BACKGROUND ART Totarol (totara-8, 11, 13-trien-13-ol ; 4b-S-trans-8, 8-trimethyl-4b, 5,6, 7,8, 8a, 9, 10-octahydro-l- isopropylphenanthren-2-ol), is an aromatic diterpenoid natural product with the molecular formula C2oH3o0. Totarol is present in relatively high concentrations-approximately 5 % by mass on a dry basis-in the heartwood of Totara (Podocarpus Totara), which is indigenous to New Zealand; and in much lower concentrations-less than 1 % by mass-in other Podocarpus, Dacrycarpus, Cupressus and Junipers species.

Totarol Totarol is remarkably resistant to oxidation and degradation and so the level in dead heartwood remains relatively constant despite the age of the timber. Totarol can be produced synthetically, by a number of routes, but the economics and suitability of these methods have not been proven to be feasible for production on a large scale, and often both stereoisomers are produced. Therefore, production of totarol for pharmaceutical, nutraceutical, cosmetic, cosmeceutical and other applications is commercially possible only by extraction of the naturally occurring compound from suitable plant raw material.

Totarol is known to be soluble in the common organic solvents hexane, acetone, ethanol and methanol. However, these solvents are not selective, and removal of the solvent residues from the extracted material is generally incomplete, which makes their use particularly

unattractive when the resulting totarol extracts are intended for use in pharmaceutical, nutraceutical, cosmetic and cosmeceutical applications.

Totarol is a potent antimicrobial agent against Gram-positive bacteria (1). JP 01-311019 describes the topical use of totarol as an anti-bacterial agent against Gram-positive bacteria.

However, totarol has not previously been reported to have any activity against Gram- negative bacteria.

Tea-tree oil is an essential oil steam-distilled from the plant Melaleuca alternifolia, which is indigenous to Australia. Tea-tree oil is a complex mixture of approximately 100 terpenes and hydrocarbons, and is one of the few known all natural extracts that has antimicrobial activity against Gram-negative bacteria.

The antimicrobial activity of plant oils and extracts may be measured as the minimum inhibitory concentration (MIC), which is the lowest concentration which results in the maintenance or reduction of inoculum viability. This is the most commonly reported method in the antimicrobial literature and may permit comparison between studies. Two methods of MIC determination are usually employed-the agar dilution method, in which various concentrations of the test substance are added to an agar medium prior to inoculation with the test organism; and the broth dilution method, in which the test organism is added to serial dilutions of the antimicrobial agent prepared in a nutrient medium.

Table 1 sets out a comparison of the antimicrobial activity of tea-tree oil and totarol produced by solvent extraction against various bacteria. However, this comparison is limited due to the insufficient amount of published MIC data for totarol against various organisms. In addition, the few investigations that have been published may not be directly comparable because of the assay methods that were used i. e. broth dilution versus agar dilution. The composition of the active components may also differ in proportion from sample to sample thus influencing the reproducibility of the results.

The limited amount of data available demonstrates that totarol produced by organic solvent extraction has greater antimicrobial activity than tea-tree oil against two micro-organisms- the Gram-positive bacteria, Enterococcus faecalis and Propionibacterium acnes. However, totarol has significantly lower antimicrobial activity than tea-tree oil against the Gram- negative bacterium, Klebsiella pneumonia.

Table 1: MIC values for tea-tree oil and solvent-extracted totarol against various bacteria Totarol Totarol Bacteria Tea-tree oil MICagar MICagar MICbroth (g/mL) (g/mL) Gram-positive bacteria Bacillus subtilis 0. 3-0. 4 % (v/v) (2,3) 1.56 (1) Brevibacterium ammoniagenes 0.78 (1) Enterococcus faecalis 0.5-0. 75 % (v/v) (2,3) 2 (4) Propionibacterium acnes 0.4-0. 5 % (v/v) (2,3) 0.39 (1) Staphylococcus aureus 0.2 % (v/v) (2,3) 2 (4) 1. 56 (1) Staphylococcus aureus ATCC 11632 0.78 (5) Staphylococcus aureus ATCC 12598 1.56 (1,5) Staphylococcus aureus ATCC 29247 0.78 (1,5) Staphylococcus aureus ATCC 25923 1.56 (5) Staphylococcus aureus ATCC 33591 MRSA 0.78 (5) Staphylococcus aureus ATCC 33592 MRSA 0.78 (5) Streptococcus mutans 0.04 pL/mL (6) 0.78 (1) Streptococcuspneumoniae 0.25 % (v/v) (3) 2 (4) Gram-negative bacteria Klebsiella pneuynonia 0.3 % (v/v) (2,3) > 32 (4)

There remains a need for a reliable and cost effective method for producing a totarol- containing extract from suitable plant material which is sufficiently selective for totarol, and which avoids the use of solvents that may result in harmful or undesired solvent residues; and for a totarol-containing extract that has antimicrobial activity against both Gram- positive and Gram-negative bacteria.

Accordingly, it is an object of the present invention to provide a process for producing a totarol-containing extract from suitable plant material; and/or to provide a totarol-containing extract ; which goes at least some way towards meeting the foregoing requirements or which will at least provide the public with a useful choice.

Other objects of the invention may become apparent from the following description which is given by way of example only.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date.

SUMMARY OF THE INVENTION In a first aspect, the present invention provides a process for producing a totarol-containing extract from suitable plant material, which process comprises the steps of : (a) contacting the plant material with a near-critical fluid to produce a near-critical fluid phase in which the totarol is dissolved; (b) separating the near-critical fluid phase from the plant material; and (c) separating the totarol-containing extract from the near-critical fluid.

In a further aspect, the present invention provides a process for producing a totarol- containing extract, which extract comprises totarol and an oil, from suitable plant material and suitable oil-containing material, which process comprises the steps of : (a) contacting the plant material and oil-containing material with a near-critical fluid to produce a near-critical fluid phase in which the totarol and oil is dissolved; (b) separating the near-critical fluid phase from the plant material; and (c) separating the totarol-containing extract from the near-critical fluid.

In a still further aspect, the present invention provides a totarol-containing extract when produced by a process of the invention.

In a yet further aspect, the present invention provides a totarol-containing extract, wherein the extract comprises one or more other compounds selected from the group consisting of : totarol hemiacetal; 7a-hydroxytotarol; 7-oxototarol ; 5-hydroxytotarol; 6,7-dehydrototarol ; and ferruginol.

In another aspect, the present invention provides an extract obtained from the heartwood of Podocarpus total-a G. Benn (Totara) or Podocarpus Hallii (Halls Totara), wherein the extract has activity against Gram-negative bacteria.

In another aspect, the present invention provides a composition comprising an extract of the invention.

