Medicinal and Aromatic Plants—Industrial Profiles


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IAN SOUTHWELL
50
Figure 10 Possible biogenetic pathways for the formation of Melaleuca terpinen-4-ol type monoterpenene constituents
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

TEA TREE CONSTITUENTS 51
terpinen-4-ol and 
γ-terpinene has been advantageous for the tea tree oil industry. Distilling
flush growth does not give a different quality oil as the precursor sabinene hydrates convert
to the end product terpinen-4-ol and 
γ-terpinene during the distillation in a similar way to
the change that takes place as the leaf matures. The inability of marjoram to carry out this
conversion as the leaf matures has meant that the quality of marjoram products varies
according to the processing method. Consequently extraction methods favour the hydrates
and distillation methods terpinen-4-ol (Fischer et al. 1987). The biosynthetic capacity of
marjoram seems confined to the synthesis of the sabinene hydrate skeleton in contrast to
that of Melaleuca which converts the thujane skeleton to the menthane skeleton as the leaf
matures. Whether this change in tea tree is a secondary enzymic transformation of thujanes
to terpinenes or the closing down of one biogenetic pathway and the concomitant initiation
of the other is yet to be confirmed. The comparative concentration of the metabolites however
suggests the former.
Cornwell et al. (1995) distilled young leaves of M. alternifolia in 
18
O-labelled water to
obtain label in the resultant terpinen-4-ol and a-terpineol. This indicated that if terpinen-4-
ol (14) was formed directly from cis-sabinene hydrate (24), the process involved hydration
of the terpinen-4-yl cation (32) rather than a 1,4-hydroxyl-shift in cis-sabinene hydrate (24)
which does not account for the non-oxygenated byproducts. In addition the ratio of these
products is similar to the ratio of the same products obtained by the acid catalysed
rearrangement of sabinene hydrate. Hence these authors conclude that the sabinene hydrate
degradation during ontogenesis is a purely chemical breakdown rather than enzymic
secondary metabolism as suggested by Southwell and Stiff (1989). This does not however,
address the comparison with marjoram where the sabinene hydrates are retained even in the
mature leaf (Fischer et al. 1987, 1988).
By applying these findings to the commercial terpinen-4-ol chemical variety of tea tree
oil, biogenetic pathways like those outlined in 
Figure 10
 are likely to be appropriate for
monoterpenoid formation.
In additionMelaleuca oil contains up to approximately 12% of sesquiterpenoids, mostly
sesquiterpene hydrocarbons. The flush growth extracts also indicated the presence of
sesquiterpene precursors which convert to aromadendrene (16), ledene (17), 
δ-cadinene
(18) etc. as either the leaf matures or the leaf is distilled. Although these precursors have not
been isolated and positively identified, a biogenetic precursor like bicyclogermacrene (38)
as suggested by Taskinen (1974) for marjoram is plausible (
Figure 11
) (Brophy et al. 1989b;
Ghisalberti et al. 1994).
The terpinen-4-ol chemical varieties of both M. linariifolia and M. dissitiflora would
be expected to display similar biogenetic pathways to M. alternifolia. The GC difference
in the 
α-thujene/α-pinene ratios (see above) for the oils of M. alternifolia and M.
linariifolia suggests that the thujane pathways differ because of the greater concentration
of 
α-thujene in the latter. A similar difference was also seen in the GC traces of the
ethanolic extracts of the flush growth of both species. In addition to the 
α-thujene/ α-
pinene ratio, M. alternifolia gave a cis: trans sabinene hydrate ratio of approximately
7:1 whereas in M. linariifolia the ratio was approximately 0.7:1. These ratios are very
similar to those obtained by Hallahan and Croteau (1989) for the sabinene hydrate
ratios when both the “natural” (-)-(3R)-linalyl pyrophosphate (36) and the “unnatural”
(+)-(3S)-linalyl pyrophosphate (37) precursors respectively were used as substrates for
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

