estonia,(E )-nerolidol, caryophyllene oxide, myrcene, (E)-�-caryopyllene and germacrene Dwere determined as the main components of the essential oils of the drug. In Estonia,the (E )-nerolidol-caryophyllene oxide, (E)-nerolidol-myrcene and myrcene chemotypesof S. herba are distinguishable [12]. As a result of investigations made in China,Iran, India, Pakistan and Japan, other compounds are mentioned among principalcomponents of essential oil of wild thyme herb [11,14–16,19]. Chemical polymorphismconserving the essential oils of the genus Thymus is a widespread phenomenon,especially in the northern species [17].

In the present work, the main components of the essential oils of wild thyme growingwild in Estonia in 2004, also thymol and carvacrol content were studied. The essentialoil content in the drug and its accordance to the EP standards was studied.

2. Results and discussion

The content of essential oil in 46 drugs collected in summer 2002–2004 varied between0.6 and 4.4mLkg–1, having an average value of 0.1–0.2mLkg–1 (table 1). The upperlimit of 3mLkg–1 stated by EP was exceeded only in the sample from Vorumaa(Southern Estonia, 4.4mLkg–1) and in the sample from Parnumaa (West Estonia,4.0mLkg–1). The average values of the contents of essential oil in samples collectedin different years were respectively 1.3, 1.2 and 1.9mLkg–1 and are not statisticallyremarkable (r¼ 0.276). The content of essential oil was not statistically differentiablein drug samples from various regions of the country.

The content of essential oil in drugs from Russia, Latvia and Armenia seems to behigher (on the avarage 3.9mLkg–1) than in S. herba growing in Estonia (tables 1 and 3).The limit of EP was not exceeded in two samples (1.9 and 2.2mLkg–1 in Russia) fromanalysed seven samples. As mentioned in literature [1], the aerial parts of floweringT. serpyllum from five sites in Armenia contained 4.5–7.4mLkg–1 of essential oil.The extremly high content of essentail oil in sample no. 3 (company Adonis,St. Petersburg) can be explained that there is Thymus pulegoides instead of T. serpyllumin the package of analysed drug. As we see from table 3, the content of thymol (42.8%)in the mentioned sample was atypically high.

We have identified and quantified 94 components of investigated essential oils ofS. herba growing in Estonia. The major components were (E)-nerolidol, geranyl acetate,

Phytochemical analysis of T. serpyllum L. essential oil 109

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caryophyllene oxide, linalyl acetate, linalool and myrcene, followed by camphene,camphor, borneol, (E)-�-caryophyllene, germacrene D, etc. (see table 2 for thequantities).

The essential oil of T. serpyllum from Estonia contains thymol and carvacrol only0–4.0%. Mentioned compounds are named as the main components of essential oil ofwild thyme [4,21]. For example the essential oil of wild thyme from India contains60–64.6% [9,11], from Pakistan 42.6% [14], from Japan 35% [16], from Iran 18.7% [15]of thymol. The content of carvacrole is usually lower than thymol content [9,11,15].

Table 1. Content of essential oil of T. serpyllum L.gathered in 2002–2004 from Estonia (mLkg�1).

Place of growth Content of essential oil

Harjumaa, Paldiski 0.6Harjumaa, Muraste 0.9Harjumaa, Keila-Joa 1.5Harjumaa, Kuusalu 1.2Harjumaa, Kareda 1.5Harjumaa, Kolga 2.6Harjumaa, Tammispea 1.1Harjumaa, Tallinn 2.5Harjumaa, Vaana 1.1Harjumaa, Kasispea 1.5Harjumaa, Tapurla 0.9Harjumaa, Laulasmaa 1.0Hiiumaa, Altsadam 0.6Hiiumaa, Tahkuna 2.1Hiiumaa, Kassari 0.6Ida-Virumaa, Riigikula 2.1Ida-Virumaa, Riigikula 1.5Ida-Virumaa, Narva-Joesuu 2.8Ida-Virumaa, Narva-Joesuu 1.9Ida-Virumaa, Kurtna 1.8Ida-Virumaa, Toila 0.6Laanemaa, Hobulaid 1.5Laanemaa, Spithami 0.8Laane-Virumaa, Palmse 0.8Laane-Virumaa, Karepa 1.4Laane-Virumaa, Mahu rand 0.9Laane-Virumaa, Laviku 2.5Laane-Virumaa, Mustpea 1.0Laane-Virumaa, Vosu 1.0Polvamaa, Taevaskoda 2.3Polvamaa, Taevaskoda 0.7Parnumaa, Kihnu 0.9Parnumaa, Tostamaa 1.1Parnumaa, Kabli 1.2Parnumaa, Treimanni 4.0a

Saaremaa, Leisi 0.8Tartumaa, Elva 1.6Tartumaa, Tartu linn 2.0Valgamaa, Puka 1.4Valgamaa, Valga linn 1.2Valgamaa, Hargla 1.0Vorumaa, Piusa 1.2Vorumaa, Roosisaar 1.2Vorumaa, Roosisaar 4.4a

aThe limit of EP is exceeded.

