lands), C. capitatus, from the island of Euboea and S. thymbra from the island of Nisyros (of the Dodecanese group). Voucher specimens of the plants collected ...
Z Lebensm Unters Forsch (1993) 197:20-23
Zeitschrift fQr
9 Springer-Verlag 1993
Original paper Composition and antioxidant activity of essential oils from Oregano plants grown wild in Greece Vasiliki Lagouri 1, George Blekas 1, Maria Tsimidou 1, Stella Kokkini 2, Dimitrios Boskou 1 1 Laboratory of Food Chemistry and Technology, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54006 Thessaloniki, Greece 2 Laboratory of Systematic Botany and Phytogeography, Faculty of Biology, Aristotle University of Thessaloniki, GR-54006 Thessaloniki, Greece Received December 17, 1992
Zusammensetzung und antioxidative Aktivit~it etherischer Ole von Oregano-Pflanzen aus Griechenland Zusammenfassung. Bei dieser Arbeit wurden vier Proben etherischer 01e aus Pflanzen gewonnen, die weit verbreitet in Griechenland sind und von 6konomischer Wichtigkeit in der ganzen Welt am Gewiirzmarkt sind. Es wurden Carvacrol und Thymol auf ihre wahrscheinliche antioxidative Aktivit~it getestet. Die vier Pflanzenarten sind
Origanum vulgare hirtum, Origanum onites, Coridothymus eapitatus und Satureja thymbra. Die etherischen Ole wurden mit Hilfe der Gaschromatographie-Massenspektroskopie charakterisiert. Sowohl die untersuchten etherischen Ole als auch Carvacrol und Thymol wurden durch TLC und auch durch Messung der Peroxid-Werte eines bei 35 ~ aufbewahrten Schweinefettes getestet. Die Ergebnisse zeigen, dab der antioxidative Effekt auf die Anwesenheit von Carvacrol und Thymol im etherischen O1 zur/ickgefiihrt werden kann.
Abstract. In this study, four samples of essential oils obtained from plant species with a wide distribution in Greece and economic importance for the world-wide spice market and also carvacrol and thymol were tested for their possible antioxidant activity. The four plant species were Origanum vulgare subsp, hirtum, O. onites, Coridothymus capitatus and Satureja thymbra. The essential oils were chemically characterised by gas chromatography-ion trap detection. All the essential oils examined and also carvacrol and thymol were found to have antioxidant activity when tested on TLC plates and by measuring peroxide values of lard stored at 35 ~ C. The results indicate that the antioxidant effect may be related to the presence of carvacrol and thymol in essential oils.
Correspondence to: D. Boskou
Introduction Synthetic antioxidants are often used in the food industry because they retard undesirable changes due to oxidation. However, in recent years some toxicity concerns and a trend among consumers toward "all natural ingredients" have resulted in a pronounced activity in the field of natural additives. Herbs and spices, used world-wide for culinary PUrposes, have gained the interest of many research groups. Various extracts obtained from such sources have been assessed as potent antioxidants in lipid systems [1-5]. Among the plants studied, those of the Labiatae (Lamiaceae), family have been often used to extract active components [6-11]. The relationship between chemical structure o f extracts obtained from various tissues of such plants and antioxidant activity has not been thorougly investigated [6, 7]. Also, very little is known about the extent to which essential oils contribute to the antioxidant activity of certain spices. Farag et al. [12-14], using simple model systems composed of refined cottonseed oil or emulsions, found that essential oils obtained from plants of the Labiatae family exhibit antioxidant activity. Although the results of these studies were not conclusive it is clear that there is a relationship between the inhibition of hydroxyperoxide formation and the presence of phenols such as thymol and eugenol in some essential oils. Their experiments, however, did not include "oregano" essential oil, which contains a high amount of carvacrol, an isomer of thymol. The vernacular name "oregano" is attributed to a vast number of species in over a dozen genera of different families. Today it is generally accepted that oregano is in fact a characteristic flavour produced by a number of plant species that yield carvacrol-rich essential oils [15-18]. In this preliminary study four samples of essential oils obtained from plant species with a wide distribution in Greece and of economic importance for the world-wide spice market were tested for their possible antioxidant activity. These four plant taxa were: Origanum vulgare L. subsp, hirtum (Link) Ietswaart (commercially known
21
with the vernacular name Greek oregano), O. onites L. (known as Turkish oregano), Coridothymus capitatus (L.) Reichenb. ill. (Spanish oregano), and Satureja thymbra L. The essential oils were chemically characterised by gas chromatography(GC)-ion trap detection [19] and their activity was related to the presence of phenols.
