Solid Phase Microextraction of Volatile Constituents from Individual Fresh Eucalyptus Leaves of Three Species

• Abstract
• Materials and Methods
• References

Abstract

Methyl polysiloxane solid-phase microextraction fibres were used for ten minutes to adsorb volatile constituents from headspace above all or part of a single cut up fresh eucalyptus leaf kept warm at 37 °C. The fibres were desorbed at 200 °C for programmed gas chromatography (40–187 °C) on a methyl polysiloxane capillary. Substances were identified by mass spectra and/or authentic sample retention. Results do not correspond to published values for steam distilled oils, being richer in sesquiterpenes, of which three are common to three different species; and also in esters in two species. Five Eucalyptus citriodora leaves from the same tree over different months gave very similar analyses to a fibre in 10min of 72.9–80.5 % citronellal, 3.5–5.4 % citronellol, 1.0–3.8 % citronellyl acetate, 9.2–11.8 % caryophyllene and 1.4–1.7 % bicyclogermacrene. Six E.nicholii leaves yielded 67.2–73.7 % cineole and 4.6–9.7 % limonene along with 10.5–16.5 % sesquiterpenes, mostly hydrocarbons, particularly bicyclogermacrene. E.globulus leaves gave only 54.0–61.3 % cineole, with 19.5–24.3 % α-pinene, 6.7–9.1 % limonene and 2.1–5.4 % α-terpinyl acetate; along with 3.6–7.7 % sesquiterpenes, particularly aromadendrene, but no bicyclogermacrene.

The results in [Table 1] show the successful analysis of whole small, or part of large, eucalyptus leaves using solid phase microextraction (SPME). They summarise single leaf assays made from May 1998 (autumn) to February 1999 (summer). Literature analyses [1] of volatile oils from the same three species are included. Eucalyptus nicholii Maiden & Blakely is a typical eucalypt with low-polarity volatiles. A fresh leaf yielded 67–74 % cineole, with 4.6–10 % limonene and 1.2–7 % of the sesquiterpene hydrocarbon (STH) bicyclogermacrene (bcg). From a new growth bright green adult leaf, there was only 59 % cineole, and reduced α-pinene, but a great increase in bcg to 21 %. However, another STH, aromadendrene, decreased to only 0.2 % from the normal leaf 2.5–4.9 %; whilst α-phellandrene increased to 5.2 % from the normal 1.3–2.8 %. These changes may be biogenetically indicative. Steam distillation greatly under-extracts the less-volatile STH, and consequently gives higher values in the oil [1] than are natural for cineole, α-pinene and α-terpineol. The unstable α-phellandrene is mostly lost.

E.globulus Labill. also yielded low-polarity volatiles, but with more terpenoids, up to 6 %. Cineole only comprised 54–61 %, with 20–24 % α-pinene and 7–9 % limonene. The STH showed no bcg, but there was 2.6 –5.0 % aromadendrene. 2.9 % of this is quoted [1] for the oil, surprisingly high, and probably the consequence of a prolonged distillation indicated by the high oil content of oxygen-containing sesquiterpenoids. The ester, α-terpinyl acetate, a distinctive minor component from fresh leaf at 2.1–5.4 %, is almost completely hydrolysed during steam distillation so that α-terpineol is increased over tenfold in the oil. This also has increased α-pinene like E.nicholii and p-cymene; but with reduced cineole and limonene. A juvenile leaf (physically different, not new growth) had less pinenes and more α-phellandrene again, and more of some trace STH.

E.citriodora Hook. yielded quite different volatiles, although still with a small amount of cineole to indicate a eucalypt. This has not been recorded for its oil [1]. Low-polar STH were still there, about 1.6 % bcg, with 9–12 % caryophyllene. The main polar constituent was 73–81 % citronellal, along with 3.5–5.4 % citronellol and 1.0–3.8 % of its acetate. Again, the ester is almost completely hydrolysed by steam distillation [1]. This also gives almost 12 % isopulegols, which are recognised decomposition products of citronellal, in distinction to the less than 2.3 % by SPME. Up to a sixth of eucalypt headspace oil vapour can consist of STH, much more than from steam distillation.

