Tea Tree Constituents

IAN SOUTHWELL

Wollongbar Agricultural Institute, Wollongbar, NSW, Australia

INTRODUCTION

As with most genera, Melaleuca contains a variety of primary and secondary metabolites. The volatile oil constituents of M. alternifolia and closely related species are the compounds responsible for the commercial development of Melaleuca as a medicinal and aromatic plant. Consequently the most frequently investigated aspects of the chemistry of tea tree concern the identification of these volatile oil constituents. As considerably less is known about the genus's primary metabolite tannins, polyphenols, waxes, amino acids and betaine constituents these will only receive brief mention. The constituents of the other Melaleuca species will be outlined in later chapters in this volume.

OIL CONSTITUENTS Chronological Perspective

At the same time that Melaleuca linariifolia var. alternifolia (Maiden and Betche) was being raised to species status as M. alternifolia (Maiden and Betche) Cheel (Cheel 1924), the first chemical investigations of the taxon were being undertaken (Penfold 1925). Earlier, Baker and Smith (1906, 1907, 1910, 1911, 1913) had investigated the oils of M. thymifolia, M. uncinata, M. nodosa, M. genistifolia (M. bracteata), M. gibbosa, M. pauciflora and M. leucadendron in what must be considered, by todays standards, a most superficial way. Penfold (1925), investigated M. linariifolia and M. alternifolia concurrently and concluded that their essential oils were "practically identical". This investigation included measurement of oil yields (1.5-2.0%), specific gravity, optical rotation, refractive index, solubility in alcohol, fractional distillation and identification of chemical constituents by the preparation of derivatives and comparison of melting points and mixed melting points with authentic materials. In this manner pinene (2), a-terpinene (7), 7-terpinene (12), p-cymene (10), sabinene (?) (3), cineole (11), terpinen-4-ol (14) and sesquiterpenes including cadinene (18) were identified from M. linariifolia and all of these except sabinene identified from M. alternifolia. The methods involved were qualitative rather than quantitative for all constituents except 1,8-cineole (11) which was estimated to be present at 16-20% and 6-8% in M. linariifolia and M. alternifolia respectively.

In subsequent decades, Jones investigated M. linariifolia more thoroughly, discovered both high cineole (61%) (Jones 1936) and low cineole (Davenport et al. 1949) chemical

varieties. Also a-thujene (1), ^-pinene (4), myrcene (5), terpinolene (13), a-terpineol (15) and aromadendrene (16) were detected in the low cineole variety and dipentene (limonene) (8) in the high cineole form. These workers were able to provide an estimate of the percentage contributions made by each component identified that was remarkably close to the gas chromatographic measurements of recent workers (Southwell and Stiff 1990; Kawakami et al. 1990) (Table 1). The structures of these major components of tea tree oil are shown in Figure 1.

The next thorough investigation of tea tree oil chemistry involved reduced-pressure spinning-band column distillation and gas chromatographic (GC) analysis of resulting fractions (Laakso, 1966). This study did not report any new constituents. It does, however, seem to be the first published report on the GC examination of tea tree oil even though routine GC quality control of tea tree oil at the Museum of Applied Arts and Sciences in Sydney had commenced around 1960 (Museum of Applied Arts and Sciences, unpublished records). Laakso's study also seemed to be the first to describe a terpinolene(13)-rich chemical variety of M. alternifolia. Soon after this investigation Guenther (1968) reported a similar fractionation-GC study from the Fritzsche Brothers laboratories.

Table 1 Estimate of the percentage composition of the oil of the terpinen-4-ol chemical variety of M. linariifolia (Davenport et al. 1949) compared with the gas chromatographic results of Southwell and Stiff (1990) and Kawakami et al. (1990)

Constituent

Davenport et al. (1949)

Southwell and Stiff (1990)

Kawakami et al. (1990)t

a—thujene (1)

<1

4.1

4.2

a-pmene (2)

1-2

2.1

2.0

sabine ne (3)

nr

2.7

10.9

ß-pinene (4)

<1

0.6

0.5

myrcene (5)

<1

1.0

1.6

Cí-phcllandrene (6)

nr

0.4

nr

a-terpinene (7)

40a

9.3

10.1

limonene (8)

nr

1.3

1.1

ß-phellandrene (9)

nr

0.9

0.8

p-cymene (10)

<1

1.4

0.8

1,8-dneole (11)

4

6.0

6.0

J^terpinene (12)

40a

18.9

17.2

terpinolene (13)

4

3.5

3.5

terpinen-4-ol (14)

37

38.2

32.1

a-terpineol (15)

4

1.5

2.7

aromadendrene (16)

6b

0.5

nr

ledene (viridiflorene) (17)

6b

0.4

tr

5-cadinenc (18)

6"

0.9

0.5

globuld (19)

2.5C

0.5

nr

vïridiflorol (20)

2.5C

0.2

nr

fClone 1, now known to be M. linariifolia (Southwell et al. 1992). nr, not recorded; tr, trace; a,b,cestimated collectively.

fClone 1, now known to be M. linariifolia (Southwell et al. 1992). nr, not recorded; tr, trace; a,b,cestimated collectively.