Materials and methods Plant material. Aerial parts of flowering plants were collected from the wild. In particular, O. vulgare subsp, hirtum was collected from Central Peloponnese, O. onites from the island of Paxi (Ionian islands), C. capitatus, from the island of Euboea and S. thymbra from the island of Nisyros (of the Dodecanese group). Voucher specimens of the plants collected are kept at the Herbarium of the Institute of Systematic Botany and Phytogeography.
Chemicals. Butylhydroxytoluene (BHT) and linoleic acid were purchased from Fluka (Buchs, Switzerland). Chloroform (proanalysis), ethanol (absolute), acetic acid (glacial) and r-carotene were from Merck (Darmstadt, Germany). Thymol (purum) was from Sigma (St. Louis, Mo, USA) and carvacro198% from Aldrich (Milwaukee, Wis., USA). Isolation of the essential oil. The plant material was air-dried and submitted to distillation (1.5 h) using a Clevenger apparatus. The essential oil content expressed in ml/100 g air-dried material was: O. vulgare subsp, hirtum 4.5, O. onites 3.3, C. capitatus 2.2 and S. thymbra 3.7.
Screening for antioxidant activity. The antioxidant activity of the four essential oils, carvacrol, thymol and BHT was tested on silica gel TLC plates. Each solution (25 p,1 of 100 mg/ml in ethanol) was spotted on plates that were sprayed with a solution of r-carotene and linoleic acid according to a method described by Pratt and Miller [20]. The plates were exposed to daylight until the background colour disappeared. Spots having antioxidant activity remained yellow coloured for a longer period.
Results and discussion
Screening tests carried out on TLC plates showed a remarkable antioxidant effect for all the essential oils examined and also for carvacrol and thymol. This antioxidant effect was confirmed by measuring peroxide values of lard stored at 35 ~ C (Figs. 1 and 2). These results indicate that the antioxidant effect may be related to the presence of phenols in the essential oil and that, besides thymol and eugenol [12-14], other phenols present in essential oils may also have an antioxidant effect. The composition of the four "oregano" oils as determined by GC and combined GC-MS is shown in Table 1. Thirty-five compounds were detected in the oils: C. capitatus oil consisted of 30 components, whereas a lower number, 22 components, was found in O. vulgare subsp. hirtum. Eighteen constituents were common in all the samples examined. Cis-pinane was present only in C. capitatus whereas cis- and trans-ocimene and e-caryophellene were found in S. thymbra oil. Carvacrol was in all cases the main component. The highest percentage of carvacrol, 82.3%, was determined in C. capitatus. Thytool (the crystallisable isomer) was constantly present but its percentage varied from 0.1% in S. thymbra up to 13.7% in O. vulgare subsp, hirtum oil. In spite of some differences in the total phenol content among the four samples, the antioxidant activity did not seem to vary at the 1000 mg/kg level. In lower concentrations no conclusive results were obtained. It appears that both isomers, carvacrol and thymol, are equally effective (Fig. 2). These findings indicate that the antioxidant activity demonstrated for some spices [1, 2, 4, 7] should be partly attributed to components present in the essential oil, which may be important in suppressing lipid oxidao
8O
Stability of lard. Each essential oil to be tested and also carvacrol, thymol and BHT were dissolved in ethanol and added to lard which that melted at 80 ~ C and further purified by filtration. Samples of lard (1 g each) containing 1000 mg/kg of each essential oil, carvacrol, thymol and 200 ppm BHT were transferred to a series of open transparent glass bottles (8 cm 3 volume and 3 cm 3 cross-section), which were then stored in an incubator at 35 ~ C in the dark. Peroxide values were determined periodically according to the AOCS method [21].
