AU2006272366B2

AU2006272366B2 - Therapeutic honey and method of producing same

Info

Publication number: AU2006272366B2
Application number: AU2006272366A
Authority: AU
Inventors: Peter Taylor
Original assignee: Honey Res & Development Pty Ltd
Current assignee: Honey Research & Development Pty Ltd
Priority date: 2005-07-19
Filing date: 2006-07-19
Publication date: 2011-09-22
Anticipated expiration: 2026-07-19
Also published as: AU2006272366A1

Abstract

A honey comprising the following features: (i) possesses non-peroxide based antimicrobial activity; and (ii) is derived from .

Description

WO 2007/009185 PCT/AU2006/001024 1 "Therapeutic Honey and Method of Producing Same" Field of the Invention The present invention relates to a novel honey and a dispensing means for topically delivering the honey. The present invention also relates to a method for producing a honey and to methods of treating microbial infections with the honey Background Art The antimicrobial and therapeutic activity of honey has been known for some time. Presently, there are a range of medical grade honeys available that are suitable for various applications, but with varying activities. The antimicrobial activity of honey is mediated via: (i) its ability to generate hydrogen peroxide when added to a site of infection such as a wound i.e. peroxide based antimicrobial activity; and (ii) plant derived additives in the honey i.e. non-peroxide based antimicrobial activity. The peroxide based antimicrobial activity in honey is derived from the activity of the enzyme glucose oxidase in the honey that originates from bees and causes the production of hydrogen peroxide at the site of application. This activity is catalase sensitive. Thus, when honey is applied to a wound its peroxide based antimicrobial activity is attenuated by the enzyme catalase that is produced by tissue at the site of application and breaks down the hydrogen peroxide. Peroxide based antimicrobial activity is also particularly sensitive to dilution by body fluids at the site of infection that further reduces the antimicrobial activity. The non-peroxide based antimicrobial activity of honey is derived from plant based additives that are incorporated into the honey during the natural honey making process. These additives are not hampered by the limitations associated with peroxide based antimicrobial activity. Thus, honey with non-peroxide based antimicrobial activity is more desirable as it is more useful for therapeutic purposes.

2/1 Manuka honey is one type of honey that has non-peroxide based antimicrobial activity. Manuka honey is derived from plant Leptospermum scoparium and a similar honey has been produced using the plant Leptospermum polygalifolium, that was located in Australia following an extensive and systematic screening process. Although there are two known sources for honey with non-peroxide based antimicrobial activity, the demand for these medical honeys vastly outweighs supply. Thus, there is a need for another source of honey with nonperoxide based anti-microbial activity. Summary of the Invention The present invention provides a honey comprising the following features: (i) possesses non-peroxide based antimicrobial activity; and (ii) is derived from Leptospermum subtenue. The invention further provides a honey comprising the following features: (i) possesses non-peroxide based antimicrobial activity; and (ii) is derived from Leptospermum subtenue wherein the honey has been stored for a period of time and under conditions necessary to increase the non-peroxide based anti-microbial activity of the honey, and wherein the honey is stored at room temperature. The present invention also provides a dispensing means for topically delivering an effective amount of a honey comprising the following features: (i) possesses nonperoxide based antimicrobial activity; and (ii) is derived from Leptospernum subtenue, the dispensing means comprising the effective amount of the honey or formulation and a membrane that, when the dispensing means is in use, is located between the honey or formulation and the intended site of application. The present invention also provides a method for producing a honey that: (a) possesses non-peroxide based antimicrobial activity; and (b) is derived from Leptospermum subtenue comprising the steps of (i) placing a hive or other population of bees in proximity to a Leptospermum subtenue and (ii) collecting honey produced by the bees.

2/2 The present invention also provides a method for producing a honey that: (a) possesses non-peroxide based antimicrobial activity; and (b) is derived from Leptospermum subtenue comprising the steps of (i) placing a hive or other population of bees in proximity to a Leptospermum subtenue; (ii) collecting honey produced by the bees; and (iii) storing said honey for a time and under conditions necessary to increase the non peroxide based anti-microbial activity of the honey, wherein the honey is stored at room temperature.

