AU742170B2 - Hydroxycitric acid concentrate and method of making - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT *e a Applicant(s): RENAISSANCE HERBS, INC.

VITTAL MALLYA SCIENTIFIC RESEARCH FOUNDATION Invention Title: HYDROXYCITRIC ACID CONCENTRATE AND METHOD OF MAKING The following statement is a full description of this invention, including the best method of performing it known to me/us: 1A The present application is a divisional of application no. 34129/95 filed 22 August 1995 as the Australian national phase of international application no.

PCT/US95/10707 which claims priority from US application no. 08/295281 filed 24 August 1994.

Background of the Invention Hydroxycitric acid, both free acid and lactone forms, is present in the fruit rind of Garcinia species Garcinia cambogia, Garcinia atroviridis, and Garcinia indica), which are commercially available in India.

SAs an inhibitor of the synthesis of fat and cholesterol, hydroxycitric acid has been shown to 0 significantly reduce the body weight and lower lipid 15 accumulation in rats. See, Sergio, Medical Hypothesis 27:39 (1988); and Sullivan, A.C. et al., Lipids 9:121 (1973); and Sullivan, A.C. et al., Lipids 9:129 (1973). Hydroxycitric acid is also the only known e anorectic agent found as a natural constituent of edible 20 foods consumed by humans.

Methods for the extraction and purification of hydroxycitric acid from Garcinia rind can be found in Lewis, Methods in Enzymology 13:613 (1967); and .Indian Patent No. 160753.

Summary of the Invention One aspect of this invention relates to a hydroxycitric acid concentrate prepared from the fruit rind of the Garcinia genus Garcinia cambogia, Garcinia atroviridis, and Garcinia indica). The concentrate comprises 23 to 54% (preferably, 32 to 48%; and particularly preferably, 36-45%) by weight free hydroxycitric acid, 6 to 20% (preferably, 10 to 18%; and particularly preferably, 13 to 16%) by weight lactone of hydroxycitric acid, 0.001 to 8% (preferably, 0.001 to 6%; and particularly preferably, 0.001 to by weight \\BRIS1\homeS\SherylM\Keep\Speci\P35311.doc 26/08/99 2 citric acid, and 32 to 70% (preferably, 35 to 55%; and particularly preferably, 38 to 50%) by weight water, wherein the free hydroxycitric acid, the lactone of hydroxycitric acid and the citric acid constitute 94 to 99% (preferably, 96 to 99%; and particularly preferably, 98 to 99%) by weight of total solutes dissolved in the water.

Another aspect of this invention relates to a process of enriching hydroxycitric acid from Garcinia 10 rind. The process comprises obtaining a salt-free .water extract of the Garcinia rind, loading the extract on to an anion exchange column for adsorption of o9 the hydroxycitric acid onto the anion exchange column, eluting the hydroxycitric acid from the anion exchange column with a Group IA metal hydroxide LiOH, NaOH, KOH, RbOH, CsOH or FrOH) for release of the hydroxycitric acid as a metal salt in a first solution, and loading the first solution on to a cation So exchange column for collection of the hydroxycitric acid 20 as a free acid in a second solution; wherein the extract is loaded at a capacity of 100 to 125% (more preferably, 105-115%) of the anion exchange column, and the first solution is loaded at a capacity of 50 to 90% (more preferably, 60-75%) of the cation exchange column.

The salt-free water extract used in the above process can be prepared by first extracting salted Garcinia rind and subsequently removing the salt with a water miscible organic solvent acetone or ethyl alcohol). The process may further comprise, after step reducing the volume of the second solution to form a concentrate and adding the concentrate to a food product a beverage or a snack bar).

Also within the scope of this invention is a food product, such as a beverage or a snack bar, which 3 comprises 0.17 to 23% (preferably, 0.35 to 12%) by weight free hydroxycitric acid, 0.08 to 7% (preferably, 0.15 to by weight lactone of hydroxycitric acid, and at least 0.0002% (up to a proper content, 2% by weight) by weight citric acid. Preferably, the hydroxycitric acid and its lactone are from Garcinia rind. In an embodiment, the food product further comprises 0.04 to 0.4% (preferably, 0.04 to 0.08%) by weight vitamin C and/or 0.8 to 22% by weight fiber. Note that 0.0002% by weight denotes the presence of at least a trace amount, which may not be detectable by the methods described herein.