In another aspect, the present invention provides a product, comprising an extract of the invention as an active ingredient, antimicrobial additive, or preservative.

In another aspect, the present invention provides a therapeutic formulation having activity against Gram-positive and Gram-negative bacteria, wherein the formulation comprises an extract of the invention.

In another aspect, the present invention provides a use of an extract of the invention in the preparation of a therapeutic formulation for treating a Gram-negative bacterial infection in an individual in need thereof.

In another aspect, the present invention provides a method for at least inhibiting the growth of bacteria sensitive to an extract of the invention, the method comprising contacting the sensitive bacteria with an inhibitory effective amount of the extract, or a composition comprising the extract.

In another aspect, the present invention provides a method of prophylactic or therapeutic treatment of bacterial infection in an individual in need thereof, the method comprising administering to said individual an extract of the invention, or a composition comprising the extract, in an amount effective to at least inhibit growth of the bacteria.

This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

Throughout this specification the word"comprise", or variations such as"comprises"or "comprising", will be understood to imply the inclusion of a stated element, integer or step,

or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Although the present invention is broadly as defined above, those persons skilled in the art will appreciate that the invention is not limited thereto and that the invention also includes embodiments of which the following description gives examples.

DESCRIPTION OF THE DRAWINGS The invention will now be described with reference to the Figure in which: Figure 1 is a schematic diagram of an extraction apparatus which is suitable for use in performing a process of the invention.

DETAILED DESCRIPTION OF THE INVENTION Surprisingly, the applicants have determined that an extract comprising totarol can be produced from suitable plant material by near-critical extraction, and that a carrier liquid infused with the totarol-containing extract may be produced directly by near-critical extraction. Furthermore, the applicants have determined that the totarol-containing extract has activity against both Gram-positive and Gram-negative bacteria.

Accordingly, in a first aspect, the present invention provides a process for producing a totarol-containing extract from suitable plant material, which process comprises the steps of : (a) contacting the plant material with a near-critical fluid to produce a near-critical fluid phase in which the totarol is dissolved; (b) separating the near-critical fluid phase from the plant material; and (c) separating the totarol-containing extract from the near-critical fluid.

The term"contact"as used herein generally means admixing the plant material with the near-critical fluid in a suitable extraction vessel using apparatus as are well known in the art.

Advantageously, the apparatus may be equipped with multiple extraction vessels, each of which may be removed from the flow of near-critical fluid without removing the others, thereby permitting emptying and refilling of the extraction vessels without depressurising the apparatus, enabling semi-continuous operation.

The term"separate"as used herein in relation to the near-critical fluid phase and the plant material generally means removing the stream comprising the near-critical fluid phase and the dissolved totarol from the apparatus, while excluding the plant material from the stream.

The term"near-critical fluid"as used herein means a fluid that is close to its critical point and thus includes both subcritical and supercritical fluids. Near-critical includes the reduced temperature range 0.75 < Tr < 1.25 (where Tr is the temperature divided by the critical temperature, Te of the fluid); and the pressure ranges P > Pv (where Pv is the vapour pressure) for T < Te and P > PC (where P, is the critical pressure) for T > T.

Near-critical fluids are selective solvents that have found application in various extraction processes. A near-critical fluid has a density comparable to that of a liquid while exhibiting the diffusion properties of a gas. In the near-critical region, the density of the fluid is highly dependent on temperature and pressure. The solvent power of the fluid is directly related to the density at fixed temperature, and temperature at fixed density. In the near-critical region, the density of the fluid is high and the fluid can act as a solvent. When the pressure is lowered and/or the temperature increased, the density and, therefore, the solvent power of the fluid is reduced, and the material dissolved in the fluid can be separated from it.

Preferred near-critical fluids include, but are not limited to: COz ; C2-C4 hydrocarbons, including ethane, ethylene, propane, propylene, and butane; partially and fully fluorinated C1-C3 hydrocarbons, particularly R134a (1,1, 1,2-tetrafluoroethane) ; iodotrifluoromethane; nitrous oxide; sulfur hexafluoride; dimethylether; and mixtures of any two or more of the above.

COs is widely used as a supercritical fluid as its critical temperature and pressure (31. 2°C, 73.2 bar) are attained relatively easily. Furthermore, COs is inert, non-toxic, cheap and readily available.

Preferably, the near-critical fluid used is C02. In one embodiment, liquid C02 is used (T < 31. 2 °C, pressure greater than the vapour pressure of C02 at T). In a further embodiment, the plant material is contacted with supercritical C02 at a desired pressure ( 75 bar) and desired temperature, T (> 31. 2 °C).

Near-critical C02 can be used for extractions at temperatures that do not result in thermal degradation of sensitive components. Advantageously, the use of near-critical COx as a

solvent for the extraction of totarol gives a high yield of totarol-containing extract, and leaves no solvent residues in the extract or spent wood, as compared with conventional extraction methods using organic solvents, in which considerable effort is required to reduce solvent residues to levels required to satisfy regulatory requirements.

Preferably, the plant material is contacted with the near-critical fluid at a temperature in the range 273-373 K and at a pressure which is sufficient to ensure adequate recovery of the totarol-containing extract from the plant material.

In a particularly preferred embodiment, wherein the near-critical fluid is C02, the plant material is contacted with the near-critical fluid at a temperature in the range 273-373 K and at a pressure in the range 50-500 bar. More preferably, the temperature is in the range 273- 353 K and the pressure is in the range 50-300 bar. Still more preferably, the temperature is in the range 287-333 K and the pressure is in the range 70-300 bar.

The totarol-containing extract produced in a process of the present invention may also contain other bio-active and non-active totarol derivatives and associated compounds present in the plant material. Other compounds which have been found to be present in the totarol-containing extract include: podototarin ; totarol hemiacetal; 7a-hydroxytotarol; 7-oxototarol; 5-hydroxytotarol; 6,7-dehydrototarol ; and ferruginol. The extract may also contain other compounds including, but not limited to other known totarol derivatives such as: 19-hydroxytotarol; sugiol; methyl podocarpate; podocarpic acid; p-sitosterol ; and pododacric acid (7).

A degree of fractionation of the components present in the totarol-containing extract may be achieved by appropriate selection of the pressure and temperature at which the plant material is contacted with the near-critical fluid. Accordingly, in one embodiment, the plant material is contacted with the near-critical fluid at a first pressure to produce an extract having a higher proportion of totarol, followed by contacting at a second, higher, pressure to produce an extract with a relatively higher proportion of non-totarol components.

Suitable plant material includes the wood, bark, roots and leaves of those species known to contain totarol (7). These species include various Podocarpus, Dacrycarples, Cupressus and Junipers species.

