WO2014137231A2

WO2014137231A2 - Totarol extract formulations and uses thereof

Info

Publication number: WO2014137231A2
Application number: PCT/NZ2014/000031
Authority: WO
Inventors: Douglas Anthony Mende; Bernard KIMBLE; William Grayson
Original assignee: T2G Biotechnology Limited
Priority date: 2013-03-07
Filing date: 2014-03-07
Publication date: 2014-09-12
Also published as: WO2014137231A3

Abstract

The invention provides an aqueous totarol extract formulation. More specifically, provided herein is an aqueous based totarol extract formulation that has utility in treating human and/or animal infections such as mastitis and metritis; wounds and which formulations can be used in sprays, shampoos, sanitizers and the like.

Description

Totaroi Extract Formulations and Uses Thereof

Technical Field

Provided herein is an aqueous totaroi extract formulation. More specifically, provided herein is an aqueous based totaroi extract formulation that has utility in treating human and/or animal infections such as mastitis, metritis and wounds.

Background

Totaroi is a terpene and a natural phenol having the structure

(4bS,8aS)-1-isopropyl-4b,8,8-trimethyl-4b,5,6,7,8,8a,9,10-octahydrophenanthren-2-ol; also known as

(4bS)-fraA7s-8,8-Trimethyl-4b,5,6,7,8,8a,9,10-octahydro-1-isopropylphenanthren-2-ol

Totaroi is a naturally produced phenolic diterpene that is bioactive as (+)- totaroi. Totaroi extract was first isolated from the heartwood of Podocarpus totara, a yew tree found in New Zealand. Podocarpus totara was investigated for unique molecules because of the tree's increased resistance to rotting. Studies have shown that (+)-totarol extract has unique antimicrobial and therapeutic properties. Consequently, (+)-totarol extract is a promising candidate for treating infection.

New Zealand patent NZ 530834 describes a method of extracting totaroi from suitable plant material using a super critical fluid extraction technique. The contents of patent NZ 530834 is hereby incorporated in its entirety. Totarol extract is known to be difficult to formulate in a stable micro-emulsion. Previous attempts to formulate totarol extract have given unstable

suspensions that settle out or give rise to an interface or phase layer between a solvent and aqueous layer. The efficacy of a totarol formulation is dependent on a stable emulsion/surfactant system to make totarol extract available to the infected site. A stable micro-emulsion is necessary to ensure that the activity of the totarol extract in a formulation is evenly dispersed and therefore effective across the entire formulation. It is an object of the present invention to provide a stable aqueous totarol extract formulation or to at least provide the public with a useful choice.

Summary of Invention

In a first aspect the present invention provides an aqueous based totarol extract formulation comprising:

(a) at least one emollient from 0.1-20% w/w;

(b) totarol extract from 0.0005-20% w/w;

(c) at least one surfactant from 0.3-40.0% w/w;

(d) at least one GRAS solvent from 0.3-35.0% w/w; and

(e) water q.s. 100.

In a second aspect the present invention provides an aqueous based totarol extract formulation comprising:

(a) at least one emollient from 0.1 -5.0% w/w;

(b) totarol extract from 0.0005-15% w/w;

(c) at least one surfactant from 0.3-1 .0% w/w;

(d) at least one GRAS solvent from 0.3-1.0% w/w; and

(e) water q.s. 100. In a third aspect the present invention provides an aqueous based totarol extract formulation comprising:

a) at least one emollient from 0.1-5.0% w/w;

b) totarol extract from 0.0005-15% w/w;

c) at least one surfactant from 0.3-1 .0% w/w; d) at least one GRAS solvent from 0.3- .0% w/w; and e) Grapefruit phytochemical extract (GFX) from 0.01 to 0.05%;

f) a GFX carrier from 0.1 to 0.5%; and

g) water q.s. 100.

In a fourth aspect of the invention, there is provided a method of preventing or treating mastitis in a mammalian subject in need thereof by applying an effective amount of a formulation as defined above externally or internally to a mammary gland of the subject.

In a fifth aspect there is provided a method of preventing or treating metritis or endometritis in a mammalian subject in need thereof by applying an effective amount of a formulation as defined above externally or internally to the uterus of the subject.

In a sixth aspect there is provided a method of preventing or treating a wound infection in a subject in need thereof by applying an effective amount of a formulation as defined above topically to the wound of the subject. In a seventh aspect there is provided a method of healing wounds in a subject in need thereof by applying an effective amount of a formulation as defined above topically to the wound of the subject.

The foregoing summary broadly describes the features and technical advantages of certain embodiments of the present invention. Further technical advantages will be described in the detailed description of the invention that follows. Novel features which are believed to be characteristic of the invention will be better understood from the detailed description of the invention when considered in connection with any accompanying examples. However, the examples provided herein are intended to help illustrate the invention or assist with developing an understanding of the invention, and are not intended to be definitions of the invention's scope. DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to provide definitions of certain terms to be used herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

Totarol extract' - means a totarol containing extract extracted or derived from a number of species, including but not limited to Podocarpus, Dacrycarpus, Cupressus, Rosmarinus and Juniperus species, especially Podocarpus

Totara. The Totarol extract can be extracted by using the method described in NZ patent 530834 the contents of which is hereby incorporated in its entirety. 'GRAS solvent' - means a solvent that is Generally Recognised as Safe by the Food and Drug Administration of the United States of America.

The term "mammalian subject" means a human or animal mammal such as a cow, horse, buffalo, goat, dog, cat or the like.

The term "subject" means a human or animal subject that includes mammals, birds and reptiles.

The term "effective amount" means an amount of a formulation as defined in the specification to prevent, diminish or alleviate at least one symptom associated or caused by the state, disorder or disease being treated or prevented.

The term "treating" means that the application of the formulation to diminish alleviate one or several symptoms of a disorder or disease or a complete eradication of a state, disorder or disease.

In a first aspect the present invention provides an aqueous based totarol extract formulation comprising: a) at least one emollient from 0.1-20% w/w;

b) totarol extract from 0.0005-20% w/w;

c) at least one surfactant from 0.3-40.0% w/w;

d) at least one GRAS solvent from 0.3-35.0% w/w; and

e) water q.s. 100.

In one embodiment the at least one emollient is selected from but not limited to fatty esters, such as isopropyl myristate isopropyl palmitate; vegetable oils such as corn oil, soya oil and rice bran oil; terpenes such as d-limonene and pine oil; phenolic essential oils such as eugenol; and benzoic acid esters.

In one embodiment the at least one emollient is selected from isopropyl myristate. In one embodiment the at least one surfactant is selected from but not limited to a non-ionic surfactant. The non-ionic surfactant isselected from ethoxylated oleyl alcohol, ethoxylated castor oil, ethoxylated fatty alcohol, ethylene oxide propylene oxide adducts and alkyl polyglucosides; or combinations thereof. In a further embodiment the at least one surfactant is selected from but not limited to an anionic surfactant, such as calcium dodecyl benzenesulphonate, sodium dodecylbenzene sulphonate, alkane sulphonate, secondary alkyl sulphonate, alkyl sulphate and fatty acid soaps; or combinations thereof. In one embodiment the at least one surfactant includes a combination of an anionic surfactant and a non-ionic surfactant.