IAN SOUTHWELL
52
sweet marjoram (Majorana hortensis) partially purified cyclase. This suggests that the
difference between the two species is significant at the enzyme level where for M.
linariifolia the “unnatural” (3S)-linalyl pyrophosphate is better accommodated even
though there is minimal difference at the end of the biogenetic pathways especially
with the steam distilled oil constituents (Southwell and Stiff 1990). Thus the sabinene
hydrate cyclase from M. linariifolia should bind preferentially to the right handed screw
form of geranyl pyrophosphate (35) which isomerises to the bound (+)-(3S)-linalyl
pyrophosphate (37) in contrast to the M. alternifolia cyclase which should bind to the
left handed screw form of geranyl pyrophosphate (34) isomerising to the bound (-)-
(3R)-linalyl pyrophosphate (36) (
Figure 10
).
Determination of oil quality in tea tree seedlings by extraction has thrown some
light on the progressive initiation of biogenetic pathways in Melaleuca. At the
dicotyledon leaf stage of development, the pinene, cineole and terpinolene pathways
are evident with no initiation of the thujane sabinene and sabinene hydrate or the
terpinene terpinen-4-ol and 
γ-terpinene pathways (Russell et al. 1997). α- and ß-
Pinene appear to be the products of one cyclase. Croteau (1987) suggested a possible
association with myrcene which is not supported by Melaleuca seedling ontogeny
Figure 11 Possible biogenetic pathways for the formation of Melaleuca terpinen-4-ol type sesquiterpene
constituents
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

TEA TREE CONSTITUENTS 53
where myrcene concentrations increase only after the formation of 
α- and ß-pinene.
1,8-Cineole is also formed immediately and varies little in the course of development
of the seedling. The other products which, like cineole, are possibly derived from
moiety (31) (
Figure 9
) are 
α-terpineol (15), terpinolene (13) and limonene (8).
Terpinolene, which can also be derived from moiety (32) (Figure 9), is present in
the dicotyledon leaves in higher proportions than in the mature leaves or the distilled
oil. This decreasing proportionality is in contrast to the constant proportion of cineole
suggesting some differences in formation pathways. On the other hand, along-the-
branch analysis of the terpinolene variety shows an inverse proportion relationship
between cineole and terpinolene suggesting that, in this variety, the two are similarly
derived (Southwell et al. 1992). Limonene (8) and 
α-terpineol (15), known to
increase in concentration as cineole increases, only appear later in seedling ontogeny
(
Figure 8
).
The most remarkable concentration changes in seedling development occur, as in mature
tree along-the-branch analysis, with the thujanes sabinene, cis- and trans-sabinene hydrate
and the terpinenes 
α-terpinene, γ-terpinene, terpinolene, and terpinen-4-ol. These
biogenetic pathways seem to be initiated in earnest at a seedling age of around three
weeks when the third leaf set begins to emerge (Figure 8). From three weeks to ten weeks
both cis-sabinene hydrate and terpinen-4-ol concentrations increase. After ten weeks, the
more mature leaves have greater concentrations of terpinen-4-ol than cis-sabinene hydrate
(
Figure 6
).
MELALEUCA OIL STABILITY
Health authorities require shelf life measurements to be made so that “use by…” dates can
be printed on health care product labels. For these purposes an understanding of the chemical
changes taking place as an oil ages is essential. The first and most obvious chemical change
in oil composition to be noted is an increase in p-cymene concentration as the menthadienes
a-terpinene (7) 
γ-terpinene (12) and terpinolene (13) oxidise (
Figure 12
). The rate at which
this change occurs is variable as some oils are remarkably stable over ten years whereas
others can oxidise after two years if storage conditions are poor (
Table 7
) (Southwell 1988;
Brophy et al. 1989b). Storage in cool, dark, dry, inert atmosphere (or minimum surface
area/volume ratio) and inert containers (stainless steel or tinted glass) ensures optimum
stability.
Some aged oils have been reported to be deep yellow in colour before depositing small
amounts of oil insoluble crystals. These crystals were found to be 1S,2S,4S-trihydroxy-p-
menthane (39) from both M. linariifolia (Jones and Oakes 1940; Davenport et al. 1949) and
M. alternifolia (Brophy et al. 1989b) terpinen-4-ol type oils. The formation of this oxidation
product occurs concurrently with but by no means as extensively as, the formation of p-
cymene from the terpinenes (Figure 12).
Shelf-life trials conducted in our laboratories have shown that the antimicrobial activity
of aged oils is not decreased as long as terpinen-4-ol levels are maintained. Indeed some
researchers have reported increased activity with aged oils in which terpinen-4-ol
concentrations have been maintained (Markham 1996). Consequently the antimicrobial
stability of aging oils can be assessed by measuring terpinen-4-ol content.
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