Preferably, the plant material is obtained from Podocarpus totara G. Benn (Totara) or Podocarpus Hallii (Halls Totara), both of which are indigenous to New Zealand. More preferably, the plant material is heartwood obtained from these species.

Advantageously, because of its resistance to oxidation and degradation, totarol may be extracted from dead Totara wood including stumps, used fence posts and building timbers, and fallen tree logs recovered from the forest or swamps.

In order to improve the efficiency of the extraction, the plant material may be ground to a selected particle size, thereby increasing the surface area to volume ratio, prior to being contacted with a near-critical fluid in a process of the invention. Preferably, the particle size is between about 0.01 and 5 mm. More preferably, the particle size is between 0.01 and 3 mm. Still more preferably, the particle size is between 0.05 and 1 mm.

Water present in the plant material may be co-extracted into the near-critical fluid phase in which the totarol is dissolved. Accordingly, the totarol-containing extract produced after separation from the near-critical fluid may contain varying amounts of water. If required, this water may be removed by variety of means as would be appreciated by a person skilled in the art.

In one embodiment, the totarol-containing extract is freeze-dried. The freeze-dried extract may also be milled to a powder.

Preferably, the plant material is partially to completely dried prior to being contacted with a near-critical fluid in a process of the invention. Partially dry plant material will typically have a moisture content of < 15 % on a weight basis, while completely dry material will have a moisture content of < 2 % on a weight basis. Advantageously, the use of completely dry plant material may render subsequent drying of the totarol-containing extract unnecessary.

The plant material may be ground to a selected particle size before drying. Advantageously, the increased surface to volume ratio after grinding will increase the rate at which the plant material dries. Alternatively, the plant material may be dried before being ground to a selected particle size. As a further alternative, the plant material may be subject to a preliminary grinding to a first selected particle size, followed by drying and a final grinding to a second selected particle size.

In one embodiment, the totarol-containing extract is separated from the near-critical fluid by transferring the near-critical fluid phase in which the totarol is dissolved into a suitable separation vessel wherein the pressure is reduced to the point where the totarol-containing extract is precipitated into the vessel.

The pressure reduction can be carried out in a single step. Alternatively, the pressure reduction may be carried out in multiple steps to give more than one fraction of the totarol- containing extract.

Generally, as the pressure is reduced, those components of the totarol-containing extract that are least soluble in the near-critical fluid will precipitate first. The most soluble components will precipitate last. Therefore, a degree of separation or fractionation of the components of the totarol-containing extract may be achieved, the fractionation being related to both the molecular mass and polarity of the individual components.

Accordingly, in a preferred embodiment, the totarol-containing extract is separated from the near-critical fluid in several sequential pressure reductions and/or temperature changes, thereby fractionating the extract into a plurality of separation vessels.

Preferably, the first pressure reduction is in a separation vessel held at a first selected pressure which is less than the pressure at which the plant material is contacted with a near- critical fluid, but greater than the pressure at which any subsequent separation vessel is held.

More preferably, where the near-critical fluid is CO2, the first selected pressure is in the range 55-120 bar. Still more preferably, the first selected pressure is in the range 55-100 bar.

Preferably, the second pressure reduction is in a separation vessel held at a second selected pressure which is less than the first selected pressure. More preferably, the second selected pressure is in the range 1-70 bar. Still more preferably, the second selected pressure is in the range 40-60 bar.

In one embodiment, the separation vessel is equipped with a basket in which the totarol- containing extract is collected. Such baskets are well-known to those skilled in the art.

Advantageously, the use of a collection basket permits convenient emptying of the separation vessel. Furthermore, the collection basket may be readily removed from the depressurised separation vessel while the pressure of the extraction vessel is maintained,

thereby permitting semi-continuous operation of the apparatus. Alternatively, one or more auxiliary separation vessels may be used to recover the totarol-containing extract, again permitting semi-continuous operation of the apparatus.

The totarol-containing extract may be subjected to a re-extraction at different near-critical conditions to provide a second totarol-containing extract in which the proportions of totarol and other components differ from those in the original extract. Typically, totarol-containing extract produced from the extraction of plant material at relatively high pressures may be re- extracted at lower pressures to yield an extract in which the proportion of totarol has increased.

In one alternative embodiment, a carrier liquid is used to separate the totarol-containing extract and the near-critical fluid.

Typically, the carrier liquid is injected into the flow of the near-critical fluid phase in which the totarol is dissolved upstream of the separation vessel/s. Advantageously, the carrier liquid dissolves the solid totarol-containing extract after the pressure is reduced to give an infusion of totarol in the carrier liquid, and prevents the solid totarol-containing extract from, for example precipitating in the lines and valves and causing blockages. The use of a carrier liquid also enables the continuous removal of the infusion from the separation vessel/s without depressurisation.

In one embodiment, the infusion may be recycled by removal from the separation vessel/s and re-injection into the flow of the near-critical fluid phase. Generally, the infusion may be recycled until the carrier is saturated with the totarol-containing extract, thereby providing a more concentrated infusion.

Suitable carrier liquids include oils but are not limited thereto. It will be appreciated that the carrier liquid must have a sufficiently low melting point such that it does not solidify in the apparatus.

In a preferred embodiment, the carrier liquid is an oil with a melting point less than about 10°C.

Advantageously, the oil may be pharmaceutically, nutraceutically, cosmetically or cosmeceutically acceptable, such that the infusion of the totarol-containing extract in the oil

is suitable for formulation with, and into, pharmaceuticals, nutraceuticals, cosmetics or cosmeceuticals.

Preferred oils include vegetable oils, seed oils and nut oils. More preferably, the oil is selected from: soya oil; linseed oil; evening primrose oil; rosehip oil; borage oil; blackcurrant oil; kiwifruit seed oil; avocado oil; olive oil; and mixtures thereof.

Therefore, in an alternative embodiment, the present invention provides a process for producing a totarol-containing extract, which extract comprises totarol and an oil, from suitable plant material and suitable oil-containing material, which process comprises the steps of : (a) contacting the plant material and oil-containing material with a near-critical fluid to produce a near-critical fluid phase in which the totarol and oil is dissolved; (b) separating the near-critical fluid phase from the plant material; and (c) separating the totarol-containing extract from the near-critical fluid.

Suitable oil-containing materials include seeds and other matter from which oil can be extracted.

It will be appreciated that the solid totarol-containing extract of the invention may also be mixed with a suitable oil to provide an infusion comprising totarol.

In a further aspect, the present invention provides a totarol-containing extract when produced by a process of the invention.

Using a process of the invention, a totarol-containing extract which is free of solvent residues may be produced. Such totarol-containing extracts are, therefore, suitable for use in the formulation of pharmaceuticals, nutraceuticals, cosmetics, cosmeceuticals and other products.

