Octanoic acid

Abstract

Oils produced from pre-flowering and flowering plants of Micromeria Juliana L. were investigated by GC/MS. The oils were qualitatively similar but differed quantitatively. After the oil fractionation by LS chromatography and GC/MS analysis, 64 compounds were identified. Important hydrocarbons were α-pinene (7.2-10.6%) with β-pinene (4.9-7.0%) as monoterpenes and β-caryophyllene (2.5-4.2%) with α-gurjunene (2.1-6.4%) as sesquiterpenes. The main oxygenated monoterpenes were linalool (4.7-7.6%) with its furanoid cis- and trans-oxides (3.5-4.6%) and borneol (2.2-3.5%). Other monoterpenes, sesquiterpenes as well as non-terpenoid compounds were present in smaller amount. The results of the present research reveal some differences in the oil composition of M. juliana of Greek origin.

Key Word Index

Micromeria juliana, Lamiaceae, essential oil composition, α-pinene.

Introduction

Microtneria juliana (L.) Bentham ex Reichenb., syn. Satureja Juliana L. (Lamiaceae) is herbaceous, decidious and perennial shrub (20-30 cm high). The plant is slightly aromatic. It grows wild in Croatia (Dalmatia) and only in some Mediterranean and sub-Mediterranean regions (1-2). This plant prefers medium loamy soil and can grow in nutritionally poor soil. Other species of genus Micromeria could also be found in Croatia such as M. thymifolia Scop. (Fritisch.), M. croatica (Pers.) Schott, M. pseudocroatica Silic, M. dalmatica (Benth.), M. kerneri Murb., M. graeca (L.) Benth., M. fruticulosa (Bertol. in Desv.) Grande and M. microphylla (D'Urv.) Bentham (3), some of which are endemic.

The leaves of M. juliana are used for food flavoring as a substitute for savory and as a diuretic. The oil of this plant has not been investigated in detail in distinction to the oils from other Micromeria species (4-7). The oil of M. thymifolia Scop. (Fritisch.), M. dalmatica Bentham, M. albanica (Grisebach ex K. Maly) and M. cristata (Hampe) Griseb. subsp. phrygia showed antibacterial and antifungal activities (8-10).

Experimental

Plant material: The plant material was collected in the Mediterranean region of Croatia, near Sinj. Two samples of the plant material were collected, one prior to flowering and other during flowering, June, 1999. The young stems with leaves and flowers (15-20 cm) were harvested and dried for 10 days at room temperature. Voucher specimens are deposited at Department of Organic Chemistry, Faculty of Chemical Technology, University of Split.

Oil isolation: The oil was isolated by simultaneous hydrodistillation-extraction in a modified Likens-Nickerson-type apparatus for 3 h. A mixture of pentane and diethyl ether (1:1 v/v) was used as solvent for extraction. The extract was dried over anhydrous sodium sulfate. The yield of oil was determined gravimetrically after removing the solvent by fractional distillation.

Oil fractionation: The oil (20 µL) was fractionated on a micro-column (silica gel 30-60 µm, 500 mg) and five fractions were obtained (11). Pentane (10 mL) was used for hydrocarbons elution (fraction I). For elution of oxygenated compounds different mixtures of pentan-ether were used: 5 mL 5% ether (fraction II), 5 mL 10% ether (fraction III), 5 mL 50% ether (fraction IV) and 5 mL pure ether (fraction V). All fractions were concentrated to 0.5 mL and tested by thin layer chromatography (TLC). The first fraction contained only nonpolar monoterpene and sesquiterpene hydrocarbons. The other fractions contained polar, oxygenated compounds according to the increasing polarity from fraction II to fraction V. These results were also confirmed by GC/MS analysis.

GC/MS analysis: All fractions of the essential oils as well as overall oils were analyzed by gas chromatography/mass spectrometry (Hewlett-Packard, model 5890, with a mass selective detector, model 5971A). Two columns with different polarity of stationary phases, HP-20M and HP-101, were used. GC operating conditions: column HP-20M (Carbowax 20M), 50 m x 0.2 mm, film thickness 0.2 µm; column temperature programmed from 70°C isothermal for 4 min, then increased to 180°C at a rate of 4°C/min; column HP-101 (Methylsilicone), 25 m x 0.2 mm, film thickness 0.2 µm; column temperature programmed from 70°C isothermal for 2 min, then increased to 200°C at a rate of 3°C/min. Carrier gas: helium, flow rate: 1 mL/min, injector temperature: 250°C; volume injected: 1 µL; split ratio: 1:50. MS conditions: ionization voltage: 70 eV; ion source temperature: 280°C; mass range: 30-300 mass units. Individual peaks were identified by comparison of their retention indices to those of authentic samples, as well as by comparison of their mass spectra with those stored in database (Wiley library) and library data (12). The percentage composition of the samples was computed from the GC peak areas without using correction factors. The overall oil composition was calculated from analysis of the fractions with an internal standard (menthol). Preliminary GC/MS analysis showed the absence of menthol as a component of M. juliana.