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Table 2. Composition of the essential oil of T. serpyllum L. from Estonia, %.

RI

Compound NB-30 SPB-5 Min–Max Mean (n¼ 33) Std. deviation

Tricyclene 920 918 0–0.4 0.062 0.108�-Thujene 924 923 0–0.2 0.047 0.070�-Pinene 929 930 0–2.6 0.649 0.849Camphene 944 944 0–10.8 2.170 2.7572-Heptenol 949 0–0.8 0.071 0.170Sabinene 965 970 0–0.6 0.187 0.192�-Pinene 970 972 0–1.9 0.374 0.4661-Octen-3-ol 968 976 0–2.0 0.724 0.5793-Octanone 983 0–3.2 0.379 0.727Myrcene 984 990 0–20.2 6.152 6.6793-Octanol 986 995 0–0.7 0.091 0.155�-Phellandrene 998 1002 0–0.4 0.016 0.071�-Terpinene 1010 1014 0–0.2 0.041 0.074p-Cymene 1013 1020 0–4.2 0.410 0.766Limonene 1024 1024 0–1.1 0.352 0.4011,8-Cineole 1022 1026 tr.–5.4 1.247 1.710(Z)-�-Ocimene 1028 1034 0–0.4 0.069 0.103(E)-�-Ocimene 1039 1044 0–3.7 0.986 1.193�-Terpinene 1050 1055 0–0.6 0.147 0.175trans-Sabinene hydrate 1058 1064 0–0.3 0.087 0.111cis-Linalol oxide 1056 1068 0–3.0 0.100 0.590Terpinolene 1080 1086 0–0.6 0.090 0.130cis-Sabinene hydrate 1087 1096 0–0.2 0.047 0.070Linalool 1089 1100 0.4–22.8 3.000 4.084�–Thujone 1090 1102 0–0.3 0.046 0.079�–Thujone 1100 1112 0–2.4 0.084 0.417(Z)-p-Mentha-2,8-dien-1-ol*1118 1121 0–0.1 0.003 0.017(Z)-p-Mentha-2-en-1-ol* 1173 1133 0–0.1 0.003 0.017Camphor 1123 1138 0.2–14.2 3.545 2.924cis-Sabinol* 1128 1142 0–1.3 0.206 0.364trans-Sabinol* 1147 0–tr. 0.0003 0.0017Isoborneol 1135 1155 0–0.3 0.028 0.062Borneol 1156 1160 0–19.0 4.667 5.853Terpinen-4-ol 1165 1172 0–1.5 0.594 0.287p-Cymen-8-ol* 1162 1184 0–0.3 0.037 0.069a-Terpineol 1177 1188 0.2–9.7 2.490 2.330(Z)-Dihydrocarvone 1181 1193 0–0.2 0.006 0.035(E)-Dihydrocarvone 1198 1205 0–1.0 0.079 0.270(E)-Carveol 1214 1221 0–1.1 0.088 0.231Nerol 1217 1230 0–2.2 0.170 0.444Neral 1220 1236 0–0.1 0.009 0.029Carvone 1221 1239 0–0.7 0.055 0.162Geraniol 1242 1253 0–4.1 0.233 0.768Linalyl acetate 1244 1254 0–31.0 1.170 5.460Geranial 1258 1270 0–0.2 0.022 0.0541-Decanol 1280 1275 0–2.5 0.118 0.441Bornyl acetate 1274 1285 0.1–4.6 1.291 0.948(E)-Sabinyl acetate 1273 1287 0–2.0 0.140 0.380Thymol 1282 1292 0–4.0 0.958 0.881Carvacrol 1292 1300 0–4.0 0.620 0.730�-Elemene 1331 1338 0–0.2 0.013 0.041�-Terpinyl acetate 1336 1348 0–0.1 0.016 0.057Neryl acetate 1345 1364 0–0.7 0.074 0.152�-Copaene 1373 1370 0–1.0 0.191 0.273�-Ylangene 1375 1378 0–2.5 0.222 0.529

(Continued)

Phytochemical analysis of T. serpyllum L. essential oil 111

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In the essential oil of wild thyme from Lithuania, thymol and carvacrol were notdetected, which is a characteristic feature of the Serpyllum species growing in theNorthern European countries [7]. These both phenols were not main components alsoin the essential oils of T. serpyllum var. serpyllum and T. serpyllum var. tanaenis fromFinland [17].

