Joseph J. Brophy and Ian A. Southwell
Introduction
The medicinal and aromatic properties of Eucalyptus are normally associated with the steam-volatile components, and so this chapter, with the exception of the last section, is devoted to the chemistry of the essential (or volatile) oils. This then generally restricts discussion to molecules of molecular weight less than 250 amu, which are either terpenoid or aromatic in structure. The ease with which volatile oils can be analysed using gas chromatography (GC) and GC coupled techniques has meant that many investigations have been undertaken and the structures of numerous constituents elucidated. Consequently, the number of volatile compounds reported from Eucalyptus far exceeds the number of non-volatile ones. The leaves are the most frequently investigated part of the plant but interesting constituents have also been isolated from the bark and the wood.
Brophy has investigated many new Eucalyptus species using GC-MS techniques (see below) and the results, together with a listing of the major components from these and previous studies of eucalyptus oils, have been published in book form (Boland et al. 1991).
The bulk of the present chapter comprises a revised and up-dated list of those Eucalyptus species for which the volatile oil has been analysed, showing oil yields and the identity and rela-tive abundance of the most significant constituents (Table 5.2). The data for this table, as earlier (Boland et al. 1991), are taken mostly from Australian sources, representing trees growing in their natural habitat. Although some non-Australian data are included, these are usually so similar to the analyses of endemic populations that the wider inclusion of such data is considered unnecessary. A few species which do not occur in Australia (e.g. E. leizhou No. 1, a natural hybrid which is used as a source of oil in China, and E. urophylla) are included in the table. Coppen and Dyer (1993) have published an extensive bibliography of Eucalyptus and its leaf oils, indexed by species and country and covering the literature from the 1920s to late 1992, which includes exotic eucalypts.
The taxonomy of such a large and complex genus as Eucalyptus is under continual revision. For example, the frequently distilled lemon-scented gum, E. citriodora, along with many other species previously placed in the Eucalyptus genus, has recently been renamed as a member of the Corymbia genus (Hill and Johnson 1995). For the purposes of this chapter, however, this species will still be considered under Eucalyptus. At the latest count there were 777 species listed (Wilcox 1997).
Early investigations
Although the first reported eucalyptus oil distillation is thought to be that of the Sydney Peppermint, E. piperita, in 1788, regular steam distillations were not frequent until the 1850s,
when Bosisto in Victoria, Australia, produced firstly experimental and then commercial quanti-ties of eucalyptus oil for the European market (Penfold 1935, Penfold and Morrison 1950, McKern 1967, 1968). At that time, organic chemistry was only an emerging science and it was not until later the same century that the principal constituent of E. globulus oil was named
‘eucalyptol’ and its structure established as 1,8-cineole (see later, Figure 5.2, structure 1) (Boland et al. 1991). Baker and Smith then systematically investigated oils from other Eucalyptus species, isolating some forty different compounds (Baker and Smith 1920, McKern 1968).
Several schools of essential oil research were established in New South Wales, Queensland, and South and Western Australia, all publishing data on Eucalyptus oils and the chemical structures of their constituents. Most of these results were included in Guenther’s series of volumes on essential oils (Guenther 1950) and, later, in Penfold and Willis’s treatise The Eucalypts (Penfold and Willis 1961).
The advent of gas chromatography and, more recently, techniques coupling gas chromato-graphy with mass spectrometry (GC-MS) and infrared spectroscopy (GC-IR), rapidly accelerated the analysis of volatile oils and the identification of their constituents and this has given rise to an extensive literature on the oils of Eucalyptus. Today, this literature continues to grow.
Sample preparation and analytical techniques
Sample preparation
As a guide to the commercial viability of production of any eucalyptus oil, laboratory distilla-tions must first be performed to determine yields and chemical composition. Oil yields, usually expressed as a percentage of the leaf weight, are measured on either a dry or fresh weight basis.1 The highest oil yield recorded in the authors’ laboratories was 12.75 per cent from the partly dried leaf of E. dives. The equipment of choice for laboratory scale distillation is the Clevenger apparatus (Clevenger 1928) or a modification of it (e.g. Hughes 1970, Whish 1996).