1 ,-,~6 0
oa~ E
~40-
High resolution gas chromatography (HRGC) - mass spectrometry (MS). Capillary gas chromatography (HRGC) was performed by means of a Carlo-Erba gas chromatograph (Carlo Erba, Hofheim, Germany) using a SE-54 capillary column (30 m long, 0.3 mm internal diameter). The carrier gas was helium with a flow rate of 2 ml/min. The samples were applied by the "on-column injection technique" at 35 ~ C. After 2 rain the temperature of the oven was raised by 40 ~ C/min to 75 ~ C, then by 4 ~ C/rain to 115 ~ C and finally by 2 ~ C/min to 250 ~ C. The final temperature was retained for 10 min. MS analyses were performed on a Finnigan ITD 800 (Finnigan MAT, Bremen) instrument coupled to the above chromatograph equipped with a fused silica J&W DB-5 capillary column (30 m x 0.3 mm i.d.). The identification of the individual compounds was based on linear retention indices (RI) calculated from the retention times, mass spectra and a LIBR (TP) search [22].
20
'~
i
8
i
12
"f~6
[
20
'
2'4
2
~lR
i
32
i
36
t(d~ys ) ~ Fig. 1. Effect of butylhydroxytoluene (BHT, 200 mg/kg) and essential oils of Origanum vulgare subsp, hirtum (+), O. onites (zx), Satureja thymbra ( x ), Coridothymus capitatus (*) (1000 mg/kg) on the autoxidation rate of lard at 35 ~ C: o, control test
22 Table 1. Qualitative und quantitative composition of the essential oils obtained from four "oregano" taxa Components
Essential oil samples: contribution
O. vulgare subsp. hirtum (%)
RI
1. 2. 3. 4.
cr-Thujene a-Pinene Camphene /3-Pinene
926 933 948 976 981 990 1003 1009 1016 1023 1028 1038 1048 1058 1067 1087 1098 1165 1177 1191 1243 1247 1285 1290 1293 1299 1313 1413 1432 1446 1467 1488 1505 1509 1523
5. cis-Pinane 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.
Myrcene c~-Phellandrene zi3-Carene c~-Terpinene p-Cymene Limonene
cis-Ocimene trans-Ocimene y-Terpinene
cis-Thujanol Terpinolene
trans-Thujanol Borneol 4-Terpineol c~-Terpineol Carvone Carvacrolmethyleter Bornyl acetate Thymol c~-Caryophyllene Carvacrol Unidentified ]3-Caryophyllene Aromadendrene cr-Humulene y-Guriunene c~-Murolene /3-Bisabolene 7-Cadinene Unidentified
0.7 0.5 0.1 0.1
O. onites (%) 0.1 0.2 0.3 tr 1.0 0.1 tr 0.7 4.9 0.6 4.0 0.4 0.2 0.3 4.6 1.6 0.4 tr 0.2 tr 0.7 73.3 0.4 1.1 0.2 tr tr 0.1 3.2 0.2
1.3 0.1 tr 1.1 7.6 0.7 11.0 0.4 0.2 0.1 0.2 tr 13.7 59.7 0.8 -
0.6 0.6 0.3
C. capitatus (%)
S. thymbra (%)
0.1 0.2 0.1
0.8 1.3 0.4 0.5
0.8 1.3 0.1 tr 0.6 5.8 0.5 2.7 0.5 0.1 1.1 0.6 0.7 0.2 0.1 tr 0.3 82.3 0.9 tr 0.2 0.2 tr -
1.6 0.2 tr 1.5 13.7 0.1 0.1 18.0 0.3 0.1 1.0 0.5 0.5 tr 0.4 0.1 0.1 0.1 53.6 4.3 0.1 0.2 0.1 tr 0.1 -
tr