The essential oils of samples from Russia, Latvia and Armenia contain (E )-nerolidol,caryophyllene oxide and myrcene in quite low amounts (table 3). For example thecontent on (E)-nerolidol was only 0.1–3.8%, but in the samples from Estonia drasticallylarge amounts such as 70.1, 61.9, 55.1% were determined.

The content of essential oil was very variable in the wild thyme herbs of Russianorigin. Rather different content of essentail oil was determined in the sample no. 2

Table 2. Continued.

RI

Compound NB-30 SPB-5 Min–Max Mean (n¼ 33) Std. deviation

Geranyl acetate 1362 1382 0–46.4 3.303 8.722�-Bourbonene 1383 1388 0–2.0 0.350 0.470�-Elemene 1385 1392 0–1.0 0.076 0.140(E)-b-Caryophyllene 1415 1413 tr.-13.3 6.222 3.636�-Ionone 1413 1422 0–0.4 0.040 0.093Bicyclosesquiphellandrene*1436 1437 0–0.2 0.013 0.041�–Guaiene* 1435 1444 0–0.3 0.021 0.069�-Humulene 1450 1450 0–1.7 0.300 0.372(E)-�-Farnesene* 1453 1453 0–0.5 0.092 0.142Alloaromadendrene 1456 1458 0–1.6 0.285 0.300�-Muurolene 1476 1470 0–0.2 0.019 0.053Germacrene D 1478 1474 tr.–12.4 5.495 3.7861-Dodecanol 1488 0–0.8 0.055 0.152�-Muurolene 1490 1495 0–1.8 0.237 0.362Bicyclogermacrene 1489 1500 0–1.1 0.200 0.298�-Bisabolene 1500 1506 0–2.5 0.873 0.613�-Cadinene 1502 1512 0–5.0 0.320 0.900�-Cadinene 1518 1520 0–2.8 0.655 0.603Selina-3,7(11)-diene* 1540 1543 0–2.4 0.600 0.656Hedycaryol* 1534 1550 0–1.0 0.120 0.015Germacrene B 1555 1562 0–1.3 0.297 0.326(E)-Nerolidol 1554 1565 1.7–70.1 24.527 18.536Spathylenol 1572 1572 0–5.0 1.220 1.380Caryophyllene oxide 1770 1574 1.4-45.0 10.288 9.648Germacren-4-ol 1576 1581 0–1.0 0.088 0.190Viridiflorol 1586 1595 0–1.0 0.170 0.310Ledol* 1587 1600 0–1.8 0.382 0.488Humulene epoxide* 1600 1605 0–0.6 0.106 0.195�-Cadinol 1627 1635 0–2.2 0.331 0.539�-Bisabolol 1622 1643 0–3.0 0.100 0.510T-Cadinol 1629 1649 0–3.9 0.773 0.899T-Muurolol 1632 1652 0–0.2 0.006 0.035�-Cadinol 1644 1663 0–3.0 0.570 0.750�-Farnesol 1653 1665 0–3.2 0.561 0.678�-Santanol 1660 1680 0–1.5 0.197 0.383�-Bisabolol 1674 1683 0–6.0 0.740 1.610n-Heptadecane 1700 1700 0–0.4 0.043 0.097n-Nonadecane 1900 1900 0–0.4 0.049 0.097n-Heneicosane 2100 2100 0–1.0 0.079 0.120

tr. – traces (<0.05%); 0 – not determined; atentatively identified. Bold indicates the principal components.

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(Pskov), where borneol, caryophyllene oxide and camphor are dominating compounds.Geraniol and linalool are main components in sample no. 5. Geraniol was also the maincomponent of essential oil (60.3%) of wild thyme herb, collected from Central Siberianbotanical garden [6]. There was maximum content of geraniol only 4.1% in the essential

oils of T. serpyllum growing in Estonia.The relatively high content of geraniol, thymol, �-terpinyl acetate and geranyl acetate

is typical for the sample from Armenia.Relatively high concentrations of p-cymene, �-terpinene, carvacrol and (E )-�-

caryophyllene are detected in essential oils of wild thyme herbs both from Latvia andEstonia, the content of mentioned terpenes was much less in samples from Russia and

Armenia.(E )-Nerolidol, caryophyllene oxide, myrcene and borneol chemothypes of wild

thyme drug are distinguishable in Estonia. Differently from the data of foreigncountries, thymol and carvacrol are not the main components of wild thyme growingin Estonia. The oil content of Estonian drugs is usually not compliant with EP

requirements.The chemical composition of T. serpyllum depends on many factors, mainly

on genetic and ecological [2]. The content of essential oil and chemical compositionof T. serpyllum from Estonia is not statistically differentiable in drug samples fromvarious regions of the country and ecological differences (habitation type). Probably

the chemical composition of analysed drug samples from Estonia and other countries(Russia, Armenia, Latvia) above all depends on genetic reasons.