The distillation process is the most time-consuming step in the assessment of essential oil quality. Consequently micro-extraction methods using a suitable organic solvent are frequently used to determine oil yield and quality, especially where large numbers of samples are involved (Ammon et al. 1985, Southwell and Stiff 1989, Brophy et al. 1989). A room temperature extrac-tion time of 30–40 h can be reduced to one hour if microwave irradiaextrac-tion is first carried out (Southwell et al. 1995). Although the product is not as clean as the steam-distilled oil, the extract accurately reflects leaf quality and, with the addition of an internal standard, can also give a reliable estimate of selected constituents of the oil. The use of a GC vial insert (0.1 ml) means that this method can be adapted to micro-analyses (Brophy et al. 1989, Chen and Spiro 1995, Spiro and Chen 1995). Consequently this is the method of choice for many laboratories (including our own) for the screening of essential oil-bearing plants, including Eucalyptus.
Soxhlet extractions are also used for Eucalyptus analysis, as well as simultaneous distillation-extraction procedures like those adapted to the Likens–Nickerson apparatus (Schultz et al.
1977). The principles of general essential oil preparation reviewed by researchers such as Koedam (1987) are most applicable to Eucalyptus leaf analysis.
1 If possible, the moisture content of the distilled leaf should be determined so that oil yields can be expressed on a dry weight basis. This then enables all yields that are determined to be compared on an equal basis. The moisture content of fresh leaf may be as high as 70 per cent but declines rapidly after harvesting. However, even leaf which has been cut and air-dried has a residual moisture content (around 5 per cent) and should not be assumed to be perfectly dry.
Although more recent techniques, including supercritical fluid extraction (Boelens and Boelens 1997), microwave extraction (Craveiro et al. 1989) and headspace analysis (Chialva and Gabri 1987) have been used to analyse plant volatiles, Eucalyptus has not received the same atten-tion as other species. There is one report of the supercritical fluid extracatten-tion of Eucalyptus (Milner et al. 1997). Other methods of isolation include vacuum distillation and this has been the method of choice in a series of papers by Bignell et al. (1994–1998) who report on the oils obtained from almost 200 species of Eucalyptus. The procedure has the advantage that it does not lead to the production of artifacts or the degradation of some of the constituents of the oil which can happen with conventional steam distillation, that is, the composition thus determined is closer to the intrinsic composition of the oil within the leaves than that obtained via steam dis-tillation. However, by the same token, the composition may not reflect what is obtained in prac-tice (or could be expected) commercially. Moreover, the results reported by Bignell et al. are derived from single trees and may not be representative either of the particular population of which the tree is part or of the wider distribution of the species. Nevertheless, they are reported here (Table 5.2) because in a significant number of cases they are the only analyses reported for the particular Eucalyptus species.
Analytical techniques
Before the advent of gas chromatography most essential oils were analysed by measuring optical rotation, relative density, refractive index and solubility in alcohol. Bench chemistry methods were also used to determine acid value, ester value (before and after esterification), carbonyl value, phenol content and the concentration of some specific isolates (e.g. 1,8-cineole). These methods still have a place today in monographs published by standards organisations such as the International Standardization Organization (ISO) and pharmacopoeias such as the British Pharmacopoeia (BP). With cineole-type eucalyptus oils, the o-cresol method of Cocking (1920, 1927) is still used as the bench chemistry method for the determination of cineole. Further information on eucalyptus oil standards is given in Appendix 5; details of how and where the standards can be obtained are given in Appendix 7.
In the main, the identification of individual eucalyptus oil constituents used to be dependent on the isolation of pure compounds, either by fractional distillation under vacuum or using alumina or silica column chromatography. This was then followed by derivatisation and mixed melting point comparison with an authentic derivative of the suspected compound. With modern chromatographic, spectroscopic and X-ray crystallographic techniques, identifications previously taking months or years can now be achieved in hours or days. Examples illustrating the use of these techniques in identifying Eucalyptus constituents are abundant in the literature (e.g. Boland et al. 1992, Ghisalberti et al. 1995, Singh and Etoh 1995). ISO standards now include a chromatographic profile and typical gas chromatographic trace in addition to physico-chemical and other data.