IAN SOUTHWELL
54
The overall chemical stability however, is best assessed by measuring a-terpinene, 
γ-
terpinene and p-cymene in addition to terpinen-4-ol. Chemical stability trials at the
Wollongbar Agricultural Institute indicated that well-stored oils, 9–13 years old, only show
a little oxidation and actually increase in terpinen-4-ol content. Poorly stored oils however,
can oxidise rapidly (even in 12 months) and one 47 year old oil contained 37.7% p-cymene
and 3.2% 1S,2S,4S-trihydroxy-p-menthane (39) while still retaining 26.7% terpinen-4-ol
(
Table 8
). 1,8-Cineole content remains remarkably constant during aging as it does during
the different stages of plant ontogeny.
PROCESS MODIFIED OILS
Typical Melaleuca oil compositions are based on steam or hydro-distillations of several
hours duration for either laboratory or commercial scale operations. It has already been
seen that this composition changes for solvent extraction (especially when flush growth or
early seedling leaf is included) (Southwell and Stiff 1989, 1990), for supercritical fluid
extraction (Wong 1997), and for headspace analysis (Southwell 1988; Kawakami et al.
1990). The compositions of these process-modified oils are shown in 
Table 9
.
Of commercial significance for oil quality is the compositional variation that can occur
using different distillation times. For example Brophy et al. (1989b) found that the first 30
minutes of a laboratory scale-distillation gave a 55.9% terpinen-4-ol oil whereas the remaining
60 minutes gave only 25.1% terpinen-4-ol (Table 9). This trend also exists in commercial-
scale distillations albeit within a reduced time-frame (Russell et al. 1997). This variation in
Table 7 Monoterpenoid composition comparison of aged oils of M. alternifolia
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

TEA TREE CONSTITUENTS
55
constituent percentages showed that the more polar constituents distil over before the less
polar, lower boiling components suggesting that hydrodiffusion acts to extract more polar
components first (Koedam, 1987). More detailed studies of a similar nature were reported
by Stiff (1996) for hydrodistillations and by Johns et al. (1992) for steam distillations.
Consequently, distillation for reduced periods of time will enhance terpinen-4-ol content
and reduce the percentage of terpene hydrocarbons that occur in a completely distilled oil,
the former enhancing the concentration of the active ingredient in Melaleuca and the latter
reducing the concentration of some possible allergenic fractions (Southwell et al. 1997).
Figure 12 The oxidation of oil of Melalecua, terpinen-4-ol type constituents
Table 8 Shelf-life tests for tea tree oil chemical stability (25–35°C) at the Wollongbar Agricultural
Institute (italics indicate composition of freshly distilled oil)
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