In a further embodiment the at least one surfactant includes both alkane sulphonate and ethoxylated oleyl alcohol.

In one embodiment the at least one GRAS solvent is selected from but not limited to alcohols, such as ethanol, isopropyl alcohol, butanol, glycerol and the like, from carboxylic acids such as propanoic acid and the like, from glycols, such as monopropylene glycol and hexylene glycol and the like; and combinations thereof.

In one embodiment the formulation further includes one or more antioxidants, such as but not limited to DL alpha tocopherol and octyl gallate.

In one embodiment the formulation further includes one or more thickening agents, such as but not limited to xanthan gum. In one embodiment the formulation further includes one or more

preservatives, such as but not limited to octyl gallate and phytic acid.

In one embodiment the formulation further includes one or more pH adjusters, such as but not limited to sodium tripolyphospahte.

In one embodiment the formulation has a pH of between 5.5 to 7.5. In one embodiment the formulation further includes glycerin. In one embodiment the formulation comprises:

Ingredients %w/w

Totarol Extract 16.70%

Isopropyl myristate (Emollient) 16.70%

Ethoxylated oleyl alcohol (Surfactant) 6.60%

Alkane sulphonate (Surfactant) 30.00%

Monopropylene glycol (Solvent) 30.00%

Water (Sterile) 100.00% In a second aspect the present invention provides an aqueous based totarol extract formulation comprising:

a) at least one emollient from 0.1 -5.0% w/w;

b) totarol extract from 0.0005-15% w/w;

c) at least one surfactant from 0.3-1.0% w/w; d) at least one GRAS solvent from 0.3-1.0% w/w; and e) Water q.s. 100.

In one embodiment the at least one emollient is selected from fatty esters, such as isopropyl myristate, isopropyl palmitate; vegetable oils such as corn oil, soya oil and rice bran oil; terpenes such as d-limonene and pine oil;

phenolic essential oils such as eugenol; and benzoic acid esters.

In one embodiment the at least one emollient is selected from isopropyl myristate.

In one embodiment the at least one surfactant is selected from a non-ionic surfactant. The non-ionic surfactant is selected from alkane sulphonate, ethoxylated oleyl alcohol, ethoxylated castor oil, ethoxylated fatty alcohol, ethylene oxide propylene oxide adducts and alkyl polyglucosides; or combinations thereof.

In a further embodiment the at least one surfactant is selected from an anionic surfactant, such as calcium dodecyl benzenesulphonate, sodium

dodecylbenzene sulphonate and fatty acid soaps; or combinations thereof.

In one embodiment the at least one surfactant includes a combination of an anionic surfactant and a non-ionic surfactant. In a further embodiment the at least one surfactant includes both alkane sulphonate and ethoxylated oleyl alcohol.

In one embodiment the at least one GRAS solvent is selected from alcohols, such as ethanol, isopropyl alcohol, butanol, glycerol and the like, from carboxylic acids such as propanoic acid and the like, from glycols, such as monopropylene glycol and hexylene glycol and the like; and combinations thereof. In one embodiment the formulation further includes one or more antioxidants, such as but not limited to DL alpha tocopherol and octyl gallate.

In one embodiment the formulation further includes one or more thickening agents, such as but not limited to xanthan gum.

In one embodiment the formulation further includes one or more

preservatives, such as but not limited to octyl gallate and phytic acid. In one embodiment the formulation further includes one or more pH adjusters, such as but not limited to sodium tripolyphosphate.

In one embodiment the formulation has a pH of between 5.5 to 7.5. In one embodiment the formulation further includes glycerin.

In one embodiment the formulation comprises:

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Water (Sterile) 98.8%

In another embodiment the formulation comprises:

Ingredients %w/w

Totarol Extract 10.0%

Isopropyl myristate (Emollient) 10.0%

Ethoxylated oleyl alcohol (Surfactant) 4.0%

Alkane sulphonate (Surfactant) 8.0%

Monopropylene glycol (Solvent) 18.0%

Water (Sterile) 40.0% In another embodiment the formulation comprises:

Ingredient %w/w

Totarol Extract 1.0%

Isopropyl myristate (Emollient) 1.0%

Ethoxylated oleyl alcohol (Surfactant) 1.8%

Alkane sulphonate (Surfactant) 1 .8%

Monopropylene glycol (Solvent) 1 .8%

Water (Sterile) 92.6%

In another embodiment the formulation comprises:

Ingredient %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Sodium tripolyphosphate (pH adjuster) 0.15%

Xanthan gum (Thickening Agent) 0.5%

Water (Sterile and filtered) 98.15%

In another embodiment the formulation comprises:

Ingredient %w/w

Totarol Extract 0.5% Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.2%

Alkane sulphonate (Surfactant) 0.87%

Monopropylene glycol (Solvent) 0.87%

DL alpha tocopherol (Antioxidant) 0.05% Xanthan gum (Thickening Agent) 0.1 %

Water (Sterile and filtered) 96.99% In another embodiment the formulation comprises:

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Xanthan gum (Thickening Agent) 0.5%

Water (Sterile and filtered) 98.3%

In another embodiment the formulation comprises:

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Octyl gallate (Antioxidant) 0.1 %

Water (Sterile and filtered) 98.7%

In a third aspect the present invention provides an aqueous based totarol extract formulation comprising:

a) at least one emollient from 0.1 -5.0%) w/w;

b) totarol extract from 0.0005-15% w/w;

c) at least one surfactant from 0.3-1.0% w/w;

d) at least one GRAS solvent from 0.3-1.0% w/w; and

e) Grapefruit phytochemical extract (GFX) from 0.01 to 0.05% w/w; f) a GFX carrier from 0.1 to 0.5% w/w; and

g) water q.s. 100.

preservatives, such as but not limited to octyl gallate and phytic acid.

In one embodiment the formulation further includes one or more pH adjusters, such as but not limited to sodium tripolyphosphate.

In one embodiment the formulation has a pH of between 4.0 to 7.5.

In one embodiment the GFX carrier is cyclodextrin.

In one embodiment the formulation further includes glycerin.

In one embodiment the formulation comprises

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Xanthan gum (Thickening Agent) 0.5%

Sodium tripolyphosphate (pH adjuster) 0.015%

Grapefruit phytochemical extract (GFX) 0.015%

Cyclodextrin (GFX carrier) 0.15%

Water (Sterile and filtered) 98.12%

In one embodiment the formulation comprises:

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Octyl gallate (Antioxidant) 0.1 %

Xanthan gum (Thickening Agent) 0.5% Sodium tripolyphosphate (pH adjuster) 0.015%

Grapefruit phytochemical extract (GFX) 0.015%

Cyclodextrin (GFX carrier) 0.15%

Water (Sterile and filtered) 98.02%

In a sixth aspect there is provided a method of preventing or treating a wound infection in a subject in need thereof by applying an effective amount of a formulation as defined above topically to the wound of the subject. In a seventh aspect there is provided a method of healing a wound in a subject in need thereof by applying an effective amount of a formulation as defined above topically to the wound of the subject.