WO 2007/009185 PCT/AU2006/001024 3 The present invention also provides a method of treating a microbial infection comprising contacting a honey according to the present invention with the infection. Brief Description of the Figures Figures 1-5 are photographs of an ulcer during treatment with honey according to one preferred embodiment of the present invention. Figure 1 is prior to treatment, Figure 2 is 7 days post treatment; Figure 3 is 21 days post treatment, Figure 4 is 28 days post treatment and Figure 5 is 63 days post treatment. Detailed Description of the Invention Honey and formulations thereof The present invention provides a honey comprising the following features: (i) possesses non-peroxide based antimicrobial activity; and (ii) is derived from Leptospermum subtenue. For the purposes of the present invention Leptospermum subtenue or L. subtenue includes all members of the species that can be used to produce honey with non peroxide based antimicrobial activity. Using the information in the present application it would be routine for a skilled person to produce honey from any L. subtenue and test it for non-peroxide anti-microbial activity. The L. subtenue may be a L. subtenue from at least one of the Apiary sites administered by the Western Australian Department of Conservation and Land Management and identified by the numbers 788, 837, 838, 839, 1063, 1064, 1065, 1066, 1075, 1077, 1078, 1079, 1082-1087, 1096, 1097, 1101-1106, 5674, 5677, 791, 792, 793, 794, 795, 796, 799, 800, 1067, 1068, 1069, 1072, 1073, 1076, 1088, 1090, 1091, 1094, 1109, 1110, 1111, 1114, 1115, 1116, 1117-1129, 1150, 1151, 1547, 5461, 5678, 1131, 1132, 1134, 1136, 1137, 1138, 1139, 1140, 1141, 1144-1149, 1152, 1153, 1154, 1156, 1157, 1336-1344, 1347, 1348, 1352, 1353, 1356, 1362,-1374, 1438, 1569, 5619 and 5620. Alternatively, the L subtenue may be the L. subtenue, a specimen of which is held at the CALM WO 2007/009185 PCT/AU2006/001024 4 Herbarium in Perth, Western Australia and identified as L. subtenue J. Thomps. PERTH 06771866. Preferably, the L. subtenue is flowering. This may be from any time from June October. In this regard, it has been noted that this time of the year is best for making the honey of the present invention as it has a positive effect on the non peroxide based anti-microbial activity. For the purposes of the present invention the phrase "derived from" when used in respect of honey means that the honey includes or otherwise incorporates an additive from L. subtenue. Whilst not wishing to be bound to any particular basis for activity, the non peroxide based antimicrobial activity of the honey of the present invention is thought to result from the presence in the honey of active agents from the L. subtenue. The additive may be a plant phytochemical or another agent or compound from L. subtenue. Such additives include microbes that may be present on L. subtenue. Preferably, the honey of the present invention is derived entirely or principally from Leptospermum subtenue. Alternatively, the honey may be derived from a plurality of plants of which L. subtenue is one. The critical factor here is that the honey is derived from L. subtenue to the extent that it has non-peroxide based antimicrobial activity. The non-peroxide based antimicrobial activity of the honey of the present invention may vary. Preferably, the non-peroxide based activity is equivalent to a solution of at least 2% phenol, more preferably at least 5%, more preferably at least 10%, even more preferably a solution of at least 14-17% and even more preferably at least 20-30% phenol, preferably as determined using the method described in the examples section hereunder. Whilst the honey of the present invention includes non-peroxide based anti microbial activity it may also contain hydrogen peroxide based anti-microbial activity. Thus, the present invention also provides a honey comprising the WO 2007/009185 PCT/AU2006/001024 5 following features: (i) possesses non-peroxide based and peroxide based antimicrobial activity; and (ii) is derived from Leptospermum subtenue. The honey of the present invention may be used as a therapeutic formulation without additives or other modification. However, when used as a therapeutic formulation it is preferred that the honey be sterilised. Thus, the present invention also provides a sterilised honey comprising the following features: (i) possesses non-peroxide based antimicrobial activity; and (ii) is derived from Leptospermum subtenue. The honey of the present invention may be sterilised in any one of a number of ways provided the sterilisation process does not unduly compromise the therapeutic activity of the honey. Preferably, gamma radiation is used to sterilise the honey. The honey of the present invention may also be provided as part of a therapeutic formulation. Thus, the present invention provides a formulation comprising (a) a honey that: (i) possesses non-peroxide based antimicrobial activity; and (ii) is derived from Leptospermum subtenue and; (b) a physiologically acceptable carrier. The formulations of the present invention may further comprise other active agents that may contribute to the efficacy of the formulation. Thus, the present invention also provides a formulation comprising (a) a honey that: (i) possesses non-peroxide based antimicrobial activity; and (ii) is derived from Leptospermum subtenue; (b) another active agent; and optionally, (c) a physiologically acceptable carrier. The physiologically acceptable carrier is optional as the honey may be used in combination with another active agent without an additional carrier. The other active agent may be varied provided it does not unduly compromise the activity of the honey in the formulation. Other possible active agents include: anti inflammatory agents and anti-microbials.