The contents of free hydroxycitric acid, lactone of hydroxycitric acid, citric acid, and non-acid solutes 15 can be determined by the methods described in Example 4 below or equivalents thereof.

Throughout this specification and the claims, the words "comprise", "comprises" and "comprising" are used in 20. a non-exclusive sense, except where the context requires otherwise.

It will be clearly understood that, although a S" number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Other features and advantages of the present invention will be apparent from the following description of the preferred embodiments, and also from the appending claims.

3a Description of the Preferred Embodiments A preferred process of this invention for enriching hydroxycitric acid from Garcinia rind includes preparing a salt-free.water extract of Garcinia rind; loading the extract on to an anion exchange resin column for adsorption of hydroxycitrate ion on the anion resin and removal of nonionizing and nonacidic impurities in the extract, such as sugar, pectins, gum and color (which pass out unadsorbed); washing the anion column with water 10 to ensure purity of hydroxycitrate ion; adding a sodium hydroxide solution to the anion exchange resin column for release of the hydroxycitrate ion in the form of sodium hydroxycitrate salt in a solution; converting the solution of sodium hydroxycitrate salt to free oo o o *0 4 hydroxycitric acid by passing the solution through a cation exchange resin column; decoloring the hydroxycitric acid solution with activated charcoal; and, finally, concentrating the hydroxycitric acid solution to a predetermined concentration.

The salt-free water extract can be prepared from salt-free Garcinia rind by cross-current or countercurrent method. It can also be prepared from salted Garcinia rind by extracting the rind with water preferably in multiple steps (by cross-current or counter-current method), treating the extract with acetone to precipitate pectin, salt and other insoluble substances, and removing acetone by evaporation.

Alternatively, one can treat the water extract of salted 15 Garcinia obtained from cross-current or counter- current method with calcium hydroxide solution to precipitate the insoluble salt of calcium hydroxycitrate, dilute the precipitate with cold water, filter it to eliminate the salt and other impurities, treat the precipitate with 20 sulphuric acid to convert the calcium hydroxycitrate to calcium sulphate and hydroxycitric acid, and finally filter out the calcium sulphate precipitate. The saltfree water extract can optionally be prepared by passing the water extract of salted Garcinia rind obtained from cross-current or counter-current method through an anion exchange column for adsorption of the chloride ion on the column.

The salt-free water extract thus prepared is then subjected to an ion exchange purification process, i.e., anion exchange followed by cation exchange, as described above.

The anion exchange column is loaded at a capacity of 100 to 125% (more preferably, 105-115%) and the cation exchange column is loaded at a capacity of to 90% (more preferably, 60-75%). The capacity of 5 loading (expressed as a percentage) is defined as the ratio of the acid or salt content in the solution charged to an ion exchange column to the theoretical capacity of the column to bind the acid or to exchange salt, and can be controlled by the amount of acid or salt loaded on to the column.

Take Example 2, infra, for instance. The anion exchange resin as specified by the manufacturer has a capacity of 1.5 meq/ml of resin. Thus, 500 ml of the 10 anion resin has a theoretical capacity of: 500 ml x 1.5 meq/ml x 208 g/3 eq 52 g (Note that hydroxycitric acid has a molecular weight of 208 daltons and has 3 eq acid groups.) The actual amount of acid loaded was 65 g. Thus, the capacity of loading is 65 g/52 g, or 125%.

Similarly, the loading capacity for the cation exchange resin in Example 2, infra, is calculated to be 52.7%.

The solution collected from the cation exchange 20 column is usually further treated by charcoal and concentrated by vacuum evaporation to about 55% by weight free hydroxycitric acid. A typical hydroxycitric acid concentrate obtained by the process of this invention is an aqueous solution of hydroxycitric acid containing to 56% by weight total acids, of which 98 to 99% is total hydroxycitric acid (whether in the free acid or lactone form) and 1 to 2% is mostly citric acid. The concentrate also contains trace amounts of other soluble solids.

Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

6 The anion exchange resin used in both Examples 1 and 2 was from Ion Exchange India Ltd with the trade name of Indion 850. It was a macro porous cross-linked polystyrene matrix of 0.3 to 1.2 mm particle size and meq/ml total exchange capacity. The cation exchange resin used in both Examples 1 and 2 was also purchased from the same manufacturer and had the trade name of Indion 225H. It was a polystyrene DVB matrix of the same particle size and 2.0 meq/ml total exchange capacity.

1 0 Example 1 Water extraction of salted Garcinia rind by the procedure commonly referred to as counter current .9oo extraction was carried out in 3 vessels marked vessel 1 to vessel 3. For the first cycle of operation, garcinia P. 15 rind of 2 to 5 mm size was added to each vessel. In each vessel, 1.25 liters of 95 0 C water was used to contact the rind for one hour and then drain the liquid. In the second and subsequent cycles of operation, fresh hot water was added only to vessel 1 and fresh rind was added only to vessel 3. In the second and subsequent cycles of operation, vessels 2 and 1 received the extracted rind of vessels 3 and 2 of the previous cycle, respectively.

Spent rind of vessel 1 was discarded.

In the second and subsequent cycles, the extracts from vessels 1 and 2 were further extracted in vessels 2 and 3 for the next cycle. The extract from vessel 3 was the final product. After four cycles, all extracts reached steady compositions. On the fifth cycle, for an input of 750 g garcinia rind, the product obtained was 850 ml of liquid.

The final product obtained from vessel 3 was 850 ml liquid containing 158.16 g acid along with all the salt originally present in the rind. The total soluble solids was 41%. The extraction efficiency was 95.85%.

7 This extract was further concentrated to 60% soluble solid in vacuum evaporation, so that it could be subjected to acetone refining.

Acetone refining was carried out in a 3-liter flask fitted with a stirrer. Each step of the extraction consisted of mixing aqueous phase with acetone for minutes, then settling for 15 minutes and collecting the top layer. The lower layer was subjected to second and third extractions and finally discarded. More •1 0 specifically, the extraction flask was charged with S""liters of aqueous extract of Garcinia rind of approximately 60% soluble solids containing 149 g of total acids. It was extracted by using one liter of pure acetone and the first extract was separated from the S 15 lower aqueous residue layer containing pectins, gums and some unextracted hydroxycitric acid. The same lower layer is subjected to second extraction using 750 ml of acetone water mixture containing 16.7% water. The top layer was removed as the second extract and the bottom ooo 20 layer was again subjected to a third extraction using 450 ml of acetone-water mixture containing 16.7% water. The third extract was removed and pooled with the other extracts. The volume of the total extract was 2,286 ml and contained 140.36 g of acid. Acid recovery was 84.9%.

The extract was evaporated under vacuum to remove all acetone and some water.

The ion-exchange process was carried out in two PVC columns of inner diameter 10.5 cm and height 92.5 cm.

A stainless-steel grid of 100 mesh was held between two flanges at the base of the column and provided a support for the resin in the column. 4.4 liters of Indion 850 and 5.5 liters of Indion 225H resins are loaded in anion exchange column and the cation exchange column, respectively. The anion column, which had a capacity of 458 g, was charged with 507 g of hydroxycitric acid, 8 giving a loading capacity of 111%. On the other hand, the anion column, which had a capacity of 762.6 g, was charged with sodium salt made from 493 g of hydroxycitric acid, giving a loading capacity of More specifically, 1.6 liters of acetone refined Garcinia extract was diluted to 6.4 liters (containing 507 g) of acid was passed through the anion exchange column. The anion exchange resin was then washed with water to remove color and water insoluble materials such 10 as pectins, gums, and the like. Subsequently, 6 liters of sodium hydroxide solution containing 292 g of sodium hydroxide was passed through the anion exchange column.

The alkali converted the acid held on the anion exchange column into a water soluble salt, sodium hydroxycitrate, which was liberated. The anion exchange column was subsequently washed with 5 liters of water to release any salt remaining in the bed.

The sodium hydroxycitrate solution was then passed through the cation exchange column where the salt was converted to free hydroxycitric acid. The material coming out of the cation exchange column was the final product, 11 liters containing 479 g of acid. 1 liter of water was then passed through cation exchange column to collect any acid remaining on the bed.