The most commonly used technique for the identification of volatile constituents is gas chromatography-mass spectrometry (GC-MS). Using GC-MS a mass spectrum can be obtained for each peak from a chromatogram and compared with a library of reference spectra in order to obtain a spectrum of best fit. The spectra of the common terpenes are in most spectral libraries (e.g. Adams 1995) and a collection of -triketone mass spectra has recently been published (Brophy et al. 1996). Mass spectral data alone are insufficient for unambiguous identification, especially for terpenoids with similar spectra, and should be supported by retention index mea-surements on two columns of different polarity (Stevens 1996) or additional information (IR, NMR, etc., e.g. Liener 1996). The use of gas chromatography-infrared spectroscopy (GC-IR) is
one way of providing such data. The critical factor in all GC techniques is the resolution obtained by the chromatographic column. For example, 1,8-cineole often co-elutes with limonene on non-polar columns and with -phellandrene on polar columns. Columns of inter-mediate polarity often give superior resolution of these key Eucalyptus constituents (Brophy et al.
1989).
Advances in computer technology and software development have encouraged the use of multivariate statistical methods for principal component and cluster analysis of constituents (e.g. Brooker and Lassak 1981, Silvestre et al. 1997).
Infraspecific variation and chemotaxonomy
Early investigations of eucalyptus oils suggested that the composition of an oil was constant within the same species (Baker and Smith 1920) and new taxa were sometimes established solely on chemical evidence (McKern 1968). It was later realised, however, that infraspecific variation could occur. Among many examples are the piperitone and cineole/eudesmol forms of E. piperita (McKern 1968) and the piperitone/phellandrene, phellandrene, piperitone/cineole and cineole forms of E. dives (Penfold and Morrison 1927a, 1929, Hellyer et al. 1969).
The recognition of infraspecific variation led to the introduction of terms such as ‘physiologi-cal form’ or ‘chemi‘physiologi-cal variety’ and the different types were designated ‘Type’, ‘Variety A’,
‘Variety B’, ‘Variety C’, etc., in order of their discovery (Hellyer et al. 1969). More modern terminology regards these as ‘chemotypes’ or ‘chemovars’. In the case of E. punctata, however, sampling seventy-one individual trees showed a wide but continuous variation in the concentra-tion of the major oil components, indicating that there are no grounds for the establishment of separate chemical types (Southwell 1973). This infraspecific variation in oil composition high-lights the importance of investigating individual trees and not assuming that the results from single trees are representative of the species generally (Boland et al. 1991).
Correlations between the constituents of Eucalyptus species and their taxonomic relationship, both within the genus and as part of the Myrtaceae family, have been attempted. Hegnauer, for example, has addressed the issue in his Chemotaxonomie der Pflanzen series (Hegnauer 1969, 1990).
With such a large number of species within the eucalypts, it is not surprising that attempts have been made to split the genus Eucalyptus into smaller genera. The latest work in this area, by Hill and Johnson (1995), has seen Corymbia, previously a sub-genus, raised to full genus status, with approximately 110 species within it. The remaining 700 species (approximately) still belong to the genus Eucalyptus, though within this genus there are major and minor groupings.
These are shown in the phylogram (Figure 5.1) adapted from Hill and Johnson (1995).
An attempt to discern trends in the essential oils of species within the major groups was car-ried out by Boland and Brophy (1993) based on analyses of the essential oils of approximately one third of the species. While not conclusive, some trends were observed. Corymbia as a genus generally produces poor oil yields and low 1,8-cineole percentages (Boland and Brophy 1993, Brophy et al. 1998b). An exception, however, is C. citriodora (Eucalyptus citriodora) which can pro-duce up to 4.2 per cent of a citronellal-rich (80 per cent) oil. Significantly more species have now been analysed and the question will be re-examined.
Within the ‘Monocalyptus’ is a large group of species comprising the majority of section Renantheria which contain significant amounts of cis- and trans-menth-2-en-1-ol and cis- and trans-piperitol, as well as, in a significant number of species, piperitone. E. radiata and E. oblonga occur in this group. Within the eucalypts this is the only place that this occurs. There is also a group of species which contain oils composed principally of -triketone (e.g. 26, Figure 5.3)
and acylphloroglucinol (e.g. 22 and 27, Figure 5.3) derivatives (Brophy et al. 1996) in this sec-tion, an occurrence not found in the rest of the eucalypts.