Results and Discussion

Simultaneous hydrodistillation-extraction in a modified Likens-Nickerson type apparatus was applied due to low volatiles content in the plant. The yield of oil for dried plant material prior to flowering was 0.07% and 0.11% during flowering.

The plants with such low essential oil content usually contain large number of compounds and possible artifacts. The components distribution is almost uniform without dominating peaks. In order to improve peak separation and identification, the essential oils were fractionated on the micro-column. One fraction of terpene hydrocarbons and four fractions of oxygenated compounds were obtained. The analysis of fractions allowed a more certain identification of components present in small amounts as well as compounds that are overlapping. All the fractions, as well as the essential oil, were analysed by gas chromatography-mass spectrometry (GC/MS) on two columns. More than 100 components were separated and the majority of them were identified. The chemical composition of the total oil calculated from the composition and content of fractions is given in Table I. Monoterpene with sesquiterpene hydrocarbons and oxygenated compounds were identified as well as small amount of non-terpenoid compounds. Sixty-four compounds were identified representing about 70% of the total oil. It is of interest to note that only 39 compounds were identified in the analysis of the oil without fractionation. The results of the present investigations reveal differences in the oil composition of M. juliana of Greek origin reported earlier (7). The isolated aroma hydrocarbon components with highest levels were: α-pinene (7.2-10.6%) with β-pinene (4.9-7.0%) as monoterpenes and β-caryophyllene (2.5-4.2%) with α-gurjunene (2.1-6.4%) as sesquiterpenes. The most important oxygenated monoterpene compounds were linalool (4.7-7.6%) with its furanoid derivatives cis- and trans-oxides (3.5-4.6%) and pyranoid derivative of translinalool oxide (0.3-0.8%). They are structurally similar, as well as borneol (2.2-3.5%), camphor (0.8%) and bornyl formate (0.1%). All these compounds favor mutual conversions by reactions of oxidation, reduction and esterification. Furthermore, trans-pinocarveol, verbenone, carvone, trans-carveol, terpinen-4-ol as well as myrtenol were detected in smaller amounts. The majority of these oxygenated compounds were not previously identified in the oil Micromeria juliana from Greece (7). However, many are present in M. graeca (6). Carvone, cis- and trans-carveol could generate from limonene (oxidation and reduction). Myrtenal may originate by oxidation of myrtenol. Other terpene compounds were detected with smaller percentages, such as δ-cadinene, ar-curcumene. The important non-terpenoid compounds were 3-octanol, 1-octen-3-ol, (E)-2-hexenal, benzyl alcohol, phenylacetaldehyde, cuminaldehyde, methyl salicylate and octanoic acid. These compounds are products of fatty and cinnamic acid catabolism (13). Other monoterpenes, sesquiterpenes and non-terpenoid compounds were present in small amounts and are listed in Table I. Nonidentified peaks mainly present sesquiterpene and diterpene compounds. Many of them have similar structures and mass spectra with low resolution.

The contents of linalool, β-caryophyllene, α-gurjunene, α-humulene and cadinene increased several times during the flowering of the plant and that of borneol, β-cubebene, arcurcumene, β-bisabolene, manool and limonene decreased several times. Before and after flowering the oil was qualitatively similar although there are quantitative differences.

By comparison of our results with those of other investigations on the oils of Micromeria species, we can conclude that M. graeca and M. Juliana from Greece have some similarity in oil composition to our sample. The oil content of our M. juliana is 1.2-2.1 times higher than M. juliana from Greece. We believe that the complexity of the oil composition of M. Juliana merits further research.

Acknowledgments

The authors are thankful for the financial support to the Ministry of Science and Technology of the Republic of Croatia (Project 0011010).

a partially presented at the 29th International Symposium on Essential Oils, Frankfurt am Main, September 1998.

References

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Josip Mastelic,* Igor Jerkovic and Danica Kustrak

Department of Organic Chemistry and Natural Products, Faculty of Chemical Technology, Teslina 10/V, 21000 Split, Croatia

* Address for correspondence

Received: December 2002

Revised: March 2003

Accepted: April 2003

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