The use of a formulation as defined above in any one of the following products:

hair care products;

mastitis, metritis and endometritis products;

cosmetics;

personal hygiene products;

sanitation products; and

spray products for wounds, mastitis or infections. In a ninth aspect there is provided a mastitis teat spray comprising formulation as defined above.

In a tenth aspect there is provided a mastitis teat spray comprising the formulation:

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Water (Sterile) 98.8%

The following Examples describe the preparation of specific formulations that have been prepared according to the present invention.

Formulation concentrate - Preparation Example 1

The formulation concentrate was prepared according to the following process: A clean stainless steel mixing vessel fitted with a heating jacket was used for manufacture of 100 gms of a concentrate formulation. The isopropyl myristate (16.70 gms) was added to the mixing vessel and heated to 60 degrees Celsius with continual stirring. The powdered totarol (16.70 gms was slowly added while mixing. The totarol could not be added rapidly otherwise the totarol would form lumps. Once all the totarol had dissolved to a clear dark brown liquid, the ethoxylated alcohol (6.60 gms) was added. The mixing was continued and the alkane sulphonate (30.00 gms) was added to the vessel, followed by the monopropylene glycol (30.00 gms). The mixture was mixed until all the alkane sulphonate had dissolved. The resulting mixture was continuously stirred while the mixture was allowed to cool to room

temperature. The cooled concentrate mixture was then packed into clean polyethylene containers that were chosen depending on the mode of dispensing that was required. Totarol Concentrate Ingredients %w/w

Totarol Extract 16.70%

Isopropyl myristate (Emollient) 16.70%

Ethoxylated oleyl alcohol (Surfactant) 6.60%

Alkane sulphonate (Surfactant) 30.00%

Monopropylene glycol (Solvent) 30.00%

Water (Sterile) 100.00%

Formulation Example 1

The following Formulation was prepared according to the following process:

To a clean sanitized mixer 12 grams of the concentrate formulation obtained from Preparation Example 1 was then combined with about 20% of 988 ml of distilled or deionized water in a glass beaker and stirred until the totarol was completely dissolved. The mixture was then stirred continuously as the remaining 80% of the 988 mis of water was added and stirred for 15 mins to give 1000 mis of Formulation Example 1 containing 0.2% totarol. The resulting formulation was then packed in suitable sterile packaging. The resulting formulation was clear in appearance and then changed to an opal liquid upon standing for 24 hours at room temperature. The resulting formulation had a pH of 4.9 ± 0.05, viscosity at 20°C of 20cP @ 20 RPM using S1 and a specific gravity at 25 °C of 1.0003. The stability of the resulting formulation was tested in stretch blow molded PET bottles and glass vials. The following

observations were noted. After 8 weeks of storage at 40 °C some limited creaming was noted. After 8 weeks of storage at 3 °C and at room

temperature, no creaming was observed. After 15 mins sterilization in glass at 1 15 °C to 120 °C the clear formulation became opaque and creamed far quicker than when un-sterilised, but complete separation did not occur even after 2 months at 40 °C post sterilization. These findings suggest that the formulation of Formulation Example 1 is quite stable.

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2% Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Water (Sterile) 98.8%

Formulation Example 2

The following Formulation was prepared according to the process described above for Formulation Example 1 to obtain a 10% totarol containing formulation.

To a clean sanitized mixer 60.00 grams of the concentrate formulation obtained from Preparation Example 1 was then combined with about 20 percent of the 40.00 gms of distilled or deionized water in a glass beaker and stirred until the totarol was completely dissolved. The mixture was then stirred continuously as the remaining 80% of the 40 gms of water was added and stirred for 15 mins to give 100 mis of Formulation Example 2 containing 10.0% totarol. The resulting formulation was then packed in suitable sterile packaging. The resulting formulation was a clear dark brown micro emulsion in appearance. The resulting formulation had a pH of 6.39± 0.05 and a specific gravity at 25 °C of 1 .0221. After 4 weeks of storage at 40 °C, 3 °C and at room temperature, the Formulation Example 2 appeared to be stable.

Ingredients %w/w

Totarol Extract 10.0%

Isopropyl myristate (Emollient) 10.0%

Ethoxylated oleyl alcohol (Surfactant) 4.0%

Alkane sulphonate (Surfactant) 18.0%

Monopropylene glycol (Solvent) 18.0%

Water (Sterile) 40.0%

Formulation Example 3

The following Formulation was prepared according to the process described above for Formulation Example 1 to obtain a 1.0% totarol containing formulation. To a clean sanitized mixer 6.00 grams of the concentrate formulation obtained from Preparation Example 1 was then combined with about 20 percent of the 94.00 gms of distilled or deionized water in a glass beaker and stirred until the totarol was completely dissolved. The mixture was then stirred continuously as the remaining 80% of the 94.00 gms of water was added and stirred for 15 mins to give 100 mis of Formulation Example 3 containing 1.0% totarol. The resulting formulation was then packed in suitable sterile packaging. The resulting formulation was a milky opalescent yellow liquid micro emulsion in appearance. The resulting formulation had a pH of 6.04 ± 0.05 and a specific gravity at 25 °C of 1.0025. After 4 weeks of storage at 40 °C, 3 °C and at room temperature, the Formulation Example 3 appears to be stable.

Ingredients %w/w

Totarol Extract 1.0%

Isopropyl myristate (Emollient) 1.0%

Ethoxylated oleyl alcohol (Surfactant) 1.8%

Alkane sulphonate (Surfactant) 1.8%

Monopropylene glycol (Solvent) 1.8%

Water (Sterile) 92.6%

Formulation Example 4

The following thickened Formulation of totarol extract was prepared according to the following process:

Water (98.2 gms) was added to a stainless steel vessel with a heating jacket and the temperature was raised to 90 °C. The mixer was started. The xanthan gum (1.0 gms) was added rapidly into the mixer vortex, allowing the gum to be drawn down quickly and dispersed in the solution. The mixing was continued until a lump free mix was obtained.

To a further clean stainless steel vessel fitted with a propeller stirrer 47.30 gms of water was added to the mixing vessel and stirred. To the water was added 1.2 gms of totarol concentrate prepared according to the Preparation Example 1. The water and totarol concentrate mix was stirred until a clear opal yellow liquid was achieved. The stirring was continued and 50.00 grams of xanthan gum/water mix prepared above was added, taking care not to introduce air. The mixing was continued until a clear smooth gel was produced. 1.5 mis of 1 % sodium tripolyphosphate freshly prepared (pH adjuster) was then added to the resulting mixture and stirred to raise the pH to between 6.5 and 7. The resulting formulation was then packed in suitable sterile packaging. The resulting formulation was an opalescent, translucent gel of pale yellowish colour. The resulting formulation had a pH of 6.6 ± 0.2. The viscosity at 20 °C was 2000 cP at 10 RPM using S2, 1200 cP at 20 RPM using S2 and 500 cP at 50 RPM using S2. All viscosity readings are ± 10%. The specific gravity was measured at 25 °C of 1.01 , which was approximate due to aeration. The following formulation was obtained.

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Sodium tripolyphosphate (pH adjuster) 0.15%

Xanthan gum (Thickening Agent) 0.5%

Water (Sterile and filtered) 98.15% Formulation Example 5

The following Formulation was prepared according to the process described above for Formulation Example 4, however the addition of 0.05 gms of DL alpha tocopherol (vitamin E) was added to the totarol concentrate before the water was added and proportionally more totarol extract was added to prepare a 0.5% w/w totarol extract final solution.