Accordingly, in another aspect the present invention provides a composition comprising a totarol-containing extract produced by a process of the invention.

The totarol-containing extracts of the invention have antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, the totarol-containing extract of the present invention is contemplated for use as an active ingredient, antimicrobial additive or preservative in a variety of products including, but not limited to: household and industrial

cleansers and disinfectants; cosmetic formulations; cosmeceuticals; pharmaceuticals; nutraceuticals; skin and body care products, such as soap, body wash, moisturiser and facial scrub; and dental care products, such as toothpaste, mouth spray and mouthwash.

In another aspect, the invention provides a method for at least inhibiting the growth of bacteria sensitive to the totarol-containing extract of the invention, the method comprising contacting the sensitive bacteria with an inhibitory effective amount of the totarol-containing extract of the invention, or a composition comprising the totarol-containing extract of the invention.

In another aspect, the invention provides a method of prophylactic or therapeutic treatment of bacterial infection in an individual in need thereof, the method comprising administering to said individual the totarol-containing extract of the invention, or composition comprising the totarol-containing extract of the invention, in an amount effective to at least inhibit growth of the bacteria.

The invention also provides a use of a totarol-containing extract of the invention in the preparation of a therapeutic formulation for treating a Gram-negative bacterial infection in an individual in need thereof.

The term"individual"as used herein includes humans, horses, dogs, cats, pigs, sheep, cattle, goats but is not limited thereto. Preferably, the individual is a human.

In another aspect, the present invention provides a therapeutic formulation having activity against Gram-positive and Gram-negative bacteria, which formulation comprises a totarol- containing extract produced by a process of the invention.

Advantageously, the totarol-containing extract of the invention comprises active non- volatile solids and, as such, is particularly suitable for use in topical formulations where it will remain on the skin, and disinfectant applications, in comparison to those using other known natural anti-microbials such as tea-tree oil which may evaporate, thereby reducing their efficacy.

Examples of therapeutic formulations in which the totarol-containing extract of the invention can be employed include orally administrable medicaments such as: capsules;

tablets; lozenges; syrups; mouthwashes; gargles; toothpastes; chewing gums ; chewable tablets; and mouth sprays, but are not limited thereto.

Contemplated topical formulations include: lotions; creams; gels; sticks; sprays; shaving creams; ointments; cleansing liquid washes and solid bars; shampoos; pastes; powders; mousses; wipes; patches; nail lacquers; wound dressings; adhesive bandages; hydrogels; films ; and make-up such as concealers, foundations, mascaras, and lipsticks.

A"therapeutic formulation"is a formulation appropriate for administration of the totarol- containing extract of the invention, to an individual in need of same. In general, therapeutic formulations of the invention comprise the totarol-containing extract of the invention and an acceptable carrier, diluent and/or excipient.

An"acceptable carrier, diluent and/or excipient"means a vehicle for delivery of the totarol- containing extract of the invention, to the individual, in which the vehicle is compatible with the extract. Acceptable carriers, diluents and excipients suitable for use in the administration of the totarol-containing extract of the invention are well known to those skilled in the art. Suitable carriers are generally inert and can be either solid or liquid.

In one embodiment, the carrier is a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers suitable for topical administration include, but are not limited to: solutions; emulsions such as microemulsions and nanoemulsions; gels; solids; and liposomes. Such carriers can be readily formulated by those skilled in the art. A variety of pharmaceutically acceptable carriers suitable for oral administration are also well known in the art (8).

The compositions may also include excipients such as tableting aids; resins; fillers; binders; lubricants; solvents; glidants; disintegrants; preservatives; buffers; flavourings ; colourings; sweeteners; and fragrances as appropriate.

The formulations and compositions of the invention may further comprise one or more secondary antimicrobial agents compatible with the totarol-containing extract of the invention.

The proportion of the totarol-containing extract in the formulations and compositions will vary depending on the contemplated end use. The formulations and compositions of the

invention typically may comprise from about 0.005 to 20% by weight of the totarol- containing extract. In those embodiments where the formulation or composition is intended for administration, in comparison to those intended for use as household and industrial cleansers and disinfectants, the composition preferably comprises between about 0.005 and 1% totarol-containing extract.

The following non-limiting examples are provided to illustrate the present invention and in no way limit the scope thereof.

EXAMPLES NEAR-CRITICAL EXTRACTION Apparatus The near-critical extractions were carried out in apparatus schematically depicted in Figure 1. C02 (or other near-critical fluid) was supplied to the apparatus by liquid supply cylinders CYL1 and CYL2. The apparatus included a high pressure pump MP3 for circulating the near-critical fluid, and two extraction vessels EX1 and EX2, each of which had a volume of 10 litres at pilot scale and 150 litres at demonstration scale. The apparatus further included two separation vessels SV1 and SV2 which enabled the recovery of the totarol-containing extract in two fractions. Pumps MP1 and MP2 were used to refill the extraction vessels. The apparatus also included a C02 recovery vessel RV1 and associated compressor RC1, together with pressure control valve CV1, back pressure regulator BPR, flow meter FM1, and various valves (V), indicators (P or T) and heat exchangers (HX).

When oil was used as a carrier liquid to remove the totarol-containing extract from the near-critical CO2, the apparatus further included an oil reservoir OT1 and an oil pump OP1.

General procedure Totara wood was ground in a knife mill to a desired particle size-typically between about 0.01 and 5 mm-and then added to a preweighed extraction basket having porous plates at either end to enable the passage of the near-critical fluid but not solvent. The basket was weighed to determine the mass of Totara wood before extraction. The basket was placed in extraction vessel EX1, which was then sealed and filled with near-critical fluid up to the cylinder pressure via valve VDF1. EX1 was then raised to the operating pressure-typically

300 bar for C02-initially using pump MP2 up to 70 bar and then using pump MP1 or pump MP3. When the desired pressure had been reached, flow was started through the vessel (s) and near-critical fluid was continuously circulated around the apparatus in a loop.

In the extraction procedure, either or both extraction vessels EX1 and EX2 were used and a separate extraction basket was used in each vessel. The near-critical fluid then followed the flow path VDFI-VOl-EX1-VD2-EX2-VI2-VDF2-CV1 if both vessels were used; and VDF1-VO1-EX1-VI1-VDF2-CV1 if only EX1 was used.