The recovery of acid was 479 g starting from 507 g of acid in the crude acetone extract, giving an acid recovery yield of 94.4%.

Example 2 Garcinia rind was obtained in the salt-free state from the forest area of Sirsi District, South Karnataka.

The rind had 14% moisture and 19.2% hydroxycitric acid.

Extraction was carried out by three-stage batch process.

More specifically, 1 kg of rind was taken in a stainlesssteel vessel along with 3 liters of demineralized water 9 and heated. After boiling for 15 minutes and settling for 15 minutes, the liquor was drained out. Similarly, in the second and third stages of extraction, the spent rind was extracted with 1.5 liters of fresh demineralized water, each boiled for 15 minutes, settled for 15 minutes and the extraction liquor was collected.

1,500 ml of the salt free extract containing 65 g of hydroxycitric acid was passed slowly through 500 ml anion exchange resin column. The impurities came off as S 10 breakthrough. The resin was washed with 2-5 column volumes of water and collected along with the breakthrough. The amount of acids present in the breakthrough was 6.53 g. In other words, 58.47 g of hydroxycitric acid was held on to 500 ml of anion exchange column. The anion resin was washed with column volumes of water.

g of sodium hydroxide in 1,500 ml of water was then passed through the anion resin. The salt, sodium hydroxycitrate, was formed, releasing the hydroxycitrate ion from the resin. The resin was washed with 2-5 column volumes of water. The effluent from the anion exchange resin was then passed through 800 ml of cation exchange resin column. Here, Na ion was held up by releasing H+ ion from the resin to give free hydroxycitric acid, which was collected in a volume of 2,000 ml. 56.55 g of hydroxycitric acid was recovered, giving a recovery percentage of 96.6%.

Example 3 In this example, a method for removing salt, which is different from acetone refining, was performed.

200 ml of Garcinia water extract, containing 61.4 g of organic acids, was precipitated with 33.4 g of CaOH to get calcium hydroxycitrate. The precipitate was then diluted with about 300 ml of cold water and filtered 10 under vacuum. The wet precipitate obtained, on drying at 600C for 16 hours, gave 83.5 g of dry calcium hydroxycitrate. The calcium hydroxycitrate was converted to hydroxycitric acid and calcium sulphate by adding 369 ml of 2.5 N sulphuric acid. Calcium sulphate precipitate was removed by centrifugation at 8000 rpm for 15 minutes.

53 g of hydroxycitric acid was present in 355 ml of supernatant and the recovery was 87.6 150 ml of solution containing 22.4 g of 10 hydroxycitric acid was passed through 200 ml of anion exchange resin to saturate the column. The column was washed with demineralized water and 240 ml of 5% sodium hydroxide solution was passed through the column to get 800 ml of sodium hydroxycitrate solution. 800 ml of the above solution was passed through 400 ml of cation exchange resin. 1240 ml of solution containing 18.84 g of hydroxycitric acid was obtained. The overall recovery of 18.84 g of hydroxycitric acid from the cation *exchange column indicated a yield of 90.5%.

The above solution after charcoal treatment and concentration under vacuum at 72 0 C to 55% by weight of hydroxycitric acid gave a hydroxycitric acid concentrate which was stable for months.

Example 4 The composition of an exemplary hydroxycitric acid concentrate prepared from Garcinia rind by the process of this invention is shown below: 11 of Total Acids of the Concentrate Free HCA 68.58 37.53 Lactone of HCA 25.38 13.89 Citric Acid 5.90 3.23 Total 99.86 54.65 Note that the content of total acids of the concentrate was 54.72% by weight (not shown in the table), as determined by direct titration against 10 standard sodium hydroxide (0.1 N) using phenolphthalein as the indicator. Determineation of the content of total acids was required as it was used with the data of FA, LA and CA obtained by HPLC so as to acquire the composition of the concentrate.