Ingredients %w/w

Totarol Extract 0.5%

Isopropyl myristate (Emollient) 0.5% Ethoxylated oleyl alcohol (Surfactant) 0.2%

Alkane sulphonate (Surfactant) 0.87%

Monopropylene glycol (Solvent) 0.87%

DL alpha tocopherol (Antioxidant) 0.05%

Xanthan gum (Thickening Agent) 0.1 %

Water (Sterile) 96.99%

pH 6.58

Formulation Example 6

The following Formulation was prepared according to the identical process described above for Formulation Example 4, however, the STPP was omitted from the mixture to give rise to a Formulation having the ingredients below.

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Xanthan gum (Thickening Agent) 0.5%

Water (Sterile and filtered) 98.3%

Formulation Example 7

The following Formulation was prepared according to the process of

Formulation 1 , except that the octyl gallate at 0.1 grams was added to the totarol concentrate before the water was added. Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36% Monopropylene glycol (Solvent) 0.36%

Octyl gallate (Antioxidant) 0.1 %

Water (Sterile and filtered) 98.7% Formulation Example 8

GFX is a citrus bioflavanoid extract that comprises compounds from citrus fruit skin that are known to provide some anti-inflammatory properties. The active components of GFX are 4H-1 -Benzopyran-4-one, 7-[[2-0-(6-deoxy- alpha-L-mannopyranosyl)- beta-D-glucopyranosyl]oxy]-2,3-dihydro-5-hydroxy- 2-(4-hydroxyphenyl) - CAS # 10236-47-2 and 4H-1 -Benzopyran-4-one, 7-[[6- 0-(6-deoxy- alpha-L-mannopyranosyl)- beta-D-glucopyranosyl]oxy]-2,3- dihydro-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl) - CAS # 520-26-3.

GFX is insoluble in most solvents and water, but is soluble in alkaline water solution. GFX will usually precipitate out of solution at neutral or acidic pH. The precipitation of GFX at neutral pH has been found to be reduced by adding cyclodextrin.

The following GFX and totarol extract Formulation was prepared according to the following process:

Filtered water (98.2 gms) was added to the a stainless steel vessel with a high shear mixer and the mixing started. The sodium tripolyphosphate (STPP) (0.15 gms was added and mixed, followed shortly by the GFX extract (0.15 gms). The mixture was stirred until all the GFX dissolved and a bright yellow liquid was obtained. The heat was then switched off and cyclodextrin (1.50 gms) was added and mixed until all was dissolved. The resulting mixture was allowed to cool to room temperature with mixing. To a further clean stainless steel vessel fitted with a conventional mixer 38.80 gms of filtered and sterile water was added to the mixing vessel and stirred. To the water was added 1.2 gms of totarol concentrate prepared according to the Preparation Example 1 . The water and totarol concentrate mix was stirred until a clear opal yellow liquid was achieved. The stirring was continued and GFX/cyclodextrin mix (10.00 gms) was added and mixed until a uniform mixture was obtained. The mixing was continued and 50.00 gms of xanthan gum mix, as prepared in Formulation Example 5 was added and mixed taking care not to introduce air. The resulting mixture was stirred until a clear smooth gel was produced. The resulting formulation was then packed in suitable sterile packaging. The resulting formulation was an opalescent, translucent gel of pale yellowish colour. The resulting formulation had a pH of 6.6 ± 0.2. The viscosity at 20 °C was 2000 cP at 10 RPM using S2, 1200 cP at 20 RPM using S2 and 500 cP at 50 RPM using S2. All viscosity readings are ± 10%. The specific gravity was measured at 25 °C of 1.01 , which was approximate due to aeration. The following formulation was obtained:

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Xanthan gum (Thickening Agent) 0.5%

Sodium tripolyphosphate (pH adjuster) 0.015%)

Grapefruit phytochemical extract (GFX) 0.015%

Cyclodextrin (GFX carrier) 0.15%

Water (Sterile and filtered) 98.12% Formulation Example 9

The following Formulation was prepared according to the following:

A concentrate of isopropyl myristate, totarol extract and monopropylene glycol was made up as described for Preparation Example 1 and then mixed with octyl gallate. Separately, the cyclodextrin and grapefruit extract mixture was prepared as described for Formulation Example 8. The xanthan gum mixture was prepared as described for Formulation Example 4. The totarol extract mix, grapefruit mix and xanthan gum mix were then blended with water to make the final formulation mixture. Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Octyl gallate (Antioxidant) 0.1 %

Xanthan gum (Thickening Agent) 0.5%

Sodium tripolyphosphate (pH adjuster) 0.015%

Grapefruit phytochemical extract (GFX) 0.015%

Cyclodextrin (GFX carrier) 0.15%

Water (Sterile and filtered) 98.02%

Formulation Example 10

The following Formulation was prepared according to the following:

A concentrate of isopropyl myristate, totarol extract and monopropylene glycol was made up as described for Preparation Example 1. The ascorbic acid was dissolved in the concentrate before diluting with water as described in

Formulation Example 1 , except that 2.5 times more totarol extract was used to achieve the 0.5% w/w concentration of totarol.

Ingredients %w/w

Totarol Extract 0.5%

Isopropyl myristate (Emollient) 0.5%

Ethoxylated oleyl alcohol (Surfactant) 0.2%

Alkane sulphonate (Surfactant) 0.9%

Monopropylene glycol (Solvent) 0.9%

Ascorbic acid (Antioxidant) 0.02%

Water (Sterile) 96.98%

It is to be understood that the Formulations prepared above can be sterilized prior to use. Such sterilization can be achieved by gamma irradiation or by autoclaving or the like. Microbiological Study - Example 11

A study was run to determine the minimum concentration of some of the Formulation Examples described above and known antibiotics that are required to inhibit the growth of Staphylococcus aureus and Streptococcus uberis in broth cultures. Both Staphylococcus aureus and Streptococcus uberis are recognized as being significant causes of metritis and mastitis in cows. Totarol is reported to be highly active against both gram positive and gram negative bacteria as described in New Zealand patent 530834. It is also one of a few extracts that is active against penicillin and methicillin resistant strains of Staphylococcus aureus. The study aimed to investigate the bioactivity of various totarol extract formulations (Formulation Example 1 , Formulation Example 5 and Formulation Example 10, against this bacterium at a range of concentrations in a MIC (Minimum Inhibitory Concentration) type experiment.

The totarol extract formulation samples and 2 antibiotics were serially diluted and the effects of each concentration on the viability and growth of

Staphylococcus aureus and Staphylococcus uberis determined after 16 hours and 24 hours of culturing.

Method: The Formulation Examples studied were serially diluted to give 1 1 different concentrations. The diluted samples were then incubated with each bacterial strain. The MIC was determined as the lowest concentration of each formulation that inhibited the growth of each bacteria as determined by measurement of OD650nm using a VersaMax 96 well plate reader.