WO 2007/009185 PCT/AU2006/001024 6 The formulation may be suitable for pharmaceutical or veterinary uses. The concentration of the honey of the invention in such formulations can very widely, i.e., from less than about 1%, usually at or at least about 10-30% to as much as 80-99% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration and intended end use. However, the physician or veterinarian will determine the dosage of the present therapeutic formulations that will be most suitable and it will vary with the form of administration, the particular subject under treatment and the affliction being treated. The physician may wish to initiate treatment with small dosages substantially less than the optimum dose of the honey and increase the dosage by small increments until the optimum effect under the circumstances is reached. Preferably, the formulation is adapted for topical application. Thus, the present invention also provides a topical formulation comprising (a) a honey that: (i) possesses non-peroxide based antimicrobial activity; and (ii) is derived from Leptospermum subtenue and; (b) a physiologically acceptable carrier. Various topical delivery systems may be appropriate for administering the compositions of the present invention depending upon the preferred treatment regimen. Topical formulations may be produced by mixing or combining the honey of the present invention in a suitable carrier. In general, any liquid, cream, or gel, or similar substance that does not appreciably react with the honey and which are non-irritating are suitable. Appropriate non-sprayable viscous, semi solid or solid forms can also be employed that include a carrier compatible with topical application and have a dynamic viscosity preferably greater than water. Suitable formulations are well known to those skilled in the art and include, but are not limited to, solutions, suspensions, emulsions, creams, gels, ointments, powders, liniments, salves, aerosols, transdermal patches, etc, which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, emulsifiers, wetting agents, fragrances, colouring agents, odour controllers, thickeners such as natural gums etc. Particularly preferred topical formulations include ointments, creams and gels.

WO 2007/009185 PCT/AU2006/001024 7 Ointments generally are prepared using either (1) an oleaginous base, i.e., one consisting of fixed oils or hydrocarbons, such as white petroleum or mineral oil, or (2) an absorbent base, i.e., one consisting of an anhydrous substance or substances which can absorb water, for example anhydrous lanolin. Customarily, following formation of the base, whether oleaginous or absorbent, the active ingredient is added to an amount affording the desired concentration. One particular ointment may comprise honey of the present invention with one or more of: acetylated lanolin alcohol, caprylic/capric triglyceride, hydroxylated lanolin, acetylated lanolin, cetyl alcohol, 2-bromo-2-nitropropane-1, 2-diol, lanolin alcohol, mineral oil light, white soft paraffin. Creams are oil/water emulsions. They consist of an oil phase (internal phase), comprising typically fixed oils, hydrocarbons and the like, waxes, petroleum, mineral oil and the like and an aqueous phase (continuous phase), comprising water and any water-soluble substances, such as added salts. The two phases are stabilised by use of an emulsifying agent, for example, a surface active agent, such as sodium lauryl sulfite; hydrophilic colloids, such as acacia colloidal clays, veegum and the like. Upon formation of the emulsion, the honey may be added in an amount to achieve the desired concentration. Gels comprise a base selected from an oleaginous base, water, or an emulsion suspension base. To the base is added a gelling agent that forms a matrix in the base, increasing its viscosity. Examples of gelling agents are hydroxypropyl cellulose, acrylic acid polymers and the like. Customarily, the honey is added to the formulation at the desired concentration at a point preceding addition of the gelling agent. The amount of honey incorporated into a topical formulation is not critical; the concentration should be within a range sufficient to permit ready application of the formulation to the affected area in an amount that will deliver the desired amount of honey to the desired treatment site. The customary amount of a topical formulation to be applied to an affected area will depend upon the nature of the infection and the concentration of the honey in the formulation.