Materials and Methods

Eucalyptus trees (Myrtaceae) in south Perth were used. Adult leaf samples were taken from a large E.citriodora, identified by its typical smooth grey bark; small urn-shaped fruits; and long, narrow, lemon-scented leaves. Half a single lamina, cut away from the midrib and weighing 0.20–0.27 g, was enough for analysis. E.globulus, although not mature enough to flower/ fruit, was identified by the peeling bark and the mix of two leaf forms: alternate, shiny, curved-lanceolate, petiolate adult leaves and paired, glaucous, cordate, sessile juvenile leaves. 0.23–0.25 g portions of both leaf types were used without petiole or thick midrib. E.nicholii was confirmed with the aid of the computer program ”Euclid” [2]. The identification sequence for this was: rough, fibrous bark persisting onto branches; lanceolate, cineole-scented adult leaves 7 mm wide, with moderately reticulate, acute veins; umbels of seven small flower buds with operculum scars. As its leaves are smaller than those of the other species, a whole leaf was used, without petiole, weighing about 0.14–0.25 g. These trees continue to grow on Campus, and dried specimens of them E1 – 3 are kept in this School.

A manual SPME syringe was obtained from Supelco, Bellefonte, PA, USA, and used with a fibre coating of 100 µm methyl polysiloxane, selected after trying five coatings of various polarities. The retracted fibre was injected through a two-layer aluminium foil top which had been secured onto a small glass media jar containing finely cut (”julienned”) fresh eucalyptus leaf. The adsorbent fibre was protruded so it was suspended above the leaf, not touching the sides of the jar, and left exposed to the headspace for ten min. Heating the fresh leaves is required to produce satisfactory chromatograms. The fibre exposure commenced after twenty min in the warm room (37 °C). The fibre was then withdrawn into its protective sleeve, and as soon as possible exposed in the hot GC injection port (200 °C) for two min. This desorbed the volatile oil constituents onto the front of the cool GC capillary (40 °C), after which the retracted fibre was withdrawn from the port.

Hewlett-Packard 5890 II gas chromatographs were used, one with a flame ionisation detector (FID) at 220 °C for headspace analyses reported on a HP 3396 II recorder/integrator set to attenuation ”2”; the other with an interfaced HP 5791 electron impact mass selective detector and computer recording using HP G1034C Chemstation software. The latter gave identifications for many of the constituents of the chromatograms against the Wiley G1035A Mass Spectral Library, assisted by literature solute retention sequences [3] and analyses of oils from Boland et al. [1]. For the analyses, a helium mobile phase flow of about 1.6 ml min^–1 (100kPa or 14.5psi) measured at the detector outlet at 200 °C was used. Retention times were taken from the beginning of the two min fibre desorption period, following which the capillary was programmed up from 40 at 6.7min^–1 to 87 °C (at 9min). The capillary was then immediately programmed up from 87 at 10.0min^–1 to 187 °C (at 19min), after which it was rapidly cooled to 40 °C for the next analysis. Without the slower initial program, limonene could not be resolved reliably from cineole. SPME headspace FID analyses were performed with a well-used 12m × 0.20 mm i. d. capillary containing a 0.33 µm film of crosslinked methyl polysiloxane (HP-1). Mass spectral runs were made on a 30m × 0.32 mm i. d. capillary containing a 0.25 µm film of 5 % phenyl, 95 % methyl polysiloxane (HP-5). The latter had difficulty resolving limonene from cineole if used for quantitative analyses.

References

• 1 Boland D J, Brophy J J, House A PN. Eucalyptus Leaf Oils. Melbourne-Sydney; Inkata 1991: pp. 59, 80 & 103
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• 2 Brooker M IH, Connors J R, Slee A V. Euclid. Eucalyptus of South- eastern Australia (CD-ROM key). Collingwood, Australia; CSIRO 1997
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• 3 Adams R P. Identification of Essential Oils by Ion Trap Spectroscopy. San Diego; Academic Press 1989
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Thomas J. Betts

School of Pharmacy

Curtin University of Technology

GPO Box U1987

Perth, Western Australia

Australia 6845

Email: tjbetts@ozemail.com.au

Fax: +61-8-9266-2769

References

• 1 Boland D J, Brophy J J, House A PN. Eucalyptus Leaf Oils. Melbourne-Sydney; Inkata 1991: pp. 59, 80 & 103
  PubMedSearch in Google Scholar
• 2 Brooker M IH, Connors J R, Slee A V. Euclid. Eucalyptus of South- eastern Australia (CD-ROM key). Collingwood, Australia; CSIRO 1997
  PubMedSearch in Google Scholar
• 3 Adams R P. Identification of Essential Oils by Ion Trap Spectroscopy. San Diego; Academic Press 1989
  PubMedSearch in Google Scholar

Thomas J. Betts

School of Pharmacy

Curtin University of Technology

GPO Box U1987

Perth, Western Australia

Australia 6845

Email: tjbetts@ozemail.com.au

Fax: +61-8-9266-2769