IAN SOUTHWELL
56
Some of the more polar components of an essential oil will always dissolve in the
condensate. For terpinen-4-ol type Melaleuca oils, these are the more polar 1,8-cineole,
terpinen-4-ol and a-terpineol components. Extraction of several batches of condensate with
diethyl ether has shown that about 0.07% of oil can be recovered from the water containing
the dissolved oil. This oil was made up of approximately 73.7% terpinen-4-ol, 13.2% a-
terpineol, 1.6% 1,8-cineole and traces (<1%) of the other alcohols (cis-, and trans-menth-
2-en-1-ol, cis-, and trans-piperitol) (Russell et al. 1997). Although distillation condensate
is dilute, it may well provide a suitable source of terpinen-4-ol for spray application (e.g.
horticultural plant pathogens as investigated by Bishop (1995) and Bishop and Thornton
(1997)).
In addition, the interface of the oil and aqueous layers of the condensate sometimes
contain distillation debris and is kept separate from the commercial oil. Analysis of this
interface oil showed enhanced sesquiterpenes and reduced terpinen-4-ol typical of the “dregs”
of a distillation (Russell et al. 1997). Extreme conditions of distillation, especially time and
pressure, can “overcook” an oil and give similarly substandard results (Table 9).
As with any distilled oil, composition can be modified by further fractional distillation.
This can be the method of choice for preparing 98% pure terpinen-4-ol, for enhancing the
terpinen-4-ol content of an oil by removing outlying volatile constituents and for removing
the potentially allergenic sesquiterpenoid fraction. 1,8-Cineole however can not be removed
in this way without adversely effecting the composition of the oil by removing quantities of
desirable constituents as well (Russell et al. 1997).
Table 9 Percentage composition of process modified M. alternifolia products compared with a typical
terpinen-4-ol type oil
a
Kawakami et al. (1990), 
b
Southwell and Stiff (1989), 
c
Russell et al. (1997), 
d
Brophy et al. (1989b).
un, unresolved; tr, trace.
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

TEA TREE CONSTITUENTS 57
METABOLISM OF TEA TREE OIL
Little is known about the metabolism of tea tree oil in mammals including humans. The
metabolism of the oil by the tea tree plantation pest Pyrgo beetle, Paropsisterna tigrina, has
been investigated by examining the frass volatiles from larvae and adults (Southwell et al.
1995). At first, no obvious metabolites were observed when the beetles fed on commercial
terpinen-4-ol type Melaleuca plantation tea tree. When higher 1,8-cineole tea tree leaf was
the sole diet, (+)-2ß-hydroxycineole (40) was isolated as the principal metabolite (Figure
13). Reinvestigation of the frass dropped when the commercial terpinen-4-ol variety was
fed, showed that traces of (+)-2ß-hydroxycineole occurred in the frass. Other paropsine
beetles when fed high cineole diets also metabolised cineole but to other isomers of
hydroxycineole. Chrysophtharta bimaculata hydroxylated cineole in the 3
α (41), 9 (42)
and 2
α (43) positions, Faex nigroconspersa larvae in the same positions in different
proportions and F. nigroconspersa adults in different proportions again (
Table 10
, Figure
13). Investigations now need to establish whether these seemingly species-specific
metabolites are being used as pheromones for insect communication.
Figure 13 The chemical structures of metabolites of 1,8-cineole
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