‘Symphyomyrtus’ is the largest group within the eucalypts, containing over 500 species.
Within it there are groups of species with high oil yields and 1,8-cineole as the major component. E. bakeri and E. kochii belong to one section of this group, E. camaldulensis to a second, E. sparsa and E. polybractea to a third, and E. globulus, E. sturgissiana and E. smithii to a fourth.
It was thought (Boland and Brophy 1993) that Western Australian species produced more aromatic compounds than did their eastern Australian relatives, this phenomenon crossing
‘group’ boundaries. More recent work, however, published in a series of papers by Bignell et al.
(in which the oil is produced by vacuum distillation rather than steam distillation) has shown that aromatic compounds are present in most of the species examined.
Commercial utilisation of eucalyptus oils
Cineole-rich oils
Apart from practical considerations such as oil and biomass yields, and the amenability of the species to appropriate and economic field management, the most important factor in determining
Arillastrum Group (4 genera / 5 species)
Angophora (17) Corymbia Fundoria (1) Corymbia Apteria (1) Corymbia Rufaria (67)
Corymbia Ochraria (17) Corymbia Blakearia (27)
Odontocalyptus Eudesmia s. str.
Fibridia
Leprolaena Baileyanae Leprolaena Miniatae Gaubaea (2)
Idiogenes (1)
Monocalyptus (170) Symphyomyrtus (500+) Telocalyptus (4)
Nothocalyptus (1)
(20+)
Figure 5.1 Phylogram of the eucalypts (after Hill and Johnson 1995). Numbers in parentheses refer to the number of species.
1,8-cineole 1 (–)-limonene 2 (+)-α-terpineol 3
(+)-citronellal 7 neral 8 geranial 9
(–)-α-phellandrene 12 (–)-piperitone 11
geranyl acetate 10
E-methyl cinnamate 13 O
1 2 3 4 5 6
7
9 8 10
OH
CHO CHO
CHO CH2OAc
O Cineole-rich oils
(+)-α-pinene
H
H
(+)-aromadendrene 5 (–)-globulol 6
H
H
OH 4
Lemon-scented/perfumery oils
Piperitone/phellandrene-rich oils E. olida
COOMe
Figure 5.2 Chemical structures of some common constituents of commercial eucalyptus oils.
the commercial value of an oil is its chemical composition. Of the large and diverse range of compounds found in eucalyptus oils (Table 5.2), the most important is the monoterpene ether 1,8-cineole (1, Figure 5.2). It is used for medicinal, flavour and fragrance purposes and has sig-nificant biological activity (e.g. mosquito repellency (Klocke et al. 1987)). It comprises over 90 per cent of the oil in some specimens of E. bakeri (Brophy and Boland 1989), E. kochii (Gardner and Watson 1947/48), E. oblonga (Baker and Smith 1920), E. plenissima (Brooker et al. 1988), E. polybractea (Boland et al. 1991), E. sparsa (Brophy and Lassak 1986, Southwell 1987) and E. sturgissiana (Boland et al. 1991). With the exception of E. polybractea, few of these are being exploited as commercial sources of 1,8-cineole.
E. globulus remains the chief source of cineole worldwide although the oil contains a lower proportion of it than E. polybractea and some other species. However, E. globulus is widely grown for its wood and for pulp production and, as a result, the ‘waste’ leaf is available for production of oil. This and other commercially produced oils with concentrations of cineole less than that in E. polybractea (e.g. E. smithii and E. radiata) are either fractionated to enhance cineole levels to the 70–75 per cent or 80–85 per cent required by monographs published by ISO and various national pharmacopoeias or sold for uses where cineole content is not so critical (e.g. aromatherapy).
The most common constituents co-occurring with 1,8-cineole are limonene (2) and
-terpineol (3), both of which can be derived from the menth-1-en-8-yl cation, the same biogenetic precursor from which cineole is thought to be derived (Croteau 1987, Croteau et al.
1994). Other biogenetic pathways then contribute the other monoterpenes, for example,
-pinene (4), sesquiterpenes such as aromadendrene (5), globulol (6) and - , - and -eudesmol (17–19, Figure 5.3), and aromatic constituents, for example, methyl cinnamate (13).
