Medicinal and Aromatic Plants—Industrial Profiles


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part of The Gordon and Breach Publishing Group.

IAN SOUTHWELL
42
Table 5 The concentrations (%) of key components in the emerging 2-week-old dicotyledon
(normal type) and ten-week-old leaf-set-ten (parentheses) leaves of seedlings of the three main
chemotypes of tea tree
terpinen-4-ol increased to 36% (
Figure 6
). As a result of this microextraction method,
recommendations were made to the industry concerning the best times to analyse tea tree
seedlings for quality before planting out. Table 5 shows the early changes in oil constituent
concentrations from emerging to 10 week old leaf set 10 leaves. Consequently leaf set 10 at
6 weeks of age will give an accurate estimate of final oil quality. Dicotyledon analysis will
predict the chemotype propagated.
Supercritical and dense carbon dioxide extractions of tea tree oil have been attempted on
laboratory scale. In one example, investigations using a variety of supercritical carbon dioxide
extraction conditions showed that although oils chemically identical to the distilled oil can
be obtained, precursor components such as cis-sabinene hydrate are likely to extract without
substantial conversion to the end products terpinen-4-ol and 
γ-terpinene (Wong 1997). As it
is difficult to harvest material devoid of flush growth, this extraction method could give a
product with low terpinen-4-ol and high precursor contents unless temperatures approaching
those of a steam distillation are used. Thus differing compositions for the product and the
higher costs and specialised equipment associated with such an extraction may discourage
further investigation.
Leaf Waxes and Proline Analogues
In contrast with the volatile constituents, the non-volatile components in Melaleuca,
especially the M. alternifolia group have received little attention. Some such isolates are
shown in 
Figure 7
.
One published analysis of leaf waxes (Courtney et al. 1983) reported the isolation of the
triterpenoid ursolic acid (26) from the leaf wax of M. quinquenervia. Studies of the leaf
waxes of the M. alternifolia group have not been reported in the literature.
L-proline (27) and its analogues N-methyl-L proline (28), 4-hydroxy-N-methylproline
(29) and 4-hydroxy-N, N-dimethylproline (30) have been isolated from various Melaleuca
species (Naidu et al. 1987; Jones et al. 1987). The structures of these constituents were
supported by X-ray crystallographic studies. Concentrations were found to increase when
the plants were subjected to water or salinity stress under laboratory conditions. The best
sources of these compounds were found to be M. uncinata, M. lanceolata, M. cuticularis,
M. populiflora and M. viridiflora. These nitrogenous constituents are under investigation as
potential stress alleviating constituents and are increasing the biomass yields of pastures
and crop species. Investigations aimed at commercialising these compounds are underway
with field trials in several locations on the east coast of Australia (Naidu 1997).
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
43
CHEMICAL VARIATION
The species known to yield the terpinen-4-ol type of tea tree oil are M. alternifolia, M.
linariifolia and M. dissitiflora (Brophy et al. 1989b). Other species may also be suitable
sources of the commercial oil. M. uncinata R. Br. sensu lato, for example has a similar
terpinen-4-ol type oil which only fails to meet standards because of a high p-cymene
concentration (17.6%) (Brophy et al. 1990; Brophy and Lassak 1992). In addition, M.
parviflora from Vietnam has been reported to yield a similar oil (Dung et al. 1994). The
identification of this species has now been revised to M. alternifolia (Dung 1996).
The above species also yield other types of tea tree oil. The most abundant is the 1,8-
cineole chemical variety (Brophy et al. 1989b). M. alternifolia also has a terpinolenerich
chemical variety (Southwell et al. 1992).
Penfold et al. (1948), before the advent of gas chromatography used the o-cresol method
for the determination of 1,8-cineole (Cocking 1920) to separate three chemical varieties of
M. alternifolia based on 1,8-cineole content. The “Type” variety contained 6–14 percent,
“Variety A” 31–45 percent and “Variety B” 54–64 percent. The first of these, the Type
form, was the only one recommended for medicinal use (Penfold et al. 1948; Guenther
1948; Lassak and McCarthy 1983). More recent investigations (Brophy et al. 1989b) have
Figure 7 Chemical structures of significant non-volatile constituents in 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.

IAN SOUTHWELL
44
noted the high 1,8 cineole Variety B form but suggest a gradation to the Type form rather
than the existence of a clear intermediate Variety A form. On the other hand Butcher et al.
(1994) propose a total of five chemical varieties: the initial three proposed by Penfold et al.
(1948) and two varieties of the terpinolene type named Variety C (15–20% terpinen-4-ol;
30–36% 1,8-cineole; 10–18% terpinolene) and Variety D (1–2% terpinen-4-ol; 17–34%
1,8-cineole; 28–57% terpinolene). More thorough surveys of individual trees including
greater numbers of samples are giving data falling between these proposed varieties
suggesting a merger of these proposed varieties as has happened with Eucalyptus punctata
(Southwell 1973).
The existence of these chemical varieties is of economic importance for nursery
suppliers or plantation owners collecting seed and raising their own seedlings. The
quality of the seed bearing parent is not a reliable guide to the quality of the seed as
cross pollination with a parent of a different variety is sometimes possible. The analysis
of seedling leaf, even as early as the dicotyledon leaf stage, is a reliable method for
determining tree quality when the onset of different biogenetic pathways is understood.
Tea tree breeding programs are seeking to establish seed orchards of high yielding,
high quality varieties of the terpinen-4-ol variety of M. alternifolia (
Chapter 7
, this
volume). Although this species is the one at present preferred by the industry, the
Australian and International Standards indicate that the oil may be sourced from M.
alternifolia., M. linariifolia and M. dissitiflora and other species of Melaleuca yielding
a comparable oil. Trials involving the production of M. linariifolia (Southwell and Stiff
1990) and M. dissitiflora oil (Williams and Lusunzi 1994) have not yet led to large
scale plantings of these species.
QUALITY ASSURANCE AND STANDARDS
Oil Quality
The existence of chemical varieties other than the commercial terpinen-4-ol type has
meant that these have been harvested from the wild or sometimes cultivated, resulting in
substandard oils being found in the marketplace. Hence oil quality is critical. As with
other essential oils, quality before the advent of gas chromatography was defined by
measuring the physical constants of refractive index, optical rotation, specific gravity
and solubility in alcohol. Measurement of these constants is still mandatory for compliance
with International, National and Pharmacopoeia Standards because of its usefulness
defining physical constant ranges for the unique combination of constituents in any one
essential oil.
The early standards for tea tree oil such as the BPC (British Pharmaceutical Codex
1949) and the Australian Standard K-175 (Standards Association of Australia 1967) were
based on these physical constants (
Table 6
). The BPC also included an alcohol
determination based on the measurement of ester numbers before and after acetylation.
As this method is not appropriate for tertiary alcohols (Guenther 1948), reliable tea tree
oil alcohol determinations were not achieved until oils were analysed by gas
chromatography. The revised Australian Standard 2782–1985 (Standards Association of
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 45
Australia 1985) for Oil of Melaleuca, Terpinen-4-ol Type set a maximum level (15%) for
1,8-cineole and a minimum concentration (30%) for terpinen-4-ol as determined by gas
chromatography (GC). Standards Australia submitted a further revision of this standard
to the International Standards Organisation (ISO) who subsequently published ISO 4730
(International Standards Organisation 1996). This submission included a Chromatographic
Profile Table listing the required ranges for fourteen constituents and was used as a basis
for French Standard T75–358 by AFNOR (Association Française de Normalisation 1996)
and the updated Australian Standard AS 2782–1997 (Standards Australia 1997). Ledene
(viridiflorene), sometimes hidden under the a-terpineol peak on polar GC stationary phases,
was a notable omission. In addition, the upper limit for sabinene (3.5%) was not high
enough to cover all M. dissitiflora terpinen-4-ol type oils (Brophy and Doran 1996).
Furthermore, the upper limit for sesquiterpene and sesquiterpene alcohol components
was based on a few extraordinary analytical results. This profile Table was then included
in the ISO Standard which also contains three typical chromatograms (for information
only) on polar (BP 20), non-polar (OV 101) and intermediate polarity (AT 35) stationary
phases. Analysis on an appropriate intermediate polarity phase (
Figure 3
) is preferable
because polar phases sometimes do not resolve a-thujene (1) from a-pinene (2) and, ß-
phellandrene (9) from 1,8-cineole (11) and non-polar phases, although separating the
former peaks, do not resolve the latter.
The tea tree oil industry is seeking to have Oil of Melaleuca relisted as a monograph in
pharmacopoeias. The German Pharmaceutical Codex has published a monograph (Deutscher
Arzneimittel-Codex 1996) which outlines a TLC and GLC method for oil analysis. The
latter is based on the published literature and includes the determination of 

3
-carene which
has not been reported in tea tree but is viewed with suspicion because of the eczematous
properties of its auto-oxidation products (Tisserand and Balacs 1995).
Table 6 Tea tree oil standards and monographs
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
46
In the market place, most tea tree oil is bought and sold on 1,8-cineole and terpinen-4-
ol gas chromatographic area percent figures alone. This ensures that the oil is sourced from
the low-cineole-high terpinen-4-ol chemical variety. A technique involving the addition of
a known weight of internal standard (usually n-tridecane, n-tetradecane or n-pentadecane)
or the measurement of the four physical constants (optical rotation, refractive index, relative
density and solubility in alcohol) ensure that the oil has not been diluted by either solvent or
non-volatile materials.
More sophisticated means of quality control have been examined. These include gas
chromatography-mass spectrometry (GCMS), nuclear magnetic resonance (NMR), infrared
spectroscopy (IR) and chiral column gas chromatography.
GCMS has been used to help identify the components in the oil (Swords and Hunter
1978; Brophy et al. 1989b; Kawakami et al. 1990) but is unlikely to be used for routine
quality control.
Similarly chiral stationary phases have been used to determine the enantiomeric excess
of the chiral constituents in tea tree oil (Leach et al. 1993). Now that these ratios have been
determined, there is little need to use chiral stationary phases routinely. In special
circumstances they may however be useful for detecting adulteration. For example, some
years ago, a blend of tea tree oil and a eucalyptus oil fraction reached the international
market. Although the eucalyptus oil fraction was rich in terpinen-4-ol, such a source of this
key terpene alcohol is characterised by a negative optical rotation. Consequently the ratio
of (+)-terpinen-4-ol to (-)-terpinen-4-ol was much lower in the blend (1:2) than in the
unadulterated (2:1) tea tree oil (Leach et al. 1993).
High field proton and carbon magnetic resonance spectroscopy (NMR) is being
increasingly used in essential oil analysis (Formacek and Kubeczka 1982). For example,
enantiomeric purities can be confirmed by the use of chiral shift reagents. Key components,
terpinen-4-ol and 
α-terpineol were examined using 
13
C and 
1
H NMR DEPT, INEPT and 
13
C-
1
H HETCOR analyses prior to the addition of lanthanide shift reagents (Leach et al. 1993).
Although the resolution of methyl signals of the enantiomers was possible in this way,
application to the analysis of a complete tea tree oil is confounded by the complex nature
of the oil and the abundance of signals in the appropriate regions.
Infrared spectroscopy (IR) although less informative than other techniques, is useful in
indicating the degree of hydroxyl absorption and the presence of the multiple fingerprint
region peaks for cineole in higher cineole oils.
Tree Quality
The tea tree oil industry has moved in the last decade from being an industry based on
wild or bush harvesting to being based on commercial plantations. There is the
requirement then to ensure that the trees involved, whether they be wild stands for
harvest, seed trees for propagation material or trees in established plantations, are of
the right chemical variety and contain acceptable concentrations of cineole and terpinen-
4-ol. For the rapid determination of tree quality, the time consuming leaf distillation
step needs to be bypassed.
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 47
To accelerate the quality control procedure, leaf extraction methods were developed
(Brophy et al. 1989b; Southwell and Stiff 1989) (see above). Hence individual leaves on
any tree can be assessed by GC in less than one hour by inserting them into a vial, adding
ethanol, irradiating with microwaves and injecting the resultant ethanolic solution into the
gas chromatograph. Fresh leaf was found to extract much more rapidly than dried leaf.
This extraction method is an ideal way of assessing the quality of a leaf, a tree or a
number of trees by varying the vial size and solvent volume according to the number of
leaves examined. The method can also be used quantitatively by adding a known quantity
of internal standard to a known weight of leaf before chromatography. The chromatographic
behaviour of the internal standard varies with the GC stationary phase with the n-alkanes
tridecane, tetradecane and pentadecane eluting close to terpinen-4-ol. This method has been
used for determining oil quantity and quality for tea tree breeding programs (
Chapter 7
, this
volume).
Seedling Quality
With the quality of trees cultivated in plantation being critical for the marketing of the
resultant oil, then the quality of the propagation material becomes equally critical. Most
plantation trees have so far been propagated from seed as the higher costs and massive
numbers of trees planted out makes the logistics of propagation by tissue culture or cuttings
unmanageable. The latter methods have been tried successfully on a small scale and do
guarantee more consistent quality. With propagation by seed, an oil quality check on the
mother tree will not necessarily determine the oil quality of the progeny as fertilisation
from a poor quality father may have occurred. Hence unless seed is collected from a region
where cross pollination from poor quality father trees is impossible, there is a chance the
seed could be of poor quality.
In addition, seed has often been bought from merchants unaware of the chemical varieties
available within individual Melaleuca species. This has been seen with attempts to establish
tea tree plantations in at least two overseas countries. In one of these, of the eight clones
described (Kawakami et al. 1990) only one (Clone II) met the requirements of the
International Standard 4730. Clone I, although containing acceptable quantities of terpinen-
4-ol and 1,8-cineole also contained excessive concentrations of sabinene which, along with
the high 
α-thujene/α-pinene ratio (Southwell and Stiff 1990) suggested M. linariifolia.
Clones III to VIII contained insufficient terpinen-4-ol with excessive concentrations of 1,8-
cineole and Clones III and VIII contained excessive quantities of terpinolene as well.
Hence there was a need for the determination of oil quality in seedlings to prevent the
large scale planting of substandard quality trees. Such a method was developed at the
Wollongbar Agricultural Institute (Russell et al. 1997) by using the extraction analysis
method described above.
With careful analysis of the first dicotyledon leaves to emerge from a seedling, it was
possible to predict whether the variety in question was the terpinen-4-ol, cineole or
terpinolene chemotype. Although oil yields were very low at this stage, ethanolic extraction
with microwave irradiation followed by GC analysis, gave a sufficiently intense profile to
type each variety. A misleading aspect of this method was that the commercial terpinen-
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
48
4-ol variety contained substantial percentages of terpinolene (approx. 12%), 
α-pinene
(12%) and ß-pinene (14%) and no terpinen-4-ol. This early stage analysis gave the wrong
idea that the terpinen-4-ol variety was actually the terpinolene chemovar because of high
terpinolene concentrations. The cineole variety contained a similar proportion (approx.
10%) while the terpinolene chemovar had much more (approx. 25%) terpinolene
(
Table 5
).
The key component for measurement is 1,8-cineole. In the commercial terpinen-4-
ol variety, cineole is usually low (0.5–5%). In the terpinolene variety it rises to
approximately 12% whereas in the cineole variety it reaches values as high as 40–70%.
Although the measurement of terpinen-4-ol means nothing in the dicotyledon leaves
(all varieties read zero), values increase as the seedling ages. With, for example, a ten
week old leaf set ten analysis (i.e. ethanolic extract analysis of the tenth leaf pair to
emerge when tested ten weeks after emergence) terpinen-4-ol proportions are
approximately 36% with the commercial variety and only about 1% with the other
chemical varieties. Terpinen-4-ol first appears as cis- and trans-sabinene hydrate (ratio
approximately 7:1 with M. alternifolia and 0.7:1 with M. linariifolia) (Southwell and
Stiff 1989, 1990) in bright green flush seedling growth as it does with the flush growth
on mature plants. These investigations of the ontogenetic variation in oil constituent
percentages (Figure 8) indicate that different biogenetic pathways (e.g. the pinene, the
cineole/ limonene/
α-terpineol, the terpinolene and the α-thujene/sabinene/sabinene
hydrate/ 
γ-terpinene/terpinen-4-ol pathways are initiated at different stages of the plant’s
ontogeny. Clearly the cineole, pinene and terpinolene pathways appear at earlier stages
of development in seedling growth than the sabinene hydrate/terpinen-4-ol pathway.
Consequently producers can reliably assess the quality of their seedlings at both early
and late stages of the seedlings development.
Figure 8 The concentration of key consitituents in M. alternifolia seedling leaves at emergence
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
49
BIOGENESIS
Although little work has been done specifically on Melaleuca oil biogenesis, the
pathways in other genera producing similar monoterpenoids have been investigated.
For example, Croteau (1987) summarised the current thinking concerning the
metabolism from acetyl-CoA through mevalonic acid pyrophosphate and isopentenyl
pyrophosphate to geranyl pyrophosphate. Recent investigations now suggest that
isopentenyl pyrophosphate is formed, not from mevalonic acid but via the alternative
triose phosphate/pyruvate pathway (Lichtenthaler et al. 1997; Eisenreich et al. 1997).
Croteau’s review (1987) continued to examine the cyclisation reactions of geranyl
pyrophosphate (34,35) and linalyl pyrophosphate (36,37) that produce cyclic
monoterpenoids such as limonene, 
α-terpinene, γ-terpinene, sabinene, 1,8-cineole, α-
pinene, terpinen-4-ol, 
α-terpineol etc. Cyclase enzymology is complex in that different
cyclases can produce the same product and an individual cyclase can produce multiple
cyclic products. Definitive investigations involving partially purified enzymes, cell-
free extracts, isotopic labelling and substrate substitution are adding gradually to our
knowledge of these pathways. Some conclusions can also be drawn by studying the co-
occurrence and concentration variation of significant metabolites at various stages of
ontogeny in different chemical varieties.
With the high 1,8-cineole variety of Melaleuca species, the 1,8-cineole concentration
increases concomitantly with limonene and 
α-terpineol suggesting that all three cyclic
products are derived from a linalyl pyrophosphate derived, enzyme bound moiety containing
(31) (Figure 9). Conversely, the terpinen-4-ol variety is richer in congeners 
α-terpinene, γ-
terpinene, terpinolene and terpinen-4-ol, all derived from a moiety such as (32) which is
easily obtained from (31) by 1,2-hydride shift. Evidence for such a hydride shift has been
provided following investigations on the formation of cis and trans sabinene hydrate in
marjoram (Hallahan and Croteau 1989). The product of this shift is now available for further
cyclisation to form the cyclopropane moiety (33) which has been implicated in pathways to
the thujanes in both marjoram (Hallahan and Croteau 1988) and tea tree (Southwell and
Stiff 1989). Tea tree flush growth contains similar monoterpenes to the sabinene hydrate
variety of sweet marjoram, Majorana hortensis (Southwell and Stiff 1989). The difference
is that in marjoram, the sabinene hydrates remain and do not appear as terpinen-4-ol and 
γ-
terpinene in the mature leaf. The fact that steam distillation causes the hydrates to convert to
Figure 9 Chemical structures of likely intermediates in the biogenetic pathways to Melaleuca oil
constituents
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,

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