The methodology for successfully growing the Streptococcus uberis was provided by the CLSI standard, Methods for Dilution Antimicrobial

Susceptibility Test for Bacteria that Grow Aerobically, Approved Standard, Ninth Edition, M07-A9, Vol. 32 No 2, January 2012, (Deviation #1).

Sample Preparation 1 . Duplicate 10% stock solutions of the Formulation Example samples were prepared in (a), Mueller Hinton II cation adjusted broth and (b) Mueller Hinton II cation adjusted broth plus 5% lysed horse blood (v/v).

2. The samples ranged from aqueous to very viscous, syrupy emulsions. For the latter samples the Gilson Microman 1 ml positive displacement pipette was used to carefully withdraw the required volume of sample. The stock solutions were mixed on the rocker roller mixer at room temperature for two hours and vortexed to mix thoroughly immediately prior to use. 3. Formulation Example 5 sterile sample. 0.5% totarol + 0.05% di-a- Tocopherol + 10% Xantham Gum. This was a pale white to yellow very opaque emulsion. Two 10% stock solutions were prepared by adding 0.8ml aliquots of the sample to 7.2ml of Mueller Hinton II cation broth and to 7.2ml of Mueller Hinton II cation adjusted broth plus 5% Lysed Horse Blood. The instructions for mixing the samples were followed as in Step 2 above.

4. Formulation Example 5 non-sterile sample. 0.5% totarol + 0.05% di-a- Tocopherol + 10% Xantham Gum. This was a pale white to yellow very opaque emulsion. Two 10% stock solutions were prepared by adding 0.8ml aliquots of the sample to 7.2ml of Mueller Hinton II cation broth and to 7.2ml of Mueller Hinton II cation adjusted broth plus 5% Lysed Horse Blood. The instructions for mixing the samples were followed as in Step 2 above.

5. Formulation Example 1 - non-sterile sample. 0.2% totarol. This was a pale white to yellow very opaque emulsion. Two 10% stock solutions were prepared by adding 0.8ml aliquots of the sample to 7.2ml of Mueller Hinton II cation broth and to 7.2ml of Mueller Hinton II cation adjusted broth plus 5% Lysed Horse Blood. The instructions for mixing the samples were followed as in Step 2 above..

6. Formulation Example 10 - sterile sample. 0.5% totarol + 0.002% Ascorbic acid. This was a pale white to yellow very opaque emulsion. Two 10% stock solutions were prepared by adding 0.8ml aliquots of the sample to 7.2ml of Mueller Hinton II cation broth and to 7.2ml of Mueller Hinton II cation adjusted broth plus 5% Lysed Horse Blood. The instructions for mixing the samples were followed as in Step 2 above.

7. Stock Solutions

The 10% stock solutions in the Mueller Hinton broth only for Experiment A were pale white, opaque solutions. The 10% stock solution in the Mueller Hinton II cation adjusted broth plus 5% Lysed Horse Blood for Experiment B was a bright red opaque solution. 8. Experimental Procedures

Characterisation of the Test System

1. Tryptic soy agar (TSA) base (Bacto Difco Media Cat. No. 236940, Becton Dickinson and company).

2. Mueller II broth cation -adjusted (BBL media Cat. No. 212332, Becton Dickinson and company).

3. Defibrinated sheep blood (Cat. No CP-SHPBLD-20 Fort Richard)

4. Lysed horse blood (Cat. No CP LYSED 100 Fort Richard

5. Staphylococcus aureus ((Mende biotech 5/9/2012 (063533983) Brogan).

6. Streptococcus uberis ((Mende biotech 5/9/2012 (063533983) Brogan).

7. Penecillin G sodium salt (Sigma Cat. No P3032)

8. Tetracycline hydrochloride (Sigma Cat. No T7660).

Methods

i.) Assay Conditions

Experiment A

1. The Staphylococcus aureus was cultured and tested against the samples and antibiotics.

2. BBLTM Mueller Hinton II Cation Adjusted broth powder was added to distilled water at 22g/L and stirred.

3. TSA agar and Mueller Hinton II cation adjusted broth solutions were boiled for 1 minute with stirring to completely dissolve the powder.

4. TSA agar and Mueller Hinton II cation adjusted broth media was autoclaved at 121 °C for 20 minutes. 5. When the TSA agar solution had cooled sufficiently (the bottle could be touched with the back of the hand for two seconds without discomfort) it was poured into a sterile 140mm petri dish and cooled to room temperature to allow solidification of the TSA agar gel.

6. The Staphylococcus aureus was streaked from -70°C cryostocks onto the appropriate agar gel and incubated for 24-48hrs at 37°C until visible single colonies had developed.

7. One colony was taken with a sterile, disposable inoculating loop and used to inoculate 40ml of Mueller Hinton II Cation Adjusted broth medium. The inoculated broth was incubated for approximately 66hrs at 37°C.

8. The broth culture was diluted with fresh, sterile Mueller Hinton II Cation Adjusted broth to an OD650nm of approximately 0.1 , equivalent to approx 105 CFU/ml prior to commencement of MIC testing. This was the inoculant that was used to inoculate the test wells in each plate. The inoculant was held at 4°C until required for plating.

9. The stock solutions of the test samples were prepared as described above.

10. Each of the two antibiotics (Penicillin and Tetracycline) was dissolved in Mueller Hinton II Cation Adjusted broth to give a final concentration of 100Mg/ml.

1 1. 96 well microtitre plates were then set up: 200μΙ of the appropriate sample stock solution (containing 10% of sample in the appropriate broth or antibiotic standard) was added to each well

12. To all other wells 100μΙ appropriate sterile broth was added.

13. Using a multichannel pipette 100μΙ of each sample in the relevant triplicate wells on the first column on each plate was sampled and transferred to the relevant wells in the second column on each plate and mixed thoroughly by pipetting up and down 5 times. Fresh tips were added to the pipette and 100μΙ of solution was transferred from the appropriate wells in column 2 to column 3, mixed thoroughly by pipetting up and down 5 times and the tips then discarded. This process was continued through to column 1 1 on each plate. From column 1 1 , 100μΙ of solution was discarded (leaving 100μΙ as the final volume in this well). This process resulted in serial doubling dilutions that ranged from 10% to 0.01 % for the totarol extract Formulation Example samples.

14. Step 12 was repeated for each of the two antibiotics. From column 1 1 , 10ΟμΙ of solution was discarded (leaving 10ΟμΙ as the final volume in this well). This process resulted in serial doubling dilutions that ranged from 100 g /ml to 0.098pg /ml for each antibiotic standard.

15. Some additional wells contained Mueller II broth only and were not inoculated with seed culture. These wells served as sterility controls and blank for each row. Additionally some wells contained the cells and served as the negative control.

16. 100μΙ of inoculant or Mueller Hinton II cation broth was added to each well. The addition of inoculant halved the totarol extract formulation concentration in each well giving final well concentrations that ranged from 5% to 0.005% for the totarol extract formulation samples and 50pg/ml to

0.049yg/ml for the antibiotic standards.

17. The plates were gently tapped to ensure even mixing of the inoculant with the sample solutions.

18. The OD650nm of each plate was read using a Versamax microtitre plate reader. This was recorded as the zero time reading.