WO 2007/009185 PCT/AU2006/001024 8 In applications for the treatment of a particular ailment or infection, formulations of the invention are administered to an afflicted subject in an amount and for a time sufficient to at least improve the condition of the patient and preferably cure the patient of the affliction. Clearly, the precise details of any particular treatment regime will be decided by a suitably qualified practitioner. In this regard, single or multiple administrations of the formulations can be carried out with dose levels and pattern being selected by the treating physician or veterinarian. In any event, the formulation of the invention should provide a quantity of the honey of the invention sufficient to effectively treat the affliction. The honey of the present invention may also be incorporated into a device for dispensing the honey in a predetermined fashion. Thus, the present invention also provides a dispensing means for topically delivering an effective amount of honey according to the present invention to a subject, the dispensing means comprising the effective amount of the honey and a membrane that, when the dispensing means is in use, is located between the honey and the intended site of application. The dispensing means may be varied and includes slow release carriers and materials impregnated with or containing the honey such as patches, bandages, cotton wool and gauze. Preferably, the dispensing means is adapted to be removably fixed to the site of application to deliver the honey over a predetermined time and/or at a predetermined dose. Thus, the present invention also provides a dispensing means for topically delivering an effective amount of the honey of the present invention to a subject, the dispensing means comprising (i) the effective amount of honey; (ii) a membrane that, when the dispensing means is in use, is located between the honey and the intended site of application and (iii) means for releasibly attaching the dispensing means to the skin of a subject. The honey and the membrane may be provided integrally. For example, the membrane may be impregnated with the honey.

WO 2007/009185 PCT/AU2006/001024 9 The means for releasibly attaching the dispensing means may also be varied and includes an adhesive member provided on at least a part of the dispensing means. The adhesive member may also be integral with the dispensing means and thus the present invention also provides: an adhesive dispensing means for topically delivering an effective amount of honey to a subject, the dispensing means comprising (i) the effective amount of honey; and (ii) a membrane that, when the dispensing means is in use, is located between the honey and the intended site of application. Methods of producing honey The present invention also provides a method for producing a honey that: (a) possesses non-peroxide based antimicrobial activity; and (b) is derived from Leptospermum subtenue comprising the steps of (i) placing a hive or other population of bees in proximity to a Leptospermum subtenue and (ii) collecting honey produced by the bees. Preferably, the method of producing honey is carried out under conditions that maximise the exposure of the bees to the L. subtenue. Thus, the method may involve the placement of the bees proximal to a relatively high density of L. subtenue. Whilst the relatively high density of L. subtenue may be present naturally, L. subtenue populations or plantations may be created for the most consistent and controlled production of the honey. Thus, the present invention also provides a method for producing a honey that: (a) possesses non-peroxide based antimicrobial activity; and (b) is derived from Leptospermum subtenue comprising the steps of: (i) cultivating a population of L. subtenue in a designated area; (ii) placing a hive or other population of bees in proximity to the population of Leptospermum subtenue and (iii) collecting honey produced by the bees. The non-peroxide based anti-microbial activity of honey produced according to the present invention can vary. However, surprisingly, applicant has determined that honey produced according to the present invention may attain non-peroxide WO 2007/009185 PCT/AU2006/001024 10 based anti-microbial activity over time and or attain enhanced non-peroxide based anti-microbial activity over time. In this regard, applicant has noted that honey made according to the present invention may initially have little or no non peroxide based anti-microbial activity but then over time develop such activity. Thus, the present invention also provides a method for producing a honey that: (a) possesses non-peroxide based antimicrobial activity; and (b) is derived from Leptospermum subtenue comprising the steps of (i) placing a hive or other population of bees in proximity to a Leptospermum subtenue; (ii) collecting honey produced by the bees; and (iii) storing said honey for a time and under conditions necessary to increase the non-peroxide based anti-microbial activity of the honey. The storage time may be varied and includes about 1-24 months, 1-12 months and 1-6 months. Alternatively, the storage time may be a time sufficient for the honey to undergo granulation or crystallisation. The granulation/crystallisation process involves the formation of dextrose crystals in the honey and can be affected by a range of variables including the moisture content and dextrose concentration in the honey. In this regard, it is hypothesised that the granulation/crystallisation process may be correlated with increased non-peroxide anti-microbial activity in the honey. The storage conditions can be varied and include storage at ambient conditions in a sealed container. Methods of treatment The honey of the present invention may be used to treat any disease or infection that is amenable to treatment with medical grade honey such as Manuka honey. These include microbial infections and the diseases and disorders caused thereby. Thus, the present invention also provides a method of treating a microbial infection comprising contacting a honey or a formulation thereof of the present WO 2007/009185 PCT/AU2006/001024 11 invention with the infection. The present invention also provides for the use of honey according to the present invention for preparing a medicament for treating a microbial infection. The microbial infection may be caused by various organisms and includes an infection caused by fungi and/or bacteria. Particular bacterial infections that can be treated according to the present invention include those that are commonly associated with wounds as well as a bacterium or species thereof selected from the group comprising: Staphylococcus such as S. aureus and MRSA, Enterococcus including VRE, Escherichia such as E. coil, Pseudomonas such as P. aeruginosa, Acinetobacter such as A. baumarii, Helicobacter such H. pylori, Neisseria such as N. meningitidis. Antibiotic resistant bacteria are particularly amenable to treatment using the present invention. Any disease or disorder that is caused or complicated by the microbial infections mentioned above may also be treated using the honey or formulations thereof of the present invention. These include but are not limited to peptic ulcers, diarrhoea, eye infections and throat infections. Honey according to the present invention or formulations thereof can also be used to treat viral infections such as cold sores and other herpes simplex infections. Skin allergies and insect bites are also amenable to treatment with the honey of the present invention as are burns, ulcers, skin, scalp and hair disorders, and other tissue conditions, including those of the eye, nose, ear and mouth. Plant Variety The present invention also provides an isolated and cultivated Leptospermum plant, known as L. subtenue, a specimen of which is held at the CALM Herbarium in Perth, Western Australia and identified as L. subtenue J. Thomps. PERTH 06771866 (hereinafter referred to as "06771866"). The botanical description of L. subtenue J. Thomps [Telopea 3: 353 (1989)] is provided hereunder.