15 In the above table, "HCA" is the abbreviation of hydroxycitric acid and refers to by weight." FA, LA, and CA were determined by the following HPLC system: C18 Supelcosil column; waters HPLC system comprising waters 510 HPLC pump, waters 484 Tunable 20 absorbance detector and Maxima 820 Chromatography Software. Instrument conditions are given below: Column: C18 supelcosil with a guard column Mobile phase: 0.5% THF in 2.5 pH water; pH was adjusted by using H 2

SO

4 Flow rate: 1.0 ml/min.

Run time: 10 min.

Injection volume: 10 ul Detector 1: 210 nm Concentrations of the samples were maintained at 1 mg/ml.

The content of non-acid solutes solutes other than FA, LA and CA) in a concentrate can be derived from brix value by weight) and the total acid 12 content by weight) as quantitated by titration and HPLC. For example, the concentrate the composition of which is shown in the above table had a Brix value of 57.

Thus, its content of non-acid solutes was 2.35% by weight.

Brix (FA+LA+CA) 57-54.65 2.35 Traditionally, brix is defined as a measure of density or concentration of sugar solutions and the brix value equals percent of weight of sucrose in the solution 10 (at 17.5 0 Determinations can be made with a refractometer or a hydrometer (solutions must be deaerated). See Foods and Food Production Encyclopedia, Ed. Considine, D.M. et al., p. 418 (1982). In this invention, a refractometer (National Hand Held Refractometer No. A-0818) purchased from Thomas Scientific, Swedesboro, NJ was used.

S..It should be pointed out that LA content increases undesirably upon gradual removal of water from the free HCA-containing solution collected from the S* 20 cation ion exchange column.

Example Preparation of fiber snack bars and natural beverages from a hydroxycitric acid concentrate of this invention involves the steps of diluting the concentrate in water, adding supplements, blending, heating, and periodic agitating.

For both snack bars and beverages, it is desirable to pre-pasteurize the concentrate in a highly diluted ratio with purified water. For example, approximately 10 ml of the concentrate can be added to 12 fluid ounces. These figures vary depending upon the types of products sought, ranging from 1% to 25% for the beverage and 1%-40% for the snack bar. After the blending step, the temperature of the vessel of which the 13 preprocessing step is taking place is heated frequently using steam as in the case of beverage manufacturing or in low heat as in the snack bars. Before heat is applied 195 0 materials such as nutrients, antioxidants, vitamins and minerals can be added.

In the production of the beverage, after the supplementation of the desired additives and achievement of homogeneity, the liquid is pumped via stainless-steel pipeline travelling many hundreds of yards in a bottling 10 factory. Steam at high temperatures is applied from the outside to the pipes which in turn maintains the temperature of the liquid as it is moving. The pipes are strategically placed over the machinery that injects the :--liquid into bottles. This mechanism operates like a valve that is turned on and off. Simultaneously, as the liquid is being pumped, it is being agitated as the beverage is transported to the bottles.

For the fiber based dietary snack bar, the preprocessing follows the same directions as the beverage. Furthermore, the environment for the development of this product is in a industrial kitchen with the use of large cooking pots. The diluted hydroxycitric acid solution is blended with water, covered and heated, bringing it to a boil for about minutes. The bubbles in the boiling process provide the agitation.

The order of added ingredients for the bar is based on their solubility in water. Once again this is done at an average temperature after the cool down phase of preprocessing pasteurization. The first ingredients added are the ones that are most easily blended into a liquid solution. After all of the ingredients of the formula have been added one step at a time, the mixture is blended for 20 to 30 minutes with an industrial Hobart blender, or in a mixing vessel. The mixture is then 14 placed into an extruder that puts forth various sizes and shapes of snack bars onto a conveyor belt for further processing, if desired.

Other Embodiments From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various 0 usages and conditions. Thus, other embodiments are also within the claims.

For example, the hydroxycitric acid concentrate of this invention can be formulated with ginger extract or licorice extract in a liquid concentrate form.

S 15 Similarly, it can be used to make lozenges with hydroxycitric acid, herbal extracts, or a variety of nutrients and flavors.