19. The plates were then incubated for 16 hours at 37°C at which point the OD650nm of each well was read and recorded as the 16hr reading.

20. The microtitre plates were then returned to the incubator for a further 8 hours and the OD650nm was read and recorded as the 24hr reading.

21. Once the OD650nm of the plates had been read the wells containing the highest dilution of the test sample (lowest concentration of test extract) without a detectable change in OD650nm in comparison to the initial reading at time zero were noted.

Experiment B

1 . The Stretptococcus uberis was cultured and tested against the samples and the antibiotics See Deviation #1 .

2. BBLTM Mueller Hinton II Cation Adjusted broth powder was added to distilled water at 22g/L and stirred. 3. TSA agar and Mueller Hinton II cation adjusted broth solutions were boiled for 1 minute with stirring to completely dissolve the powder.

4. TSA agar and Mueller Hinton II cation adjusted broth media was autoclaved at 121 °C for 20 minutes.

5. When the TSA agar solution had cooled sufficiently (the bottle could be touched with the back of the hand for two seconds without discomfort) 50ml of defribrinated sheep blood was added to give a final concentration of 5%. The agar solution was mixed and poured into sterile 140mm petri dishes and cooled to room temperature to allow solidification of the TSA agar gel.

6. The Stretptococcus uberis was streaked from -70°C cryostocks onto the blood agar gel and incubated for 24-48hrs at 37°C until visible single colonies had developed.

7. One colony was taken with a sterile, disposable inoculating loop and used to inoculate 40ml of Mueller Hinton II cation adjusted broth containing 5% Lysed Horse Blood (v/v). The inoculated broth was incubated for approximately 66hrs at 37°C.

8. The broth culture was used without dilution (Deviation #2). The initial OD was 0.3 prior to the MIC testing. This was the inoculant which was used to inoculate the test wells in each plate. The inoculant was held at 4°C until required for plating.

9. The stock solutions of the test samples were prepared as described above.

10. Each of the two antibiotics (Penicillin G, Tetracycline) was dissolved in Tryptic soy broth to give a final concentration of 100μg/ml.

1 1. 96 well microtitre plates were then set up as indicated in the plate layout diagrams below: 200μΙ of the appropriate sample stock solution

(containing 10% of sample in the appropriate broth or antibiotic standard) was added to each well in column one on the relevant plates,.

12. To all other labelled wells 100μΙ appropriate sterile broth was added. 13. Using a multichannel pipette 100μΙ of each totarol sample in the relevant triplicate wells on the first column on each plate was sampled and transferred to the relevant wells in the second column on each plate and mixed thoroughly by pipetting up and down 5 times. Fresh tips were added to the pipette and 100μΙ of solution was transferred from the appropriate wells in column 2 to column 3, mixed thoroughly by pipetting up and down 5 times and the tips then discarded. This process was continued through to column 1 1 on each plate. From column 1 1 , 100μΙ of solution was discarded (leaving 10ΟμΙ as the final volume in this well). This process resulted in serial doubling dilutions that ranged from 10% to 0.01 % for the totarol Formulation Example samples.

14. Step 12 was repeated for each of the two antibiotics. From column 11

10ΟμΙ of solution was discarded (leaving 10ΟμΙ as the final volume in this well). This process resulted in serial doubling dilutions that ranged from 100 g /ml to 0.098pg /ml for each antibiotic standard.

15. Some wells contained Tryptic soy broth only and were not inoculated with seed culture. These wells served as sterility controls and blank for each row. Some wells contained the cells and served as the negative control.

16. 100μΙ of inoculant or Tryptic soy broth was added to each well as indicated in the plate layout below. The addition of inoculant halved the extract concentration in each well giving final well concentrations that ranged from 5% to 0.005% for samples and 50 g/ml to 0.049μg/ml for the antibiotic standards.

9. The plates were then incubated for 16 hours at 37°C at which point the OD650nm of each well was read and recorded as the 16hr reading.

20. The microtitre plates were then returned to the incubator for a further 8 hours and the OD650nm was read and recorded as the 24hr reading. A final reading was also taken after 40hrs (Deviation#3)

21. Due to the unknown growth pattern of the S. uberis, an additional reading was taken after 40hrs (Deviation #3)

22. Once the OD650nm of the plates had been read the wells containing the highest dilution of the test sample (lowest concentration of test extract) without a detectable change in OD650nm in comparison to the initial reading at time zero were noted. Results

Each of the samples was tested over a 1000-fold concentration for their effects on the viability and growth of both S.aureus and S.uberis that had been isolated from infected dairy cows.

Formulation Example 5 - Sterile

When this sample was tested for its effect on S.aureus, the MIC value was 0.156% after both 16 hours and 24 hours of culturing. This can be regarded as a moderate inhibitory effect when compared with the other test samples This sample had a higher MIC value for S. uberis indicating it was a somewhat poorer inhibitor of this bacterium. After both 16 and 24 hours the MIC was 1.25%.

Formulation Example 5 - non-sterile

Compared with Formulation Example 5 Sterile, the non-sterile sample was considerably more potent as an antibiotic for both S. aureus and S. uberis. With S. aureus, Formulation Example 5 Non-sterile completely inhibited the growth at a concentration of 0.005% after 16 hours and by 73.4% after 24 hours. This indicates that the MIC value at both times is less than 0.005%. That the effect was slightly less after 24 hours compared with 16 hours suggests that is was gradually losing some of its effectiveness with time. The efficacy and potency of this sample for inhibiting S. uberis was very similar. After 16 hours a 0.005% concentration was almost completely inhibitory (93.0% inhibition) and was 100% inhibitory after 24 hours. So the MIC values here are also less than 0.005%.

The difference between these effects and those of Formulation Example 5 Sterile suggests that the sterilization caused a loss of activity. As the sterilization was by autoclaving it is possible that the heat, pressure and/or steam causes an interaction among some of the key constituents of

Formulation Example 5 resulting in a loss of activity. Possibly an alternative method of sterilization, such as gamma irradiation would not result in this loss of activity. Formulation Example 10 - Sterile

This preparation was a reasonably strong inhibitor of the growth of S. aureus. The MIC value was 0.04% after both 16 and 24 hours of culturing. This constancy indicates that the activity is reasonably stable and lasts for a period of time, although there is a slight diminution of the potency. After 16 hours it was 75.6% at the MIC value which had decreased slightly to 54.1 % after 24 hours.

Formulation Example 10 Sterile was somewhat less effective when tested with S. uberis. The MIC value was 1 .25% at both measurement times.

However, at this concentration it was a strong antagonist. It reduced the cell concentration by 82.0% after 16 hours and was completely effective at 24 hours. The activity was obviously very stable.

Formulation Example 1 - non-sterile

After 16h of culturing of S. aureus Formulation Example 1 - Sterile was a reasonably strong inhibitor. The MIC was 0.04%. At this concentration and higher concentrations it either completely or nearly completely inhibited the growth. The effect was very similar after 24h with the MIC still being 0.04%. There was a slight loss of efficacy at higher concentrations but nonetheless it still appeared to retain most of its activity over 24hours.