WO 2007/009185 PCT/AU2006/001024 12 L. subtenue J.Thomps. Frutex c. I m altus. Folia crassa angusta elliptica ad oblanceolate, 1-1.5 cm longa. Flores 8-10 mm diametro, ad apicem ramulorum tenuium dispersarum, sepalis pubescentibus vel glabratis persistentibus. Ovarium 3-loculare. Fructus decidui. HOLOTYPE: WESTERN AUSTRALIA: N. Esperance, C. Andrews, Oct. 1903 (W.E. Blackall Collection, PERTH). ISOTYPE: herb. C.A. Gardner 1267 (PERTH). Shrub c. Im tall, the bark (as seen on specimens) close; the young stems very slender, at first silky-pubescent but soon becoming glabrous, without a flange but with a cup-shaped extension below each node that remains on older branches, and with the branching at an angle of c. 450 . Leaves rather well-spaced and erect, 3-4 mm long, and 1-1.5 mm wide, elliptical, thick, especially near the obtuse apex, concave, glabrous or silky-pubescent and tapering gradually to a slender petiole. Flowers white or pink, to 12 mm in diameter, occurring singly, occasionally 2 together, each in a bract-axil, at the ends of long and slender side branches that are scattered (rather than in adjacent leaf-axils), but with most branchlets terminated by flowering shoots, and with new growth appearing to develop where flowers have been aborted rather than shed. Bracts scarious, pale red-brown, short and broad; the bracteoles red-brown, scarious, very large, broad and concave, all shed from the young bud leaving conspicuous scars. Hypanthium densely and shaggily pubescent, all over or at least on the lower part, dark-coloured and ridged, c. 3 mm long and tapering to a more or less pedicel-like base, the upper part expanded and erect, occasionally glabrous, the top of the ovary pubescent near the style but otherwise almost glabrous. Sepals persistent, c. 2mm long, broadly ovate, densely pubescent to glabrous, rather keeled and hooded, and like the hypanthium in texture apart from the thin scarious margins. Petals c. 4mm long. Stamens in bundles of 5-7, c. 1 mm long, the anther-cells c. 0.3 mm long, parallel, broad and shallow. Style rather shallowly inset, very broad based, tapering to a slender upper part, with a small stigma. Ovary 3-locular, each loculus extended upward with c. 6-12 ovules in 2 rows on a small narrow WO 2007/009185 PCT/AU2006/001024 13 and high placenta. Fruit (not seen mature) deciduous, the valves thin and raised high above the erect upper part of the hypanthium. Mature seeds not seen; immature c. 1.5mm long and narrow-oblong with little surface marking. Main flowering period: Sept.-Oct. The isolated and cultivated 06771866 can be produced via a planned and controlled breeding program with the objective of creating new Leptospermums with at least one characteristic that distinguishes the plant from other known Leptospermums and is also uniform and stable through multiple generations. 06771866 was identified west of Widgiemootha in Western Australia at locations corresponding to Apiary sites 5619 and 5620 and will now be propagated for commercial use via asexual reproduction of the new cultivar by vegetative cuttings taken from the original cultivar. As plants of the new cultivar have not been observed under all possible environmental conditions, the phenotype may vary somewhat with variations in environment such as temperature, light intensity, day length, and fertilizer rate without, however, any variance in genotype. General Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein. The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are WO 2007/009185 PCT/AU2006/001024 14 hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. The present invention will now be described with reference to a number of examples. The examples are in no way limiting on the preceding description. Examples Example 1: Antimicrobial honey Materials/Methods (A) Honey A number of bee hives with bees (Apis melifera) were located in proximity to L. subtenue J. Thomps. PERTH 06771866 plants at apiary sites 5619 and 5620 and the honey produced was harvested using standard methods/techniques. Two samples of the honey designated Al and A2 were analysed as detailed below.