Chemical Structure Cardamom
Figure 1 Chemical structures of the significant constituents in tea tree oil

The first gas chromatography-mass spectrometry (GCMS) investigation of tea tree oil (Swords and Hunter 1978) reported forty eight constituents of which only eight were unassigned. Assignments were based on GCMS data with preparative GC, liquid chromatography (LC) and infrared spectroscopy (IR) confirmation used for some identifications. It must be noted however that this oil was not a typical commercial oil as cineole content (16.5%) was above standard limits (Brophy et al. 1989b; Standards Association of Australia 1985; International Standards Organisation 1996) and _p-cymene (11.4%), a-terpinene (2.7%) and )-terpinene (11.5%) indicated substantial oxidation (Brophy et al. 1989b). Components identified in tea tree oil above trace levels (>0.1%) included a-gurjunene (0.23%), ^-terpineol (0.24%), allo-aromadendrene (0.45%), a-muurolene (0.12%) 8-p-cymenol (0.13%) and viridiflorene (1.03%). The short, packed Carbowax 20 M GC column used for the GCMS analysis had obvious deficiencies. Even the 100m Carbowax 20 M capillary column failed to resolve a-thujene from a-pinene, ^-phellandrene from 1,8-cineole and had difficulty in resolving myrcene from a-phellandrene and 1,4-cineole and terpinen-4-ol from ^-elemene.

These authors described the sesquiterpene hydrocarbon viridiflorene for the first time and proposed structure (21) (Figure 2). This structure was inconsistent with the dehydration product of viridiflorol (20) which should have structure (17) because of the absolute configuration assignments of Buchi et al. (1969) following the synthesis of (-)-aromadendrene (22), the enantiomer of naturally occurring (+)-aromadendrene (16). Viridiflorene must then have structure (17) which is also the structure of ledene, the dehydration product of ledol (23), the C10 enantiomer of (+)-viridiflorol (20). This assignment has been confirmed by nuclear magnetic resonance (NMR) by both Australian and French workers (Southwell and Tucker 1991; Faure et al. 1991). (+)-Ledene (17) has been well known as a dehydration product of viridiflorol (e.g. Birch et al. 1959) and also as a natural constituent of essential oils (e.g. Taskinen 1974). Consequently the name viridiflorene should be replaced by ledene in tea tree and other essential oil reports.

A more recent GCMS analysis of M. alternifolia oil corrected the viridiflorene structure, listed a total of 97 constituents and identified several new components (Brophy et al.

Tea Tree Chemical Composition

16 (+)-aromadendrene

17 (+)-ledene (viridiflorene)

20 (+)-viridiflorol

16 (+)-aromadendrene

17 (+)-ledene (viridiflorene)

20 (+)-viridiflorol

22 (-)-aromadendrene

Figure 2 Chemical structures of sesquiterpenoids conformationally related to ledene

22 (-)-aromadendrene

Figure 2 Chemical structures of sesquiterpenoids conformationally related to ledene

1989b). The most significant findings of this investigation included (a) determining suitable GC and column conditions for the optimal separation of tea tree oil constituents, b) the identification of the cis and trans alcohol pairs of sabinene hydrate, p-menth-2-en-1-ol and piperitol, (c) the composition of atypical and aged oils, (d) the affect of distillation time on oil composition, (e) the micro-scale analysis of a single leaf (6 mg) for quality determination and (f) the presence of oil precursors in the ethanolic extracts of tea tree flush growth.

This last finding was subsequently investigated more thoroughly (Southwell and Stiff 1989). Although individual mature leaves when immersed in ethanol gave an extract which accurately reflected the quality of the oil if the leaves were distilled, the same could not be said for the brighter green flush growth. This flush growth was found to be rich in cis-sabinene hydrate (24) which was replaced by 7-terpinene (12) and terpinen-4-ol (14) as the leaves matured. Very little of (24) and other minor precursors were detected in the distilled oil due to the lability of cis-sabinene hydrate (Erman 1985; Fischer et al. 1987, 1988).