Table 3. Composition of the essential oil of T. serpyllum L. from some various countries (%).

Russia

Compound 1a 2 3 4 5 Latvia Armenia

Camphene 0.2 9.4 1.0 – – – –Myrcene 1.1 0.5 0.9 0.4 0.5 0.4 0.2p-Cymene 1.1 1.0 12.5 2.0 2.0 16.6 0.71,8-Cineole 14.2 2.1 2.0 0.5 0.4 tr. 0.3�-Terpinene 0.3 0.6 13.0 0.8 0.3 14.5 0.5Linalool 2.0 1.9 0.4 11.3 21.8 0.8 0.6Camphor 4.6 10.5 0.1 0.5 0.1 tr. 0.1Borneol 1.2 22.8 3.7 – 0.6 0.7 0.5�-Terpineol 21.2 3.8 0.3 – 5.5 0.2 1.1Methyl thymol ether – – – – – 5.7 –Geraniol 1.9 – – 3.5 26.8 3.3 29.1

Linalyl acetate – – – 19.6 – tr. –Thymol – 0.2 42.8 18.2 6.3 3.0 17.7

Carvacrol – 0.4 3.0 1.8 2.1 11.5 7.3�-Terpinyl acetate 0.2 tr. tr. – 1.3 0.1 13.9Geranyl acetate 1.0 0.2 tr. 5.0 2.2 0.3 14.3

(E)-�-Caryophyllene 3.4 0.6 1.2 3.5 2.3 10.9 2.6Germacrene D 3.8 – 0.2 2.2 1.3 2.4 1.1�-Bisabolene 0.9 tr. 1.5 3.1 0.7 8.0 0.4(E)-Nerolidol 2.9 3.8 1.1 2.0 3.8 0,1 0.6Caryophyllene oxide 6.5 11.1 0.6 1.8 1.8 2.0 0.1Essential oil (mLkg–1) 2.2 3.8 8.2 1.9 4.0 3.0 4.1

a1 – Company Narodnaja meditsina, Saint Petersburg; 2 – gathered near Pskov; 3 – Adonis, Saint Petersburg; 4 – Fito-M,Ural; 5 – Faros-21, Yekaterinodar; Latvia – Planta Medica Ltd, Turkums; Armenia – Mospharm, Yerevan. tr. – traces. Boldindicates the principal components.

Phytochemical analysis of T. serpyllum L. essential oil 113

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3. Experimental

3.1. Plant materials

The whole-dried S. herba was used as the research material. The samples were gatheredin July and August of 2001–2004 from 46 different places of growth (sands, alvar andpine forest) in Estonia (table 1). The plants were identified on the basis of macroscopiccharacteristics; the drug was dried in the temperature range of 30–40�C.

Plant materials (commercial T. serpyllum L. – S. herba) were obtained also from retailpharmacies in Russia (or gathered near Pskov), Latvia and Armenia in years 2001–2004(table 3). The growing places of mentioned drugs are therefore not known. Voucherspecimens have been deposited at the Institute of Pharmacy, University of Tartu,Estonia.

3.2. Isolation of essential oil

The essential oil was isolated from dried drug of wild thyme by the distillation methoddescribed in the European Pharmacopoeia [3] using 50 g of cut drug, a 1000mL round-bottomed flask, 500mL of water as the distillation liquid. Quantity of 0.2mL of xylenein the graduated tube was added to take up the essential oil. The distillation time was2 h at a rate of 2–3mLmin–1.

3.3. Capillary gas chromatography conditions

GC analysis was carried out using a Chrom 5 chromatograph with FID on two fusedsilica capillary columns with bonded stationary phases SPB-5 (30m� 0.25mm,SUPELCO) and NB-20M (50m� 0.20mm, NORDION, Finland). Film thicknessof both stationary phases was 0.25mm. Carrier gas helium with split ratio 1 : 150 and theflow rate 20–25 cm s–1 was applied. The temperature program was from 50–250�C at2�Cmin–1, the injector temperature was 200�C. A Hewlett-Packard Model 3390Aintegrator was used for data processing.

The identification of the oil components was accomplished by comparingtheir retention indices (RI) on two columns with the RI values of reference standards,our RI data bank and with literature data. The results obtained were confirmed byGC/MS [5,18].

The percentage composition of the oils was calculated in peak areas usingnormalization method without correction factors. The relative standard deviationof percentages of oil components of three repeated GC analysis of single oil sampledid not exceed 5%.

Acknowledgement

Financial support for the work reported here was provided by the Estonian ScienceFoundation (grant no. 4332).

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