WO 2007/009185 PCT/AU2006/001024 15 (B) Assay The agar well diffusion method used was adapted from the punch plate assay for inhibitory substances described in the Microbiology Standard Methods Manual for the New Zealand Dairy Industry (1982). (i) Inoculum Preparation A freeze-dried culture of Staphylococcus aureus (ATCC 9144) was reconstituted in Trypticase Soy broth (Merck 1.05459) according to the instructions supplied, and incubated at 370C for 18 hours. A loopful of the broth culture was subcultured onto blood agar plates (Blood Agar Base, Merck 1.10886, with 5% sheep's blood) incubated for 24 hours at 370C and used to inoculate 7 x Microbank vials ( Pro Lab Diagnostics PL.160 ) for long term storage at -70*C. Working cultures were obtained by placing one bead from the preserver ampoule stock into 10 ml of Trypticase Soy broth and incubating for 18 hours at 37"C. The culture was then adjusted to an absorbance of 0.5 measured at 540nm using sterile TS broth as a blank and a diluent and a cuvette with a 1 cm pathway. A volume of 100pl of the culture adjusted to 0.5 absorbance was used to seed 150ml nutrient agar to make the assay plates. (ii) Culture quality control A new freeze dried culture was obtained every 6 months. At the end of 6 months the new culture was reconstituted and placed on beads as above. This was then tested and compared with the previous culture to ensure compatible results. (iii) Plate preparation To prepare the assay plates 150 ml nutrient agar (23g/l BBL 4311472) was sterilised then held at 500C for 30 minutes before seeding with 100 pl of S. aureus culture adjusted to 0.5 absorbance as above. The agar was swirled to mix thoroughly and poured into a large square assay plate (Corning 431111 - WO 2007/009185 PCT/AU2006/001024 16 245x245x18mm) that had been placed on a level surface. As soon as the agar was set the plates were placed upside-down at 4"C overnight before using the next day. Using a quasi-Latin square as a template, 64 wells were cut into the agar with a flamed, cooled 8mm cork borer and removed with an inoculating needle. The template was prepared on black card 230 x 230 mm. A 25 mm grid was drawn on the card, 27.5 mm away from the sides, and the wells were centred on the intersections of the grid. The intersections were numbered using a white china pencil just above the intersection using a quasi-Latin square that enabled the samples to be placed randomly on the plate. (iv) Catalase solution A 2 mg/ml solution of catalase from bovine liver (Sigma C9322 2800 units/mg) in distilled water was prepared fresh each day. (v) Sample preparation A primary honey solution was prepared by adding 10g of well mixed honey to 10ml of distilled water in universals and placed at 37"C for 30 minutes to aid mixing. To prepare secondary solutions, 1ml of the primary honey solution was added to 1 ml of distilled water in a bijou for total activity testing and 1 ml of the primary honey solution was added to 1 ml of catalase solution for non-peroxide activity testing. The density of honey, which is 1.35 g/ml (average value for Manuka honey), is allowed for in the final calculation. (vi) Sample and Standard Application Each sample was tested in quadruplicate by adding 100 pl to each of 4 wells with the same allocated number on the assay plate.

WO 2007/009185 PCT/AU2006/001024 17 (vii) Plate incubation After application of samples and standards the plates were incubated on individual racks i.e. not stacked on top of one another, for 18 hours at 370C. (vii) Zone measurement The plates were placed back over the black quasi-Latin square to measure the diameter of the zones of inhibition with digital calipers (Mitutoyo 500.321) using the points of the prongs used to measure inside diameters of tubes. (viii) Preparation of phenol standards Standards of 2%, 3%, 4%, 5%, 6%, and 7%, were prepared from a 10% w/v solution of phenol BDH A.R. in water. These solutions were kept at 4'C for one month before making fresh standards and brought to room temperature in the dark before use. Each standard was placed in two wells to test in duplicate. (ix) Calculation of antibacterial activity of honey The mean diameter of the clear zone around each phenol standard was calculated and squared. A standard graph was plotted of % phenol against the square of the mean diameter of the clear zone. A best-fit straight line was fitted using Cricket Graph software and the equation of this line was used to calculate the activity of each diluted honey sample from the square of the mean measurement of the diameter of the clear zone. To allow for the dilution and density of honey this figure was multiplied by a factor of 4.69 and the activity was then expressed as the equivalent phenol concentration (% w/v). Results Al & A2 both displayed non peroxide activity in the range equivalent to 14.9 to 16.8% phenol when tested initially and equivalent to 14.5% phenol when retested a year later.

WO 2007/009185 PCT/AU2006/001024 18 Example 2 - Antimicrobial honey Materials/Methods (A) Honey A number of bee hives with bees (Apis mellifera) were located in proximity to L. subtenue J. Thomps. PERTH 06771866 plants in the following locations: B1 apiary site 1063, B2- apiary site 1127, B3- apiary site 1063, Cl- apiary site 1362, C2- apiary site 1362 and the honey produced was harvested using standard methods/techniques. Samples of various honeys were analysed as detailed above in Example 1 and the results are provided hereunder in Table 1. Results Honey Code Date of Sample Non-peroxide antimicrobial activityA B1 August 2004 0 (initial test) September 2005 17.80 April 2006 22.70 June 2006 23.07 B2 August 2004 0 (initial test) November 2005 13.9 B3 2004 Not tested initially November 2005 12.66 June 2006 20.5 C1 September 2005 0 (initial test) April 2006 13.00 C2 October 2005 0 (initial test) April 2006 17.00 A - equivalent to phenol solution of a given strength WO 2007/009185 PCT/AU2006/001024 19 Example 3: Use of antimicrobial honey to treat ulcers Materials/Methods An 89 year old female was admitted to an aged care facility with a chronic right ankle ulcer. Her ulcer had been managed with a number of treatments over the past 11 years. However, the ulcer remained ulcerated and very painful, resulting in the loss of mobility. The ulcer was cleaned with warm water and the honey described above in Example 1 was applied to a non-adherent dressing with combine and secured with a bandage daily. The ulcer was assessed regularly. Photographs were taken as well as measurements of the ulcer's area over the course of the treatment. Results The time course of the treatment is illustrated in Figures 1-5 that show the ulcer during treatment. Table 1 hereunder details the approximate area of the ulcer during treatment. Table I - Area of ulcer during treatment Day 0 7 14 21 28 35 42 63 77 99 (post treatment) Area 480 456 380 372 272 260 292 236 4 0 (mm2) Other positive outcomes from the treatment were that: WO 2007/009185 PCT/AU2006/001024 20 (i) the dressing could be removed with less distress to the subject and damage to new tissue was reduced; (ii) pain gradually subsided and this allowed a reduction and eventual cessation of analgesia; and (iii) the subject became happier and was able to once again weight bear. A number of other patients have been treated with honey made according to the present invention including AI/A2 honey. Anecdotal evidence shows the successful treatment of a range of disorders including: Rosea, ulcers, skin lesions/blemishes and skin cancers.

Claims

1. A honey comprising the following features: (i) possesses non-peroxide based antimicrobial activity; and (ii) is derived from Leptospermum subtenue wherein 5 the honey has been stored for a period of time and under conditions necessary to increase the non-peroxide based anti-microbial activity of the honey, and wherein the honey is stored at room temperature.

2. A honey according to claim 1 wherein the non-peroxide based antimicrobial activity is equivalent to a solution of at least 2-10% phenol. 10

3. A honey according to claim 1 wherein the non-peroxide based antimicrobial activity is equivalent to a solution of at least 14-17% phenol.

4. A honey according to claim 1 wherein the non-peroxide based antimicrobial activity is equivalent to a solution of at least 20-30% phenol.

5. A honey according to any one of the preceding claims further comprising 15 peroxide based antimicrobial activity.

6. A formulation comprising a honey according to any one of the preceding claims and a physiologically acceptable carrier.

7. A formulation according to claim 6 further comprising another active agent.

8. A formulation according to claim 7 wherein the other active agent is an anti 20 inflammatory agent or an anti-microbials.

9. A topical formulation according to any one of claims 6 to 8.

10.A dispensing means for topically delivering an effective amount of a honey according to any one of claims 1 to 5 or a formulation according to any one of claims 6 to 9 to a subject, the dispensing means comprising the effective 25 amount of the honey or formulation and a membrane that, when the - 22 dispensing means is in use, is located between the honey or formulation and the intended site of application.

11.A dispensing means according to claim 10 adapted to be removably fixed to the site of application to deliver the honey or formulation over a predetermined 5 time and/or at a predetermined dose.

12.A method for producing a honey that: (a) possesses non-peroxide based antimicrobial activity; and (b) is derived from Leptospermum subtenue comprising the steps of (i) placing a hive or other population of bees in proximity to a Leptospermum subtenue (ii) collecting honey produced by the 10 bees and (iii) storing said honey for a time and under conditions necessary to increase the non-peroxide based anti-microbial activity of the honey, wherein the honey is stored at room temperature.

13.A method for producing a honey that: (a) possesses non-peroxide based antimicrobial activity; and (b) is derived from Leptospermum subtenue 15 comprising the steps of: (i) cultivating a population of L. subtenue in a designated area; (ii) placing a hive or other population of bees in proximity to the population of Leptospermum subtenue (iii) collecting honey produced by the bees and (iv) storing said honey for a time and under conditions necessary to increase the non-peroxide based anti-microbial activity of the honey, 20 wherein the honey is stored at room temperature.

14.A method according to any one of claims 12 or 13 wherein the storage time is at least 1-12 months.

15.A method according to any one of claims 12 to 14 wherein the storage time is a time sufficient for the dextrose in the honey to crystallise. 25

16.A method of treating a microbial infection comprising contacting a honey according to any one of claims 1 to 5 or a formulation according to any one of claims 6 to 9 with the infection. - 23

17.Use of a honey according to any one of claims 1 to 5 or a formulation according to any one of claims 6 to 9 for preparing a medicament for treating a microbial infection.

18.A method according to claim 16 or a use according to claim 17 wherein the 5 microbial infection is caused by a bacterium or species thereof selected from the group comprising: Staphylococcus such as S. aureus and MRSA, Enterococcus including VRE, Escherichia such as E. coli, Pseudomonas such as P. aeruginosa, Acinetobacter such as A. baumarii, Helicobacter such H. pylori, Neisseria such as N. meningitidis. 10

19.A method according to claim 16, a use according to claim 17 or a method according to claim 18 wherein the microbial infection is associated with a disease or disorder selected from the group consisting of: peptic ulcers, diarrhoea, eye infections and throat infections.

20.A method according to claim 16, a use according to claim 17 or a method 15 according to claim 18 wherein the microbial infection is a viral infection such as cold sores and other herpes simplex infections.