The effects of this sample on the viability of S. uberis were rather similar. After 16h of culturing the MIC value was 0.04% with this bacterium also and it was completely inhibitory. After a further 8h the effect was identical - the MIC was 0.04% and there was complete inhibition at all higher concentrations. So this formulation was a significant inhibitor of this bacterium and it is a stable preparation as the efficacy appeared to be maintained over 24 hours.

Penicillin

After 16 hours, the MIC against S. aureus was 1 .56pg/ml. This had decreased to 0.78pg/ml after 24 hours. It was potently inhibitory at concentrations at or above 1 .56pg/ml at both times but quite suddenly lost most of its effect at 0.78pg/ml. When incubated with S. uberis the MIC for penicillin was 0.78pg/ml at both 16 hours and 24 hours. So to that extent, its effect was similar to that with S. aureus. However, at the higher concentrations its effect was much less with inhibition being only 30% or less.

Tetracycline

Compared with penicillin, tetracycline was a more potent antagonist of S. aureus. After both 6 hours and 24 hours, the MIC was less than 0.049pg/ml. Down to this concentration it was a potent inhibitor producing more than 70% reduction in the cell concentration.

Tetracycline had a MIC value of 0.195pg/ml after 16 hours and 0.39l g/ml after 24 hours when incubated with S. uberis. However, it was noticeable that at higher concentrations this antibiotic was not a particularly strong inhibitor of the growth of this bacterium. For example, at 50pg/ml the inhibition was 37.7% after 16 hours and 45% after 24 hours.

Example 12 a pilot study of the use of sterile totarol formulations in lactating dairy cows

A pilot study was carried out to determine whether the sterile totarol formulations were safe and effective to use when administered as an intra- mammary dose of one 10 ml syringe in each quarter for three consecutive 24- hour treatments.

The test formulations studied were autoclaved samples of Formulation Example 1 , Formulation Example 5 and Formulation Example 10. Prior to the study, a milk specimen was collected from each udder quarter of cows ID1 and ID 152 for somatic cell count (SCC) analysis and inhibitory substance (IS) analysis. The greater the SCC, the higher the level of inflammation in the tissue. So, SCC in milk is an important indicator of the inflammation status of the udder. Ideally, uninfected mammary glands should have somatic cell counts of 50,000 cells/ml or less. In practice, composite milk samples (from all four quarters) of less than 200,000 cells/ml are taken as indicating the absence of infection. As cell counts increase so does the chance that mastitis is present. The milk yield was recorded and the udder palpitated.

Three quarters of cow ID1 and all four quarters of cow ID 152 were eligible for inclusion in the study because the somatic cell count was less than 250,000 cells/ml and the palpitation score was less than or the same as 2 and the inhibitory substance analysis was less than 0.003 lU/ml.

The cows were milked twice daily and milk samples were collected, milk yield was recorded and udder palpatations were performed at each milking from immediately before test formulation administration for as long as considered necessary to monitor the effects of treatment.

Results with sterile Formulation Example 1

Cow ID 152 was treated with Formulation Example 1 in each quarter at the morning milking over three consecutive days. The effects of treatment were monitored at each milking and for an additional 2 mornings post treatment.

No clots were observed in the first strips of milk at any time after treatment.

Somatic cell count, milk yield and udder palpitation scores appeared to be unaffected by treatment. Inhibitory substances were not detected in any milk samples.

Results with sterile Formulation Example 5

Cow ID 1 was treated with Formulation Example 5 in three eligible quarters at the morning milking over three consecutive days. The effects of treatment were monitored at each milking and for an additional 10 mornings post treatment. Somatic cell count increased after treatment from a base-line of 10,000 cells/ml to a peak of 4.5 million cells/ml at 33 hours after the first treatment. There was a slow recovery of SCC over the next 8 days and the SCC remained elevated in the afternoon milkings (more than 400,000 cells/ml). Clots were notices in the first strips of milk from 33 hours after first treatment and at most milkings for the next four days. Milk yield and udder palpitation scores appeared to be unaffected by treatment. Inhibitory substances were not detected in any milk samples.

Results with sterile Formulation Example 10

Cow ID 152 was treated with Formulation Example 10 in each quarter at the morning milking over three consecutive days. The effects of treatment were monitored at each milking and for an additional 5 days post treatment. There was a small increase in somatic cell count after treatment from a base-line of 16,000 cells/ml to a peak of 227,000 cells/ml at 57 hours after the first treatment. SCC returned to low levels by 2 days after the last treatment. Small clots were noticed in the first strips of milk from 24 hours after first treatment and intermittently for the next four days. Milk yield and udder palpitation scores appeared to be unaffected by treatment. Inhibitory substances were not detected in any milk samples.

A test product is considered safe for use as an intra-mammary treatment if SCC was not greater than 500,000 cells/ml for the treated animal for the duration of the study. Using this definition of safety, Formulation Examples 1 and 10 would be considered safe for intra-mammary use and formulation Example 5 would not. The somatic cell count of cow ID 1 did not recover sufficiently after treatment with Formulation Example 5 for testing of further formulations in this cow. The measurement of inhibitory substances in milk samples after treatment of cows with the Formulation Examples was collected, not to determine the safety of the formulations, but to gather additional information from the study to aid with formulation development.

Applications for the Totarol Formulations of the present invention

It is to be appreciated that the totarol extract formulations of the present invention can be used to prepare a number of different solutions with differing totarol concentrations for different applications. While an intra-mammary application in cows is described above, it is also expected that mastitis could be treated in cows and an intra-uterine treatment for metritis in the likes of cows or other animals including humans could also be developed. For example it is envisaged that a mastitis teat spray using a formulation of the invention could be used to treat or prevent mastitis in a dairy herd.

Hair care products such as shampoos for animals and/or humans could also be developed using the formulations described above.

Wound care products such as sprays for preventing infections can also be developed using the formulations described above. The present invention and its embodiments have been described in detail. However, the scope of the present invention is not intended to be limited to the particular embodiments of the invention described in the specification. Various modifications, substitutions, and variations can be made to the disclosed material without departing from the spirit and/or essential characteristics of the present invention. Accordingly, one of ordinary skill in the art will readily appreciate from the disclosure that later modifications, substitutions, and/or variations performing substantially the same function or achieving substantially the same result as embodiments described herein may be utilized according to such related embodiments of the present invention. Thus, the following claims are intended to encompass within their scope modifications, substitutions, and variations to the embodiments of the invention disclosed herein.

Claims

Claims:

1 An aqueous based totarol extract formulation comprising:

(a) at least one emollient from 0.1 -20% w/w;

(b) totarol extract from 0.0005-20% w/w;

(c) at least one surfactant from 0.3-40.0% w/w;

(d) at least one GRAS solvent from 0.3-35.0% w/w; and

(e) water q.s. 100. 2 An aqueous based totarol extract formulation comprising:

(a) at least one emollient from 0.1 -5.0%» w/w;

(b) totarol extract from 0.0005-15% w/w;

(c) at least one surfactant from 0.3-1.0% w/w;

(d) at least one GRAS solvent from 0.3-1.0% w/w; and

(e) Water q.s. 100.