What is claimed is: *e

Claims (24)

1. A hydroxycitric acid concentrate prepared from Garcinia rind, said concentrate comprising 23 to 54% by weight free hydroxycitric acid, 6 to 20% by weight lactone of hydroxycitric acid, 0.001 to 8% by weight citric acid, and 32 to 70% by weight water, wherein said free hydroxycitric acid, said lactone of hydroxycitric acid and said citric acid constitute 94 to 99% by weight of total solutes dissolved in said water. 1. 0 2. The hydroxycitric acid concentrate of claim 1 comprising 32 to 48% by weight free hydroxycitric acid, 10 to 18% by weight lactone of hydroxycitric acid, 0.001 to 6% by weight citric acid, and 35 to 55% by weight water, in which said free hydroxycitric acid, said 15 lactone of hydroxycitric acid and said citric acid constitute 96 to 99% by weight of total solutes dissolved in said water.

3. The hydroxycitric acid concentrate of claim 2 comprising 36-45% by weight free hydroxycitric acid, 13 20 to 16% by weight lactone of hydroxycitric acid, 0.001 to 3% by weight citric acid, and 38 to 50% by weight water, wherein said free hydroxycitric acid, said lactone of hydroxycitric acid and said citric acid constitute 98 to 99% by weight of total solutes dissolved in said water. 16

4. A process of enriching hydroxycitric acid from Garcinia rind comprising: obtaining a salt-free water extract of said Garcinia rind, loading said extract on to an anion exchange column for adsorption of said hydroxycitric acid onto said anion exchange column, eluting said hydroxycitric acid from said anion exchange column with a Group IA metal hydroxide for release of said hydroxycitric acid as a metal salt in a first solution, and loading said first solution on to a cation exchange column for collection of said hydroxycitric acid as a free acid in a second solution; wherein said extract is loaded at a capacity of 100 to 125% of said anion exchange column, and said first solution is loaded at a capacity of 50 to 90% of said cation exchange column.

5. The process of claim 4, wherein said water extract is loaded at a capacity of 105 to 115% of said anion exchange column.

6. The process of claim 4, wherein said first solution is loaded at a capacity of 60 to 75% of said cation exchange column.

7. The process of claim 5, wherein said first solution is loaded at a capacity of 60 to 75% of said cation exchange column.

8. The process of claim 4, wherein said Group IA metal hydroxide is NaOH or KOH.

9. The process of claim 4, wherein said salt-free water extract is prepared by first extracting salted 17 Garcinia rind and subsequently removing salt with a water miscible organic solvent. The process of claim 9, wherein said solvent is acetone or ethyl alcohol.

11. The process of claim 4, after step (4) further comprising reducing the volume of said second solution to form a hydroxycitric acid concentrate and adding said concentrate to a food product. C .i

12. The process of claim 11, wherein said food 0 product is a beverage.

13. The process of claim 11, wherein said food product is a snack bar.

14. A food product comprising 0.17 to 23% by weight free hydroxycitric acid, 0.08 to 7% by weight 15 lactone of hydroxycitric acid, and at least 0.0002% by weight citric acid.

15. The food product of claim 14 comprising 0.35 to 12% by weight free hydroxycitric acid, 0.15 to 4% by weight lactone of hydroxycitric acid, and at least 0.0002% by weight citric acid.

16. The food product of claim 14, further comprising 0.04 to 0.4% by weight vitamin C.

17. The food product of claim 15, further comprising 0.04 to 0.4% by weight vitamin C.

18. The food product of claim 16, further comprising 0.04 to 0.08% by weight vitamin C. 18

19. The food product of claim 17, further comprising 0.04 to 0.08% by weight vitamin C. The food product of comprising 0.8 to 22% by weight

21. The food product of comprising 0.8 to 22% by weight claim 14, fiber. claim 15, fiber. further further *r *e C C C

22. The food product is

23. The food product is

24. The food product is

25. The food product is 15 26. The food product is

27. The food product is food product a beverage. food product a beverage. food product a beverage. food product a snack bar. food product a snack bar. food product a snack bar. of claim 15, of claim 17, of claim 21, wherein said wherein said wherein said of claim 15, wherein said of claim 17, wherein said of claim 21, wherein said

28. A process for enriching hydroxycitric acid from with Garcinia rind substantially as hereinbefore described reference to the examples. Dated this 25th day of October 2001 RENAISSANCE HERBS, INC. and VITTAL MALLYA SCIENTIFIC RESEARCH FOUNDATION By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia