Medicinal and Aromatic Plants—Industrial Profiles

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strong lifting equipment. In a tightly packed still, particularly if left for some time,
endeavouring to pull the charge out in one lot might result in lifting the still off its base
before the leaf moves from the still. The use of two or more slings overcomes this
problem. A tractor with a lifting device is ideal for emptying the still. If this is not
available, a stayed post with jib and winch is adequate. This allows the charge to be
lifted and swung clear of the still.
The still described so far is suitable for small operations. For large-scale plantation
operations, while the principles of distillation are the same, a major consideration becomes
the economic handling of the biomass including harvesting and distilling and disposal of
the extracted material. In order to extract the oil from the leaf it is necessary to harvest the
biomass, put it into a vessel and pass steam through it. There are several machines that
will harvest effectively, without ill effect to the tree, and will elevate the leaf into a vessel.
Using this vessel as the still eliminates the necessity to transfer the material to another
vessel, and also allows the extracted leaf to be dumped as required, without being
transferred. At this stage of the industry’s development, mobile stills (
Plates 11


appear to be the most economical way of producing tea tree oil. This technique is well
established in the production of essential oils, including eucalyptus, lavender, mint and
Steam is injected into the mobile still at a suitable location where there is a good water
supply and where a boiler, or steam generator, condenser and separator are located. The
essential points, as in the small still, are that the leaf is uniformly and firmly packed in the
still, that a steam-tight lid is clamped on, that the steam is uniformly distributed through the
leaf, and that the condenser-duct and the condenser are of appropriate size. Cohobation is
possible, but calls for the distillate water to be stored and adjusted before being returned to
the boiler.
The tall cylindrical mobile still devised by E.F.K.Denny to distil lavender has the added
merit of evenly packing the plant material by rotating the cylinder as the material is fed into
the still by the harvester. Commonly used mobile stills in the tea tree industry are rectangular
and some care is required to ensure that the leaf is evenly packed into the still by the harvesting
A mobile still suitable for distillation of eucalyptus oils was developed by Mr. L.J.Davis
of G.R.Davis Pty. Ltd. Improvements over 25 years have resulted in an efficient still for
distillation of not only eucalyptus oils but also tea tree, peppermint, fennel and other oils.
The latest “Davis” type still, used widely in Australia, consists of a metal box
approximately 2m wide by 4m long by 1.8m deep mounted permanently on a trailer. The
trailer to which the still is fixed consists of a rigid frame with the main axle about one metre
from the rear and the front steering wheels being a standard tractor front wheel assembly
mounted in front of the still. This machine is suitable for operating where the equipment
needs to be turned sharply such as in natural stand harvesting or in small plantations with
limited headland room. Where long distance hauling at higher speed is required the same
still mounted on a single axle with tandem wheels on a rocking-bar suspension is more
suitable (Plate 17).
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.

The box, of 3mm mild or 1.5mm stainless steel, is supported by 100 by 50mm
rectangular hollow section vertical members with a similar RHS top frame on the front
and sides. The sides slope 80mm from the bottom outwards towards the top. This
facilitates unloading and allows a narrower track for the main wheels so that the still
can be towed on normal tracks. The front slopes outwards 30cm from bottom to top. A
still of these dimensions will carry about 3tonnes of leaf material, and the apparatus
will weigh about 2tonnes. If a heavy forage harvester is used, a minimum 70hp tractor
is required.
A standard towbar at the front is required to connect the still to the tractor or harvester,
and a towing device is also necessary at the back to allow two or more stills to be towed
A completely open top allows loading from the chute of the harvester (
Plate 11
Hinged boards increasing the height of the rear half of the side walls and the back wall
of the still minimise loss in windy conditions. The rear extension board should be of
mesh so that air and dust can move through while leaves are retained. A ladder is built
on the outside of the still, and footholds placed on the inside—these footholds should
not have vertical members as steam will move rapidly up them, reducing distillation
A rear-opening door on offset hinges must be able to swing completely clear to allow
removal of spent leaf material. This door is secured by over-centre locking clamps.
The lid consists of braced 3mm mild steel or 1.5mm stainless steel plate with a 75mm
inverted channel around the edge. The lid is suspended so that, when the full still is towed
into the still-house, the channel fits closely over the top edge of the still. Nitrile-bonded
cork is placed in the channel to ensure a steam-tight seal between lid and still. The lid is
secured by over-centre clamps attached to the lid itself.
From a 15cm diameter outlet near the centre of the lid (
Plate 17
) a flexible vapour duct
leads directly to a multi-tube condenser with circulating water. This duct needs to be flexible
enough to allow the lid vertical movement of at least 20cm. The lid is supported from a
single central point to allow movement in any direction. The lid can be shaped to some
extent for strength and rigidity.
The vapour duct needs to be at least 15cm in diameter to allow rapid distillation.
This is not compatible with a single tube coil-in-tank type condenser when steam from
a boiler is used, as excess pressure will build up in the still during the early phases of
distillation. Equipment with mobile stills and multi-tube condensers is not designed to
work at pressure. The required capacity of the condenser will depend on the rate of
steam application.
In the floor at the rear of the still, near one side, a 5cm socket and cock is placed to act as
a drain. This drain can be kept open so that excess condensed water can flow out. Air is also
expelled, in the early stages of the process, thus reducing pressure build up. Oil is not lost
through this drain.
Steam is introduced from a wood or spent leaf-fired boiler through a 50mm flexible
steam line with a female Kamlock fitting. It is desirable to have about 0.5m of flexible pipe
in the horizontal plane and another 0.5m in the vertical plane for ease of connection.
It is also necessary to ensure that steam is evenly distributed beneath the leaf. For this
purpose steam is injected through a 50mm diameter tube running transversely across the
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.

floor at the front from which a series of smaller tubes about 300mm apart run down the
length of the floor. The smaller tubes have holes of about 6mm diameter in the sides at
about 300mm intervals so that steam is released at points 300mm apart under the whole
charge. Dust and small pieces of leaf tend to clog these pipes, consequently it is necessary
to be able to clean them periodically. This can be done by having caps on the end which can
be removed to allow steam to be blown through, or in the case of 18mm or less diameter
tubes, leaving the ends open.
Stainless steel flexible tubing, most suitable for the vapour duct and steam lines, is
expensive. White nitrile rubber tubes are adequate, particularly for the vapour duct. These
are less expensive but have shorter lifetimes.
This is a modified version of a previously described mobile still (Davis and House
Oil is collected from the condenser in a receiver as in the simple still.
Of the various machines tried in harvesting eucalypts, heavy forage harvesters have proven
the most satisfactory. The type of machine where the flails create sufficient draft to elevate
the harvested material is preferable to a machine which elevates by an auger. As most forage
harvesters are designed to harvest soft herbage, only some will stand operating on woody
material. Standard or modified standard machines are far more economical than specially
designed machines, both in initial cost and running and repair costs.
While flail type harvesters are suitable for tea tree harvesting in dry conditions, in wet
and soft soil there is a risk of beating the tree out of the ground instead of just removing the
above-ground portion. Therefore, young trees and trees in soft soil are best harvested by a
machine with horizontal cutters near ground level. Some standard types of corn harvesters
are effective for this purpose.
Unloading is achieved by attaching two chains, each 11m long, to rear-facing hooks on the
floor of the still near the door. The chains are laid along the floor to the front and carried up
over the front wall, the remaining length (enough to reach the back wall on top of the leaf
charge) being held on brackets mounted on the outside of the front wall. When the still is
full, the chains are laid along the top of the charge.
To empty the charge, the door is opened, the chains secured to a fixed point about 3m
above the ground and the still towed forward. The leaf charge is thus rolled out.
An alternative to pulling the leaf out is to tip it out. This calls for more expensive equipment
Plate 15
) but provides very rapid emptying. It also removes the necessity for a back door,
thus allowing a more rigid still and less potential for steam leaks.
Successful distillation of many essential oils can be achieved with simple equipment. The
technology is also simple, materials cheap, and the quality of oil produced high, provided
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.

the standards of construction and operation of the still are adequate. Mechanisation improves
efficiency and makes the whole operation more cost effective, but at a greater initial financial
outlay. For new oil ventures, the approach will depend largely on the scale of production
Davis, G.R. and House, A.P.N. (1991) Still design and distillation practice. In D.J.Boland, J.J.Brophy
and A.P.N.House (eds.), Eucalyptus Leaf Oils, Inkata Press, Sydney.
Guenther, E. (1965) The production of essential oils. Methods of distillation, enfleurage, maceration,
and extraction with volatile solvents. In Guenther, E. (ed.), The Essential Oils, Van Nostrand Co.
Inc. (Reprint), Princeton, New Jersey, Vol. 1, p. 92.
International Standards Organisation (1996) Oil of Melaleuca, terpinen-4-ol type (Tea Tree Oil).
International Standard ISO 4730:1996(E), International Standards Organisation, Geneva, 8pp.
Standards Australia (1997) Oil of Melaleuca, terpinen-4-ol type (Tea Tree Oil). Australian Standard
AS2782–1997, Standards Australia, Homebush, 12pp.
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.

Centre for Biostructural and Biomolecular Research, University of Western Sydney,
Hawkesbury, Richmond, NSW, Australia
Captain Cook is reported to have used the leaves of the tea tree to brew a spicy tea and Sir
Joseph Banks, the botanist with Cook’s expedition, included samples of the plant in his
collection. Exactly which plants were used is not known as the name ‘tea tree’ has been
used for a number of similar plants in the genera Melaleuca and Leptospermum. Whilst
essential oils from several species possess antimicrobial activity, the oil which is widely
used today for its antiseptic properties is the oil of Melaleuca alternifolia.
The natural habitat of Melaleuca alternifolia is the Bungawalbyn region of north-eastern
New South Wales, Australia and Drury (1991) reports that the Bundjalung Aborigines, who
lived in this area, treated skin infections by crushing leaves of the tea tree over the injury
and covering it with a warm mudpack. It is possible that early settlers also used this remedy.
Scientific studies on tea tree oil began in the 1920s at the Museum of Technology and
Applied Sciences in Sydney. Penfold and Grant (1925) reported that the oil was a non-toxic
and non-irritating antiseptic, which was more effective than phenol, the standard for
comparison. Following this demonstration of the effectiveness of tea tree oil in laboratory
tests, evidence of its therapeutic use began to accumulate. Humphrey (1930) tested a water-
miscible preparation of tea tree oil and reported its value at concentrations of 2.5–10% for
the treatment of a variety of conditions, such as wounds, peronychia (infection of the nails),
coryza and sore throats, and suggested it would have potential applications in the treatment
of vaginal infections and burns. In addition to its efficacy as an antiseptic, several other
features of the oil were cited. These included the ability of the oil to dissolve pus in infected
wounds, to cleanse dirty wounds and to resolve chronic infections, particularly of the nail,
without any apparent damage to healthy tissue.
During the 1930’s the reputation of tea tree oil as ‘a medicine chest in a bottle’ continued
to develop. The major producers of the oil, Australian Essential Oils Ltd, published a report
in 1936 on the medical and dental applications of Ti-trol, the name used for the neat oil and
of Melasol, a water-miscible preparation (cited in Lassak and McCarthy 1983). The list of
applications in external conditions continued to grow, and all reports stressed the non-toxic
and non-irritating properties of the oil. The ability of the pure oil to penetrate unbroken skin
was also reported (Drury 1991).
A reputation for tea tree oil as a safe, effective antiseptic had been established by the
time of the outbreak of the Second World War, and its record impressive enough that cutters
and producers were exempted from military service. In addition, the oil was incorporated in
army and navy first-aid kits for use in tropical regions (Drury 1991) and added to machine
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.

cutting oils to reduce infection following injury to the hands by metal filings and turnings
(Lassak and McCarthy 1983).
A number of factors led to a decline in the industry following the Second World War. As
well as competition in the market-place from newly-developed synthetic agents, the product
was inadequately promoted and the supply was inconsistent, both in quality and quantity.
The natural stands of trees, located in swampy areas, were difficult to harvest and different
chemotypes, with oils of variable composition, were difficult to distinguish in a simple
During the 1970’s the interest in ‘natural’ products was renewed, and over the last twenty
years the production of oil has continued to increase and so have the applications. Tea tree
oil is incorporated as the active antimicrobial or as a natural preservative in a wide range of
pharmaceutical, cosmetic and ‘cosmeceutical’ products including antiseptic creams, face
washes, pimple gels, vaginal creams, veterinary skin care products, tinea preparations, foot
powders, shampoos and conditioners. It is claimed to provide rapid soothing relief when
added to burns blankets and creams. Alongside this, there has been a recognition by the
industry of the need to support the early anecdotal claims about the efficacy of the oil with
scientific data which will enable registration of the oil as a pharmaceutical product. This
chapter will review the published studies which have examined in vitro and in vivo activity
of the oil.
The major therapeutic claims for tea tree oil involve its antimicrobial activity and its
effectiveness against a wide range of bacteria and fungi has been demonstrated. The methods
used by various authors vary both in principle and detail and it is relevant here to review the
methods and evaluate their usefulness in the testing of essential oils.
Test Methods
Methods commonly used for determining antimicrobial activity of a substance fall into
two categories: those which measure whether the agent is microbiostatic (inhibits growth)
and those which determine whether it is microbiocidal (kills the target organism).
Inhibition of test organisms can be determined by diffusion tests or by measurement of
the Minimum Inhibitory Concentration (MIC) in broth or agar dilution tests, while
suspension tests, such as the Rideal-Walker test and the TGA disinfectant tests, measure
a lethal effect.
Diffusion Tests (Disc or Well)
Diffusion tests involve placing a sample of the test compound on a disc or in a well in the
centre of a plate containing a nutrient medium which has been seeded with the test organism.
Water soluble compounds diffuse into the seeded medium producing a continuous
concentration gradient of the agent. Following incubation, a clear zone, called the zone of
inhibition, develops around the disc or well if the organism is sensitive to the agent. The
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.

size of the zone provides some indication of the relative activity of the substance, however,
a number of factors including the volume and type of medium, the concentration and age of
inoculum, the incubation conditions and the size, charge and conformation of the active
ingredient(s) will all affect the result (Lorian 1986). This technique has been standardised
for antibiotic testing to give reproducible results, which correlate with MIC values, but
because of the different diffusion properties of various substances, it is only useful for
comparing sensitivity of different organisms to a single antibiotic and not appropriate for
the direct comparison of a range of antibiotics.
The method has been widely used to screen antimicrobial activity of essential oils,
including tea tree oil (Kar and Jain 1971; Yousef and Tawil 1980; Janssen et al. 1985;
Deans and Svoboda 1988; Biondi et al. 1993; Williams et al. 1993; Gundidza et al. 1994;
Bagci and Digrak 1996). However, there are a number of disadvantages when the method
is applied to the testing of water-insoluble essential oils and inconsistent results and a
lack of correlation with MIC values have been reported (Morris et al. 1979; Janssen et al.
1986; Carson and Riley 1995). The lack of water solubility of the components of tea tree
oil limit their diffusion through the agar. Only the more water soluble components, such
as terpinen-4-ol, 1,8-cineole and a-terpineol, diffuse into the agar from the disc;
hydrocarbon components remain on the disc or evaporate (Southwell et al. 1993).
Consequently, the contribution of these components to the activity of the oil cannot be
assessed and it is possible that the activity of the oil will be underestimated. Whilst the
method is easy to perform and requires only small volumes of the agent, its use as a
screening tool continues, but it is not appropriate where more quantitative measurements
are required, or where the antimicrobial activity of oils of different composition are being
Determination of Minimum Inhibitory Concentration
MIC measurements have been used extensively to quantify the antimicrobial activity of
essential oils. This type of test is more useful than the diffusion test for comparing both the
activity of oils of different chemical composition and the sensitivity of a variety of organisms,
and a number of variations of the method have been published (Beylier 1979; Villar et al.
1986; Kubo et al. 1991; Patkar et al. 1993; Southwell et al. 1993; Chand et al. 1994; Dellar
et al. 1994; Nguyen et al. 1994; Carson et al. 1995b; Griffin et al. 1998). These include
broth (macro and micro) and agar methods, with or without the use of an indicator of cell
viability to determine the endpoint.
The principle of the assay is that the test organism is added to a series of dilutions
of the agent prepared in a nutrient medium, and presence or absence of growth is
determined after a period of incubation. The Minimum Inhibitory Concentration is
recorded as the lowest concentration of the agent which inhibits growth. This definition
lacks precision as it does not clearly define the preparation and concentration of the
inoculum, the test medium, the contact time or the method of determining the end-
point. Due to different interpretations of these variables, it is difficult to directly
compare the results of various authors, and there is a strong need for the tea tree oil
industry to standardise procedures.
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.

Broth Dilution Methods
Broth methods, especially those carried out in microtitre trays, have the advantages of
lower workloads for a larger number of replicates and the use of small volumes of the test
substance and growth medium. In broth methods, turbidity of the oil-water emulsion can
interfere with the reading of the end-point, particularly in microtitre assays. For this reason,
indicators such as fluorescein diacetate (Chand et al. 1994), p-iodonitrotetrazolium violet
(Dellar et al. 1994) and triphenyl tetrazolium chloride (Carson et al. 1995b) have been
used. However, autofluorescence is a problem with FDA (unpublished observations) and
Carson and Riley (1994) report that colour changes of TTC do not correlate exactly with
MIC. An alternative method using the redox dye, resazurin, has been developed (Mann
and Markham 1998). The reported advantages of this method are the ease of end-point
determination associated with a colour change from blue to pink and the reliability of
prediction of MIC.
Broth methods can also be adapted to test Minimum Bactericidal or Fungicidal
Concentrations by transferring aliquots from tubes or wells which show no growth after the
incubation period, to fresh medium. Organisms are considered to be non-viable if there is
no growth after this second period of incubation. A microtitre method modified for the
testing of anaerobic organisms has also been reported (Shapiro et al. 1994).
Agar Dilution Methods
Agar dilution methods, in which various concentrations of the test substance are added to
the agar medium prior to inoculation with the test organisms, overcome the turbidity problem
associated with broth methods. Studies in our laboratory have shown excellent replication
using Isosensitest agar and concentrations of Tween 20 of 0.25–0.5% (Griffin et al. 1998).
This method has the advantage that with modifications to the growth medium and incubation
conditions it can be adapted to suit any organism, but it is not readily adapted to the inclusion
of MBC measurements. Some authors have reported that MIC values from agar dilution
tests tend to be higher than those obtained by broth dilution tests (Atkinson and Brice 1955;
Walsh and Longstaff 1987).
Measurement of Microbiocidal Activity
Although the methods above can determine Minimum Lethal Concentrations of agents
they involve prolonged contact (generally 24–48 hours) between the agent and the test
organism and give no evidence of the rate of kill. Because antiseptics and disinfectants
are generally required to reduce the microbial load rapidly, kill rate or suspension tests
are more relevant to their intended use. In principle, such tests involve the addition of an
appropriate volume of inoculum to a clinically relevant concentration of the agent followed
by the testing of the viability of the culture after set periods of incubation. This basic
procedure can be adapted by changing variables such as inoculum density, concentration
of the test agent, contact time and presence of potential inactivators, to satisfy the testing
requirements. In a study of the death kinetics of Staphylococcus aureus, Candida albicans
and Aspergillus niger treated with samples of thyme oil, it was reported that variations
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