Comprehensive GCMS analyses were performed on eight tea tree clones propagated at the University of California (Kawakami et al. 1990). These clones, originally claimed to be M. alternifolia stock, have since been shown to contain at least one M. linariifolia variety (Southwell et al. 1992). Oils were obtained by both simultaneous purging and extraction (SPE) to give headspace analysis and steam distillation and extraction (SDE) to give oil analyses. Only two of the distilled oils were from terpinen-4-ol type clones. The headspace (SPE) analysis concentrated the more volatile monoterpene hydrocarbons especially sabinene, a-thujene, a-terpinene and 7-terpinene at the expense of the less volatile oxygenated terpenoids especially terpinen-4-ol. These compositions were similar to the composition of the initial vapour cloud that emerges from the still condenser prior to condensation (Southwell 1988). Although this investigation reported for the first time a number of new sesquiterpenoids, none exceeded 0.6% of the total oil.

The two commercial terpinen-4-ol type tea tree oils sourced from M. alternifolia and M. linariifolia are distinguishable on the grounds of oil chemistry (Southwell and Stiff 1990). The former when analysed by GC gives a smaller peak for a-thujene than for a-pinene and the latter gives the converse. As these peaks are always the first two significant chromatographic peaks, a glance at the GC trace will tell which oil has been analysed as long as a-pinene and a-thujene are resolved. The mean a-thujene: a-pinene ratio was 0.33 (n=521) for M. alternifolia and 1.49 (n=180) for M. linariifolia. Nonpolar or intermediate polarity stationary phases are best for this analysis as resolution is greater. The a-thujene: a-pinene ratio is upset as the percentage of cineole in the oil increases. As a-thujene is associated with the terpinen-4-ol biogenetic pathway, higher cineole means less terpinen-4-ol and a corresponding lower a-thujene: a-pinene ratio. Hence the test is only suitable for the terpinen-4-ol chemical varieties. A similar way of distinguishing the two species is to measure the cis-sabinene hydrate (24) to trans-sabinene hydrate (25) ratio in the ethanolic extract of the flush growth of both species. The mean cis-sabinene hydrate: trans--sabinene hydrate ratio was 7.1:1 (62 extracts from 11 trees) for M. alternifolia and 0.7:1 (29 extracts from 6 trees) for M. linariifolia (Southwell and Stiff 1990).

Terpinolene varieties of Melaleuca have, from time to time, received passing mention in the literature (Laakso 1966; Brophy et al. 1989a; Kawakami et al. 1990). Their existence was formally acknowledged and data documented by Southwell et al. (1992) and Butcher et al. (1994). Both M. alternifolia and M. trichostachya were found to have varieties with from 10-57% terpinolene, high proportions of cineole (13-56%) and insufficient terpinen-4-ol (1-20%) for the tea tree oil market.

With the advent of chiral GC columns, the enantiomeric ratios of tea tree oil constituents were determined (Russell et al. 1997; Leach et al. 1993; Cornwell et al. 1995). Seven monoterpenes were resolved in this way and their percentages and enantiomeric ratios shown in Table 2. These ratios provide valuable criteria for checking the authenticity of tea tree oil especially blends with (-)-terpinen-4-ol from Eucalyptus dives that have been detected in the past. Similar chiral resolutions were achieved using lanthanide shift reagents on the individual tea tree oil constituents terpinen-4-ol and a-terpineol (Leach et al. 1993). The complexity was however too great for these shifts to be meaningful for an entire oil. The 58% enantiomeric excess obtained for a standard (99%) sample of terpinen-4-ol ([a]D+29°) was consistent with both the 30% enantiomeric excess for terpinen-4-ol ([a]D+16%) fractionated from tea tree oil (Russell et al. 1997) and the maximum rotation values (+47°-+48°) reported on enantiomerically pure samples (Naves and Tullen 1960; Ohloff and Uhde 1965; Verghese 1966).

Because of the efforts of these investigators over the years, the chemistry of tea tree oil is now, well established. There is a need to clarify some of the minor and trace constituent assignments and to establish the enantiomeric composition of the sesquiterpenoids. Then efforts can concentrate on which of these constituents are beneficial or detrimental (to the commercial uses of the oil) and how to maximise or minimise their contribution to commercial oils.

Table 2 The enantiomeric composition of the seven tea tree oil constituents resolved by chiral gas chromatography on ^-cyclodextrin

Constituent

—)-a-pinene + )-or-pinene •—J-a-phel I andren e + )-a-phdIafldfcnc —)-limonenc + )-Utnonene + )-j3-phe)landrene + )-/?-pheHandrenL' +)-linilool + )-lmaiool + )-terpnicn-4-ol —)-terpinen-4-ol —)-a-terpineol + )-«-terpineol

1007 1017 1058 1058 1079 1082 1092 1095 1254 1257 1334 1340 1381 1385

0.51

tr tr a a a a, insufficient resolution to allow accurate quantitation; tr, trace.

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