Other commercial oils
Although cineole-type (medicinal) eucalyptus oils dominate world production (estimated by Coppen and Hone (1992) at between 2500 and 3100 t in 1991, and by Lawrence (1993) at 3700 t), lemon-scented (perfumery) oils are also produced. The worldwide production of citronellal (7) oils from E. citriodora and the citral (i.e. neralgeranial, 8, 9) oils from E. staigeriana was estimated at 320 and 70 t respectively for 1984 (Lawrence 1985). A chemotype of E. citriodora rich in citronellol (16, Figure 5.3) also exists (Penfold et al. 1951). Geranyl acetate (10) has been produced in the past from the leaf and bark of E. macarthurii and used as a fragrance constituent (Lassak 1988) although it is not believed to be in current production.
There has been a smaller market for piperitone (11) and -phellandrene (12)-rich oils (e.g.
E. dives Type). Piperitone has been used in the synthesis of menthol for flavouring and phellan-drene as a fragrance.
The most recent example of a new eucalyptus oil product is that of E-methyl cinnamate (13) from E. olida. This natural flavouring ingredient was discovered in near pure form in high yield for the first time in E. olida in 1985 (Curtis et al. 1990) and its production has subsequently been commercialised in Australia, from where it is exported.
Oils of potential commercial use
Many other eucalyptus oils have the potential to serve as sources of chemical isolates, that is, they contain chemicals with potential commercial application (Figure 5.3).
Food flavour companies have expressed interest in -phenylethyl phenylacetate (24) which is sometimes the major component of the leaf and bark oil of E. crenulata (Lassak and Southwell 1969, Boland et al. 1991) and the leaf oil of E. aggregata (Hellyer et al. 1966, Boland et al. 1991).
The leaf oil of E. crenulata is also rich in methyl eudesmate (methyl 3,4,5-trimethoxybenzoate, 23). Oils from other species of Eucalyptus provide excellent sources of the eudesmols (17–19), the ketones agglomerone (14), jensenone (22), tasmanone (26) and torquatone (27), isobicyclo-germacral (21), spathulenol (25), benzaldehyde (15) and many other isolates. The best Eucalyptus sources of chemicals 14 –27 are shown in Table 5.1.
MeO O
CHO
CH2OH
OH OH OH
OH CHO
O CHO
OHC
OH
OH HO
COOMe
MeO
OMe OMe
PhCH2CH2OCOCH2Ph
OH H
.
O O
OH MeO
O OMe
OMe MeO
agglomerone 14 benzaldehyde 15 citronellol 16
α-eudesmol 17 β-eudesmol 18 γ-eudesmol 19
guaiol 20 iso-bicyclogermacral 21 jensenone 22 methyl eudesmate 23
β-phenylethyl phenylacetate 24
spathulenol 25 tasmanone 26 torquatone 27
OH O
Figure 5.3 Chemical structures of some constituents with the potential to be sourced as isolates from euca-lyptus oils not yet in commercial production.
The commercial development of an oil need not be dependent on the major constituents. For example, the grandinol-type -triketones from E. grandis possess powerful root and photosyn-thetic electron transport inhibition properties (Crow et al. 1971, 1976, Yoshida et al. 1988, Yoneyama et al. 1989) and the minor constituents of E. citriodora and E. camaldulensis oils have been investigated for mosquito repellent activity (Nishimura et al. 1986, Nishimura 1987, Watanabe et al. 1993).
Biogenesis of eucalyptus oil constituents
The formation of the mono- and sesquiterpenoids in Eucalyptus follows the general principles of isoprenoid biosynthesis (Erman 1985, Croteau 1987). The finer details of these principles advance as the frequency and complexity of labelling experiments increases. For example, recent investigations now suggest that in higher plants isopentenyl pyrophosphate is formed not from mevalonic acid but via the alternative triose phosphate/pyruvate pathway (Lichtenthaler et al.
1997, Eisenreich et al. 1997). These early stages of isoprenoid biosynthesis seem to have been more often studied than the finer details of the coupling and cyclisation steps to form mono-terpene, sesquimono-terpene, diterpene and sesterterpene end products.
The Croteau group, working principally on the monoterpenoids, has isolated cyclases and
The Croteau group, working principally on the monoterpenoids, has isolated cyclases and