IAN SOUTHWELL
58
OTHER USES OF TEA TREE
General Uses
Most of the other additional uses of Melaleucas are based on the physical or aesthetic
rather than the chemical properties of the wood (used for construction), branches (for broom
fences), bark (for bark paintings, art) or entire tree (in windbreak, landscaping, swamp
reclamation) (Wrigley and Fagg 1993).
Proline Analogues
The discovery of substantial quantities of proline analogues (
Figure 7
) in the leaves of some
Melaleuca species highlights a potentially commercial source of industrial chemical
compounds. Coupled with the recent interest in biochemical changes in plants suffering
water and salinity stress, is the ability of drought resistant species to accumulate proline and
proline analogues including glycinebetaine. Investigations have shown that, when subject
to water or salinity stress under glass house or laboratory conditions, proline (27) and trans-
4-hydroxy-N-methyl-L-proline (29) in M. lanceolata and trans-4-hydroxy-N-methyl-L-
proline (29) and N, N’-dimemyl-trans-4-hydroxy-L-proline (30) levels in M. uncinata
increased (Naidu et al. 1987).
Further investigations are now translating these laboratory findings into field situations
as trial plantings of M. bracteata, M. uncinata, M. lanceolata, M. populiflora and M.
viridiflora are being established in eastern and north eastern Australia (Naidu 1997). The
best species for the commercial production of proline analogues will be chosen for the
production of natural agricultural chemicals which will alleviate stress and increase biomass
yields in pastures and crops.
REFERENCES
Association Française de Normalisation (1996) Huile essentielle de Melaleuca type terpinène-4-ol.
Huiles essentielles. Tome 2 (5
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 édition), AFNOR, Paris La Défense, pp. 529–536.
Baker, R.T. and Smith, H.G. (1906) Essential oil of Melaleuca thymifolia. J. Proc. Roy. Soc. NSW,
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Baker, R.T. and Smith, H.G. (1907) Essential oil of Melaleuca uncinata and M.nodosa. J. Proc. Roy.
Soc. NSW, 41, 196.
Table 10 The proportions (%) of 2
α (43), 2ß (40), 3α (41) and 9 (42) hydroxycineoles detected in the
frass volatiles of paropsine beetles feeding on high cineole Melaleuca leaf
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

TEA TREE CONSTITUENTS
59
Baker, R.T. and Smith, H.G. (1910) Essential oil of Melaleuca genistifolia Sm. (M.bracteata). J. Proc.
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Baker, R.T. and Smith, H.G. (1911) Essential oil of Melaleuca genistifolia, M. gibbosa and M.
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Baker, R.T. and Smith, H.G. (1913) Essential oil of Melaleuca leucadendron. J. Proc. Roy. Soc. NSW,
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Bishop, C.D. (1995) Antiviral Activity of the Essential Oil of Melaleuca alternifolia (Maiden & Betche)
Cheel (Tea Tree) Against Tobacco Mosaic Virus. J. Essent. Oil Res., 7, 641–644.
Bishop, C.D. and Thornton, I.B. (1997) Evaluation of the Antifungal Activity of the Essential Oils of
Monarda citriodora var. citriodora and Melaleuca alternifolia on Post-Harvest Pathogens. J. Essent.
Oil Res., 9, 77–82.
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193–203.
Brophy, J.J., Davies, N.W., Southwell, I.A, Stiff, I.A. and Williams, L.R. (1989b) Gas Chromatographic
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Brophy, J.J, Lassak, E.V. and Boland, D.J. (1990) Steam volatile leaf oil of Melaleuca globifera R. Br,
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Brophy, J.J. and Lassak, E.V. (1992) Steam volatile leaf oils of some Melaleuca species from Western
Australia. Flav. Frag. J. 7, 27–31.
Buchi, G, Hofheinz, W. and Paukstelis, J.V. (1969) The synthesis of (-)-aromadendrene and related
sesquiterpenes. J. Am. Chem. Soc., 91, 6473–6478.
Butcher, P.A, Bell, J.C. and Moran, G.F. (1992) Patterns of genetic diversity and nature of the breeding
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alternifolia (Myrtaceae). Biochemical Systematics and Ecology, 42, 419–430.
Cheel, E. (1924) Notes on Melaleuca, with descriptions of two new species and a new variety. J. Proc.
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from the H
2
18
O steam distillates of Melaleuca alternifolia (Tea Tree). J. Essent. Oil Res., 7, 613–
620.
Courtney, J.L., Lassak, E.V. and Speirs, G.B. (1983) Leaf wax constituents of some Myrtaceous species.
Phytochemistry, 22, 947–949.
Croteau, R. (1987) Biosynthesis and catabolism of monoterpenoids. Chem. Rev., 87, 929–954.
Davenport, J.B, Jones, T.G.H. and Sutherland, M.D. (1949) The essential oils of the Queensland flora.

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