The pressure was reduced to a selected pressure-typically between 120 and 55 bar for C02 through valve CV1 to effect separation of the bulk of the totarol-containing extract from the near-critical fluid. The main totarol-containing extract fraction was precipitated into separation vessel SV1. The near-critical fluid and remaining totarol-containing extract then passed through pressure control valve BPR1 and heat exchanger HX3 where the pressure was reduced to the cylinder pressure-typically between 40 and 60 bar for C02- and the temperature was raised-to approximately 313 K for C02. Water, a small amount of totarol-containing extract, and essential oil was precipitated into vessel SV2. The near- critical fluid, now a gas, was then recycled back to the main pump (MP1 or MP3) via heat exchanger HX4, water-trap WT1 and condenser-subcooler HX5 or HX1, respectively.

When liquid C02-at 70 bar and 287 K-was used as the near-critical fluid, the first separation vessel SV1 was not used. At the end of an extraction, the extraction vessels were vented firstly through the separation vessels and then through recovery vessel RV1.

Sampling from vessels SV1 and SV2 was attempted at regular time intervals, and the mass collected was compared with the mass of COa used over the same time period to determine approximate solvent loadings. It was found that the totarol-containing extract solidified in valve EV1 and was difficult to remove from SV1.

Preliminary trials determined that propane-at 40 bar and 313 K-was also suitable for use as a near-critical fluid for producing a totarol-containing extract from Totara wood chips.

These extractions utilised the same apparatus as described above for liquid C02 extractions.

The separation vessel SV1 was used in continuous mode wherein the totarol-containing extract was removed through valve EV1, or in batch mode wherein a collection basket (a canister with an open top) was placed in the vessel to collect the extract. When SV1 was used in batch mode, it was depressurised at a given time interval, the basket holding the

totarol-containing extract was removed and replaced with an empty basket, and the vessel was repressurised. The flow through the apparatus, which was halted during depressurisation and repressurisation of SVl, was then restarted.

When oil was used as a carrier liquid to dissolve the totarol-containing extract in site and prevent blockages in the apparatus, it was injected either at valve VOP1-thereby yielding the totarol-containing extract in SV1 as an oil infusion which could be continuously removed from that vessel; or at valve VOP2-thereby yielding the totarol-containing extract in SV2 as an oil infusion which could be continuously removed from that vessel.

Analysis of the totarol-containing extract and the oil infusion was carried out by gas chromatography (GC). Volatile analysis and reversed phase high performance liquid chromatography (RPLC) were also conducted according to the methods described below.

Elemental analyses were carried out using a Carlo Erba Elemental Analyser EA 1108.

Additional analyses were carried out by'H and 13C nuclear magnetic resonance (NMR) and negative ion electrospray ionisation mass spectrometry (ESI-MS) for identification of individual components in the totarol-containing extract.

ANALYTICAL METHODS Volatile analysis An accurately weighed sub-sample (about 1 g) was held at 100 °C for 12 hours in an open vial, allowed to cool in a desiccator, and then weighed again. The volatiles were calculated as the as a percentage by weight of the original sub-sample weight.

RPLC Analysis Solutions of sub-samples in far UV acetonitrile (Scharlau)-containing 0.5 mg/ml of Cl 1 anilide (9) as an internal standard-were prepared according to standard techniques. The solutions were analysed on a Waters High Performance Liquid Chromatography System equipped with a Merck LiChrosphere 100 RP-18-end capped (5am) column and a Merck LiChrosphere 100 RP-18 4-4 guard column using 75% MeCN/25% H20, both containing 0. 1% trifluoroacetic acid, as the mobile phase, at a column temperature of 40°C. Detection was at 210 nm and the method was calibrated using totarol purified by RPLC and pure by IH NMR and RPLC-UV (MeOH) X. 207 nm (log s 4.39). The quantities of the minor

components isolated were insufficient to determine response factors, so quantification of these compounds assumed the same response factor as measured for totarol. The proportion of each compound in the sub-sample was calculated as a percentage by weight.

ANTIMICROBIAL ACTIVITY MIC for totarol-containing extract and tea-tree oil Stock solutions of the totarol-containing extract and tea-tree oil (20 it) in ethyl alcohol (95%) were prepared. Doubling dilutions were made of the totarol-containing extract and tea-tree oil separately in micro-titre plates in Mueller Hinton (MH) broth-concentrations <BR> <BR> ranged from 0 to 10 tig or uL (respectively) /mL. Each strain was resuscitated from frozen stocks, then grown overnight in MH broth. 10 uL of each of the bacterial strains was added to the test substances, and the resulting mixture incubated at 30°C overnight. The following day, absorbance readings were taken to determine the MIC for each combination of bacterial strain and test substance. All test substances were analysed in duplicate.

MIC for Totarol-containing extract combined with tea-tree oil.

The testing protocol was as described above, except that the totarol extracts and tea-tree oil <BR> <BR> were combined at concentrations ranging from 0 to 10 u. g or uL (respectively) /mL, and a kinetic assay was performed overnight in a micro-plate reader, set at 30°C. Each combination was analysed in duplicate.

EXAMPLE 1 Effect of particle size on extract yield Totara wood chip obtained from fence posts was subjected to near-critical extraction at a pilot scale according to the general procedure described above. Extractions were carried out without carrier liquid, at a variety of particle sizes, extraction pressures and temperatures. A two-stage separation was used, except where the near-critical fluid was liquid C02 at 70 bar and 287 K. The extraction conditions and yields of the totarol-containing extract are shown in Table 2.

Table 2: Extract yield as a function of particle size and extraction conditions Chip C02 Particle Extract yield (%) mass mass size ex sep T (K) st nd (g) (kg) (mm) (bar) I separation 2 separation vessel vessel 3386. 0 49. 49 0. 5-1 70 287 NU 3. 78 3383. 3 81. 78 0. 5-1 300/55 318 7. 61 0. 6 2600. 0 70. 13 1-2 300/100 313 3. 91 1. 34 2600. 0 39. 16 2-3 300/92 313 2. 98 1. 18 Notes: NU = not used; Pext = extraction pressure; and Psep = separation pressure.

The results shown in Table 2 demonstrate that that yield of extract decreases when the particle size of the wood chips increases. In the first two trials shown in Table 2, the extract was recovered continuously from the separation vessels. This became increasingly difficult as the trials proceeded, leading to valve blockages in the bottom of the separation vessel. In the final two extractions shown in Table 2, a collection basket was used in the first separation vessel.

The use of liquid C02 as the near-critical fluid yielded a totarol-containing extract having a higher proportion of totarol-at around 55 % by mass; but led to operational problems, such as blockages in the depressurisation valve BPR1. The use of supercritical COx resulted in higher extract yields having a lower proportion of totarol-at around 40 % by mass.

A large amount of water, proportional to the amount of COa used, was also extracted from the wood chips. The amount of water extracted using liquid C02 was lower than that at supercritical conditions, due to the higher solubility of water in supercritical C02.

EXAMPLE 2 Extraction of pre-dried wood chips Totara wood chips obtained from fence posts with a chip size between about 5 and 10 mm were partly to fully dried in metal trays in a convection oven at 80°C. The weight loss of the tray as a function of time was used to determine the percentage of moisture removed. Partly dried (moisture content of 5.8 %) and fully dried Totara wood chips (moisture content of 1.0 %) were then extracted in two separate trials at a pilot scale. The Totara wood chips

comprised approximately 10 % volatile matter before drying, most of which was water. The wood chips were further ground in a knife mill to a particle size between about 0.5 and 1 mm after drying. The results of the extractions-both of which were carried out at 310 K- are summarised in Table 3.

Table 3: Effect of moisture content on totarol-containing extract yield Chip Moisture mass Pcxt Psep lSt separation vessel 2 separation vessel mass (%) mass (bar) p (g) (kg) % yield % totarol % yield % totarol 3153. 3 5. 8 47. 54 300/85 3. 44 46 1. 76 72 2802. 0 1. 0 47. 25 300/85 3. 46 40 2. 07 68

Notes: Pext = extraction pressure ; and Psep = separation pressure.

The results in Table 3 show that varying the moisture content of the Totara wood chips to be extracted leads to a minimal difference in the yield of totarol-containing extract obtained in each separation vessel, and only a small difference in the proportion of totarol. The extracts were free of water when fully dried chips were used, which removes the need for a freeze- drying step after near-critical extraction. Totarol is not degraded by heat during the wood drying process.

EXAMPLE 3 Oil infusion (separation vessel 1 and separation vessel 2) This example shows how a carrier liquid-typically an oil-can be injected into the CO2 stream flowing in the apparatus, prior to one of the separation vessels. The oil dissolves the totarol-containing extract after it exits the depressurisation valve thereby preventing blockages. The use of oil as a carrier liquid also enables the continuous removal of the extract from the separation vessels, and yields an oil infusion comprising the totarol- containing extract.

Three pilot scale trial extractions and one demonstration scale trial extraction were performed at 313 K on Totara wood chip (obtained from fence posts)-ground to a particle size between about 1 and 2 mm-using vegetable oils as carrier liquids for the extract obtained in either the first or second separation vessel. The oils used were commercial soya oil or linseed oil. The results of these trials are summarised in Table 4.

Table 4: Use of oil as a carrier liquid in separation vessel 1 or separation vessel 2

Chip C02 Oil lest separation vessel 2" separation vessel mass Scale mass mass ex sep (bar) % yield % totarol % yield 2710. 1 pilot 38. 82 1374 300/92 2. 53 NM Oil used 528000 demo 10716 recycle 300/85 4. 94 40 Oil used 3000. 0 pilot 46. 35 1589 300/90 Oil used NM 1. 09 3000. 0 pilot 46. 61 916 300/90 Oil used NM 1. 36 Notes: NM = not measured; Oil mass = oil injected into the flowing C02 just prior to the specified separation vessel; Pext = extraction pressure; and Psep = separation pressure.

The totarol-containing extracts were able to be continuously removed from the separation vessel downstream of the point of injection of the oil into the CO2. The infusion- comprising the oil and the totarol-containing extract-obtained from the second separation vessel was yellow to brown in colour, and contained water which separated out upon standing. The totarol-containing extract was fully dissolved in the oil.

When used to prevent blockages, the oil infusion could be recycled, and re-injected prior to the separation vessel, until a totarol concentration of approximately 10 % by mass was reached.

The main extract from the demonstration scale extraction was freeze-dried in a tray freeze- drier.

EXAMPLE 4 Re-extraction of totarol-containing extract for purification This example shows that the totarol-containing extract produced at relatively high pressures can be re-extracted at different near-critical extraction conditions to yield an extract with a higher proportion of totarol. A totarol-containing extract-comprising 43 % totarol by mass-produced according to the procedure described in Example 1-at an extraction

pressure of 300 bar and temperature of 313 K-was re-extracted at a laboratory scale with supercritical C02 at 313 K and at pressures between 80 and 150 bar.

At 85 bar, 3.96 kg of C02 was used to obtain 0.71 g of an extract comprising 70 % totarol.

At 90 bar, 3.85 kg of CO2 was used to obtain 1.51 g of an extract comprising 71 % totarol.

EXAMPLE 5 Extraction at two pressures This example shows that Totara wood chips obtained from fence posts can be extracted at a relatively low extraction pressure to yield a totarol-containing extract having a higher proportion of totarol than the extract produced at 300 bar, and that all of the totarol can be extracted from the wood chips. This example further demonstrates the use of collection baskets in the separation vessels, and their exchange without depressurising the extractor.

This example also shows that, following an extraction at relatively low pressure, the pressure can be raised to extract non-totarol compounds from the wood chips.

3378.7 g of Totara wood chips-ground to a particle size between about 0.5 and 1 mm- were extracted with supercritical C02 at 150 bar and 333 K for three 80 minute periods. A collection basket was held in the first separation vessel, which was operated at 70 bar and approximately 313 K. Under these conditions, almost all of the totarol-containing extract was recovered in the first separation vessel and mainly water was recovered in the second separation vessel.

At the end of each 80 minute period, the extraction was stopped, and the first separation vessel was depressurised. The extraction vessel was held at the extraction pressure. The collection basket in the first separation vessel was then removed and replaced with a fresh basket, the separation vessel was repressurised, and the extraction was re-started. The extraction was stopped at the end of the third 80 minute period. The extraction and separation vessels were then depressurised, the extraction basket was reweighed, and the collection basket was replaced. The bed of wood chips (now 3059.1 g) was then re- extracted at 300 bar and 313 K for 80 minutes. The results for the first three 80 minute extractions at 150 bar and the re-extraction at 300 bar are shown in Table 5.

Table 5: Changes in the proportion of totarol in the extract with extraction time Time P C02 mass Total extract mass Dry extract mass Yield Totarol (min) (bar) (kg) * (g) (g) (%) (%) 80 150 30. 43 127 62. 9 3. 76 59.6 160 150 62. 30 83. 3 19. 3 2. 47 44.9 240 150 94. 40 53. 3 4. 8 1.58 15.9 360 300 124. 56 74. 1 26. 1 2. 19 2. 8 Notes: * = cumulative C02 used ; # = including water; and + = excluding water.

The results in Table 5 show that the yield of totarol-containing extract, and the proportion of totarol in the extract, decreases with extraction time and COa usage, whereas the amount of water decreases more slowly. The proportion of totarol in the totarol-containing extract obtained upon re-extraction of the wood chips was even lower at only 2.8 %, showing that totarol extraction was almost complete at the lower pressure.

EXAMPLE 6 Demonstration scale extraction and analysis This example demonstrates the semi-continuous near-critical extraction of Totara wood chips. The demonstration scale extraction of Totara wood chips-with a particle size between about 1 and 2 mm-obtained from fence posts was carried out using supercritical C02 at 150 bar and 60°C (333 K) without the use of a carrier liquid. 335.4 kg of wood chips were processed with 8347 kg of C02 to give 14.35 kg of a mixture of totarol-containing extract and water in the first separation vessel, and 3.18 kg of a mixture comprising mainly water and a small amount of totarol-containing extract in the second separation vessel. The extraction was carried out using three extraction vessels, one of which was being emptied and refilled at any time, and the other two of which were in the C02 flow. Each vessel was on-line for four hours, and off-line-being emptied and refilled-for two hours. Each of the extraction vessels held between approximately 40 and 50 kg of Totara wood chips. The first separation vessel was equipped with a basket to collect the extract. The separation vessel was operated for six hours before it was depressurised, the basket removed and replaced, and then repressurised. Free water was removed from the main product by filtration prior to

weighing the extract. The main product was then freeze-dried in a tray drier to give 10.05 kg of totarol-containing extract. The composition of this extract is shown in Table 6.

Table 6: Detailed compositional analysis of totarol-containing extract

Property Method Unit Results Appearance--Yellow powder Elemental analysis Combustion % w/w C 81.4, H 10.1, 08. 5 Volatiles Loss on drying % w/w 0.3 Totarol RPLC % w/w 623 Totarol hemiacetal RPLC % w/w 7. 0 7a-Hydroxytotarol RPLC % w/w 3. 2 7-Oxototarol RPLC % w/w 3. 9 5-Hydroxytotarol RPLC % w/w 2.2 6, 7-Dehydrototarol RPLC % w/w 0. 7 Ferruginol RPLC % w/w 0. 4 Total (Volatiles + RPLC) % w/w 84. 9* Notes: Unidentified peaks have been omitted from the table. * = the sample also contained <1% podototarin which was identified by silica gel chromatography fractionation and ESI-MS, but was not eluted during RPLC analysis.

EXAMPLE 7 Production of totarol-containing extract from fallen tree logs This example shows that a totarol-containing extract having a relatively high proportion of totarol can be produced from the logs of fallen trees recovered from the forest floor or swamp. A kiln-dried swamp log was coarsely ground in a wood chipper. The large chips were then ground in a knife mill to a particle size between about 1 and 2 mm. 3107.7 g of the ground chips were then extracted using supercritical C02 at a pressure of 150 bar and temperature of 333 K for a total of three hours. A collection basket was placed in the first separation vessel, which was operated at 70 bar and 333 K, and the second separation vessel was operated at 55 bar and 313 K.

The extraction was carried out according to the method of Example 5, with some modifications. The first extraction was carried out for 60 minutes after which the first

separation vessel was depressurised, the collection basket removed and replaced, and the separation vessel repressurised. The extraction vessel was maintained at full pressure with no flow during the exchange of collection baskets in the separation vessel. Extraction was then carried out for a further 60 minutes at the same conditions, after which the collection basket in the separation vessel was again removed and replaced. The extraction was then carried out for a further 60 minutes, and the collection basket in the first separation vessel was again removed and replaced.

Both the extraction vessel and the first separation vessel were then depressurised and the extraction basket was reweighed, and the collection basket replaced. The extraction pressure was then raised to 260 bar, and extraction was carried out for a further 60 minutes. The pressure in the first separation vessel was also increased to 90 bar. Some blockage problems were then encountered in the pipework leaving the first separation vessel. The yield of totarol-containing extract and the proportion of totarol in the extract were measured. A comparative experiment was carried out where 3112.7 g of Totara wood chips from the fallen tree log were extracted for three hours at 120 bar and 333 K without opening the separation vessel. The results for all of these extractions are shown in Table 7.

Table 7: Extract yield and proportion of totarol from the extraction of Totara fallen tree log heartwood Time P C02 mass Total extract mass Dry extract mass Yield Totarol (min) (bar) (kg) * (g) (g) (%) (%) 60 150 17. 66 82. 0 45. 2 1. 46 84.9 120 150 38. 71 83. 7 32. 0 1. 03 70. 2 180 150 60. 18 62. 8 16. 8 0. 54 61.9 240 260 80. 36 87. 8 40. 7 1. 31 44.8 0-180 150 51. 72 184. 2 88. 3 2. 84 80. 4 Notes: * = cumulative C02 used; # = including water ; and + = excluding water.

The results in Table 7 show that the proportion of totarol in the totarol-containing extract obtained from fallen tree log heartwood is substantially higher than that in the extract obtained from fence posts. The results also show that the yield of totarol-containing extract, and the proportion of totarol in the extract, decreases with extraction time and COa usage, whereas the amount of water decreases more slowly. The proportion of totarol in the

totarol-containing extract obtained upon re-extraction of the wood chips at higher pressure was even lower at only 44. 8 %.

EXAMPLE 8 Removal of totarol-containing extract from separation vessels with an auxiliary separation vessel This example shows how an auxiliary separation vessel can be used to recover the totarol- containing extract from the first separation vessel, thereby enabling semi-continuous extraction without the use of oil as a carrier liquid, or the use of collection baskets.

Two identical additional separation vessels equipped with a valve at the bottom, an inlet dip tube that passes into the interior of the vessel, an exit tube that suitable for connection to the base of the second separation vessel, and a branch tube and valve off this exit tube that enabled venting to the atmosphere, were used to recover the extract from the first separation vessel. Extraction of 2469.2 g of Totara wood chips-obtained from a fallen tree log-was carried out at 150 bar and 333 K with the additional auxiliary separation vessel connected to the bottom of the first separation vessel (70 bar, 313 K) via valve EV1. The vent line from the auxiliary separation vessel was connected to the second separation vessel via valve EV2.

The auxiliary separation vessel was initially filled to the pressure of the second separation vessel-approximately 55 bar-via valve EV2, which was then left open. After the extraction had proceeded for 60 minutes, valve EV1 was opened to induce flow of extract into the auxiliary separation vessel. When the outlet pipe from the auxiliary separation vessel became hot, valves EV1 and EV2 were closed, and the gas in the auxiliary separation vessel was then slowly vented to atmosphere. The auxiliary separation vessel was then removed and replaced with a second auxiliary separation vessel. The totarol-containing extract was recovered from the first auxiliary separation vessel by solvent extraction with acetone, followed by evaporation of the solvent under vacuum.

The second auxiliary separation vessel was then connected to the apparatus, and filled to the second separation vessel pressure of approximately 55 bar. The auxiliary separation vessel was filled with extract in the same manner as the first auxiliary separation vessel after 120 minutes of extraction, and then the extraction was completed. The second auxiliary separation vessel was removed as above and washed with acetone to recover the totarol-

containing extract. The first separation vessel was opened at the end of the extraction and was found to be empty of totarol-containing extract, confirming that all of the extract had passed into the auxiliary separation vessels.

The trial was then repeated with 2796.5 g of finely ground Totara wood obtained from fence posts. Again, all of the totarol-containing extract that had precipitated into the first separation vessel was recovered in the auxiliary separation vessels.

EXAMPLE 9 Minimum inhibitory concentration (MIC) against Gram-positive bacteria The totarol-containing extract produced as the main extract in the demonstration scale extraction described in Example 3 was tested for activity against a range of Gram-positive bacteria using the agar dilution method. The test samples were dissolved in ethanol, and then further diluted in water to give final concentrations, in a doubling dilution series, from 0.004 to 64 p. g/mL. The agar used was Mueller Hinton agar. An inoculum of approximately 1 x 104 organisms of each bacterial strain was replicated onto the agar plates containing the totarol-containing extract. The MIC for each bacterial strain is shown in Table 8.

Table 8: MIC of totarol-containing extract against Gram-positive bacteria Bacterial strain Antibiotic resistance MIC (g/mL) Staphylococcus aureus Acc2243 Not resistant 8 Enterococcus faecalis Acc2244 Not resistant 8 Staphylococcus aureus MRS02/2215 Epidemic MRSA 4 Staphylococcus aureus MRS02/2214 Multiresistant MRSA 4 Staphylococcus aureus MRS/02/2249 Community MRSA 8 Enterococcus faecalis Acc2244 High-level gentamicin 8 Enterococcusfaecalis Acc2244 Vancomycin 4 Streptococcuspyogenes ARL02/752 Erythromycin 4 The results shown in Table 8 demonstrate that the totarol-containing extract has very high antibacterial activity against a wide range of antibiotic-resistant Gram-positive bacterial strains.

EXAMPLE 10 Minimum inhibitory concentration (MIC) against Gram-negative bacteria This example shows that the totarol-containing extract produced by supercritical extraction has antimicrobial activity similar to, or better than, tea-tree oil.

The MIC against four Gram-negative bacteria-Salmonella menston ; Escherichia coli ; Enterobacter aerogenes ; and Pseudomonas aeruginosa-was determined for the totarol- containing extract produced in Example 6, tea-tree oil and a mixture of the totarol- containing extract and tea-tree oil. The results are shown in Table 9.

Table 9: Comparison of the minimum inhibitory concentration (MIC) for totarol- containing extract, tea-tree oil, and a mixture of the extract and tea-tree oil against four Gram-negative bacteria Bacterial Strain MICtxtraet (llglmL) MICoi g/mL) MICmixture (g/mL) Salmonella menston 0.625 0.555 1. 11 Escherichia coli 0. 625 0. 555 0. 555 Enterobacter aerogenes 1.25 1. 11 0.555 Pseudomonas aeroginosa 0.313 0.278 0.555 The results in Table 10 show that the totarol-containing extract is a very effective antimicrobial against a range of Gram-negative bacteria. The MIC for each bacterial strain tested is approximately 0.3-1. 25 ug/mL, and surprisingly, this figure is the same for each strain, whether subjected to the totarol-containing extract or tea-tree oil. Tea-tree oil is known to have activity against Gram-negative bacteria, whilst pure totarol has not been reported to have any activity against Gram-negative bacteria. Other components of the totarol-containing extract may be responsible for the activity against Gram-negative bacteria.

The antimicrobial effect of the totarol-containing extract is enhanced when combined with tea-tree oil only for Enterobacter aerogens. A concentration of 0.3-0. 6 ug/mL of the totarol-containing extract was sufficient to stop the growth of the other three Gram-negative strains tested. This concentration was not decreased by the addition of tea-tree oil, nor was the MIC of tea-tree oil affected by the addition of totarol-containing extract. The results

obtained for tea-tree oil are in accordance with those found in the literature, where the MIC for tea-tree oil ranges between 0.2 and 2.0 % (v/v), which equates to between 2.0 and 20 IlL/mL.

EXAMPLE 11 Topical formulation The totarol-containing extract produced according to the methods described above was used to prepare a solution, suitable for topical application, and having the following composition: Component Quantity Totarol-containing extract 5. 0081 g Ethyl alcohol (96%) 700.01 g Propylene glycol 100.01 g FDC Blue No. 1 0. 53 mg Purified water qs Total 1 L

A stock solution of FDC Blue No. 1 was prepared by weighing 10.5 mg into a 50 mL volumetric flask. The dye was dissolved in a small quantity of purified water and the solution made up to the mark with purified water.

The totarol-containing extract was dissolved in the ethyl alcohol and the propylene glycol added to the resulting solution. A portion of the FDC Blue No. 1 solution (2.54 mL) was then added to give a solution with a green colour. The purified water was then added in a sufficient quantity to give a total volume of 1 L. The resulting solution was transferred into applicator bottles.

It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention (as set out in the accompanying claims).

REFERENCES (1) 1. Kubo et al."Antibacterial activity of totarol and its potentiation"J. Nat. Prod. 55 (1992) pp. 1436-1440.

(2) J. Markham"Antimicrobial effectiveness of tea-tree oil"Australian Tea-tree Export and Marketing Limited Conference (1996).

(3) Tea-tree Oil Growers of Australia, Main Camp Tea-tree Oil Group.

(http://www. avreskincare. com/teatreeoil/teatreeoilextracts. html) (4) G. B. Evans et al."The synthesis and antibacterial activity of totarol derivatives (Part 1)"Bioorg. Med. Chem. 7 (1999) pp. 1953-1964.

(5) H. Muroi and I. Kubo"Antibacterial activity of anacardic acid and totarol, alone and in combination with methicillin, against methicillin-resistant Staphylococcus aureus" J Appl. Bacteriol. 80 (1996) pp. 387-394.

(6) L. J. Walsh and J. Longstaff"The antimicrobial effects of an essential oil on selected oral pathogens"Periodontology 8 (1987) pp. 11-15.

(7) J. G. Bendall and R. C. Cambie"Totarol: a non-conventional diterpenoid"Aust. R Chem. 48 (1995) pp. 883-917.

(8) Remingtoc's Pharmaceutical Sciences, l8th ed. , Gennaro (ed. ) 1990, Mack<BR> Publishing Co. , Easton, Pa.<BR> <P>(9) N. B. Perry et al. "Fatty acid anilides as internal standards for high performance liquid chromatographic analyses of Valeriana officinalis L. and other medicinal plants"Phytochemical Analysis 7 (1996) pp. 263-268.