3 An aqueous based totarol extract formulation comprising:

(a) at least one emollient from 0.1 -5.0% w/w;

(b) totarol extract from 0.0005-15% w/w;

(c) at least one surfactant from 0.3-1.0% w/w;

(d) at least one GRAS solvent from 0.3-1.0% w/w; and

(e) Grapefruit phytochemical extract (GFX) from 0.01 to 0.05%;

(f) a GFX carrier from 0.1 to 0.5%; and

(g) water q.s. 100.

4 The formulation as claimed in any one of claims 1 , 2 or 3 wherein the at least one emollient is selected from fatty esters, vegetable oils, terpenes phenolic essential oils such as eugenol; and benzoic acid esters. 5 The formulation as claimed in any one of claims 1 to 4 wherein the at least one emollient is selected from isopropyl myristate, isopropyl palmitate; corn oil, soya oil, rice bran oil, d-limonene, pine oil, eugenol and mixtures thereof. 6 The formulation as claimed in any one of claims 1 to 5 wherein the at least one surfactant is a non-ionic surfactant.

7 The formulation as claimed in claim 6 wherein the non-ionic surfactant is selected from ethoxylated oleyl alcohol, sorbitan laurate, glyceryl caprylate, ethoxylated castor oil, ethoxylated fatty alcohol, ethylene oxide propylene oxide adducts and alkyl polyglucosides; or combinations thereof.

8 The formulation as claimed in any one of claims 1 to 5 wherein the at least one surfactant is an anionic surfactant.

9 The formulation as claimed in claim 8 wherein the anionic surfactant is selected from calcium dodecyl benzenesulphonate, sodium dodecylbenzene sulphonate, alkane sulphonate, secondary alkyl sulphonate, alkyl sulphate and fatty acid soaps; or combinations thereof.

10 The formulation as claimed in any one of claims 1 to 9 wherein the at least one surfactant includes a combination of an anionic surfactant and a non-ionic surfactant.

1 1 The formulation as claimed in claim 10 wherein the at least one surfactant is a combination of alkane sulphonate and ethoxylated oleyl alcohol. 12 The formulation as claimed in any one of claims 1 to 1 1 wherein the at least one GRAS solvent is selected from alcohols, carboxylic acids, glycols, and combinations thereof.

13 The formulation as claimed in claim 12 wherein the GRAS solvent is selected from ethanol, isopropyl alcohol, butanol, glycerol, propanoic acid, monopropylene glycol, and combinations thereof.

14 The formulation as claimed in claim 12 or claim 13 wherein the GRAS solvent is monoproylene glycol. 15 The formulation as claimed in any one of claims 1 to 14 wherein the formulation further includes one or more antioxidants. 16 The formulation as claimed in claim 15 wherein the one or more antioxidants are selected from DL alpha tocopherol, lecithin and octyl gallate or combinations thereof.

17 The formulation as claimed any one of claims 1 to 16 wherein the formulation further includes one or more thickening agents.

18 The formulation as claimed in claim 17 wherein the thickening agent is xanthan gum. 19 The formulation as claimed in any one of claims 1 to 18 wherein the' formulation further includes one or more preservatives.

20 The formulation as claimed in claim 19 wherein the one or more preservatives are selected from octyl gallate and phytic acid.

21 The formulation as claimed in any one of claims 1 to 20 wherein the formulation further includes one or more pH adjusters.

22 The formulation as claimed in claim 21 wherein the pH adjuster is sodium tripolyphospahte.

23 The formulation as claimed in any one of claims 1 to 22 wherein the formulation has a pH of between 5.5 to 7.5. 24 The formulation as claimed in any one of claims 1 to 23 wherein the formulation further includes glycerin.

25 The formulation as claimed in any one of claims 1 to 24 comprising: Ingredients %w/w

Totarol Extract 16.70%

Isopropyl myristate (Emollient) 16.70%

Ethoxylated oleyl alcohol (Surfactant) 6.60%

Alkane sulphonate (Surfactant) 30.00%

Monopropylene glycol (Solvent) 30.00%

Water (Sterile) 100.00%

26 The formulation as claimed in any one of claims 1 to 25 comprising

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Water (Sterile) 98.8%

27 The formulation as claimed in any one of claims 1 to 25 comprising comprises:

Ingredients %w/w

Totarol Extract 10.0%

Isopropyl myristate (Emollient) 10.0%

Ethoxylated oleyl alcohol (Surfactant) 4.0%

Alkane sulphonate (Surfactant) 18.0%

Monopropylene glycol (Solvent) 18.0%

Water (Sterile) 40.0%

28 The formulation as claimed in any one of claims 1 to 25 comprising: comprises:

Ingredient %w/w

Totarol Extract 1 .0% Isopropyl myristate (Emollient) 1 .0%

Ethoxylated oleyl alcohol (Surfactant) 1.8%

Alkane sulphonate (Surfactant) 1.8%

Monopropylene glycol (Solvent) 1 .8%

Water (Sterile) 92.6%

The formulation as claimed in any one of claims 1 to 25 comprising:

Inqredient %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Sodium tripolyphosphate (pH adjuster) 0.15%

Xanthan gum (Thickening Agent) 0.5%

Water (Sterile and filtered) 98. 5%

The formulation as claimed in any one of claims 1 to 25 comprising: Inqredient %w/w

Totarol Extract 0.5%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.2%

Alkane sulphonate (Surfactant) 0.87%

Monopropylene glycol (Solvent) 0.87%

DL alpha tocopherol (Antioxidant) 0.05%

Xanthan gum (Thickening Agent) 0.1 %

Water (Sterile and filtered) 96.99%

The formulation as claimed in any one of claims 1 to 25 comprising

Totarol Extract

Isopropyl myristate (Emollient)

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Xanthan gum (Thickening Agent) 0.5%

Water (Sterile and filtered) 98.3%

32 The formulation as claimed in any one of claims 1 to 25 comprising:

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Octyl gallate (Antioxidant) 0.1 %

Water (Sterile and filtered) 98.7%

33 A method of preventing or treating mastitis in a mammalian subject in need thereof by applying an effective amount of a formulation as defined above in any one of claims 1 to 32 externally or internally to a mammary gland of the subject.

34 A method of preventing or treating metritis or endometritis in a mammalian subject in need thereof by applying an effective amount of a formulation as defined in any one of claims 1 to 32 externally or internally to the uterus of the subject.

35 A method of preventing or treating a wound infection in a subject in need thereof by applying an effective amount of a formulation as defined in any one of claims 1 to 32 topically to the wound of the subject.

36 A method of healing a wound in a subject in need thereof by applying an effective amount of a formulation as defined in any one of claims 1 to 32 topically to the wound of the subject. 37 A mastitis teat spray comprising a formulation as defined in any one of claims 1 to 32.

38 A mastitis teat spray comprising the formulation:

Ingredients %w/w

Totarol Extract 0.2%

Isopropyl myristate (Emollient) 0.2%

Ethoxylated oleyl alcohol (Surfactant) 0.08%

Alkane sulphonate (Surfactant) 0.36%

Monopropylene glycol (Solvent) 0.36%

Water (Sterile) 98.8%

39 The use of a formulation as claimed in any one of claims 1 to 32 in any one of the following products:

hair care products;

mastitis, metritis and endometritis products;

cosmetics;

personal hygiene products;

sanitation products; and

spray products for wounds, mastitis or infections.