Title of Invention

METHOD FOR PREPARING HYDROXYCITRIC ACID COMPOSITIONS, DIETARY SUPPLEMENTS AND FOOD PRODUCTS COMPRISING THE SAME

Abstract

There is disclosed a method for manufacturing a hydroxycitric acid composition for reducing body weight, said method comprising formulating a composition by mixing calcium salt of hydroxycitric acid having approximately 14 - 26% by weight of calcium and at least one of the potassium salt and sodium salt of said hydroxycitric acid, in proportions 24-40% by weight and 14-24% by weight respectively, all calculated as a percentage of the total hydroxycitric acid content of said composition.

Full Text

-1A-Technical Field The present invention relates to method for manufacturing hydroxycitric acid compositions, dietary supplements and food products containing such compositions, and to the use of such compositions and products to reduce body weight in mammals. This application is divided out of Indian Patent Application No. 66CAL2OO1 whih is visional of 1223CAL98 Background of the Invention Hydroxycitric acid has been known for many years to be beneficial for the control and reduction of mammalian body weight. In particular, a specific stereoisomer of hydroxycitric acid, the (-)hydroxycitric acid isomer and derivatives thereof, is known to inhibit fatty acid and cholesterol synthesis and to function as a natural anoretic agent in mammals. The stereoisomers of hydroxycitric acid are related structurally to citric acid wherein a hydroxy group is substituted for one of four methylene hydrogens. Thus, there are four possible stereoisomers of hydroxycitric acid. Of these four stereoisomers, the (-)hydroxycitric acid isomer has been found to substantially inhibit fatty acid synthesis in biological systems in profused organs and intact mammals, and particularly in non-ruminant mammals. -1B- It has also been known that the particular stereoisomer of interest, in both free acid and lactone forms, is found in the rind of the fruits of Garcinia species, for example, Garcinia cambogxa, Garcinia arroviridis and Garcinia indica, which are native to the Indian subcontinent. The hydroxycitric acid component can be obtained by isolation from the fruit of Garcinia species using known procedures, for example Lewis, Y.S. "Methods in Enzymology" (J.M.Lowenstein.Ed., Vol.13, pg.613) (Academic Press, N.Y. 1969), and U.S. Patent No. 5,536,516. As an inhibitor of the synthesis of fatty acids and cholesterol, hydroxycitric acid has been shown to significantly reduce the body weight and lower lipid accumulation in - 2 - rats. Sec. e.g. Sergio. W.Medical Hypothesis 27:39(1998), Sullivan, A-C.et al., Lipids 9:121(1973), and Sullivan, A.C., et.al, Lipids 9:129(1973). However, in order to formulate the compositions containing hydroxycitric acid into dietary supplements and food products a number of desirable properties are sought. First, as most such supplements and food products are administered orally, the composition should have negligible odor and taste. Second, in order to increase the bioavailability of the hydroxycitric acid, the composition should be soluble in water. Solubility in water, as well as clarity in solution, are also important properties in many food and beverage applications. In addition, it is highly desirable that the composition be non-hygroscopic, in order to facilitate storage and formulation into dietary supplements and food products. Another desirable property is the hydroxycitric acid potency of the composition, where high potency levels are desirable. DISCLOSURE OF THE INVENTION The present invention provides method for manufacturing hydroxycitric acid compositions, food products made therefrom and methods for their use in reducing body weight. 2A Accordingly, the present invention provides a method for preparing a hydroxycitric acid composition for reducing body weight, wherein said method comprises mixing the calcium salt of hydroxycitric acid with at least one other salt of hydroxycitric acid selected from the potassium salt of hydroxycitric acid and the sodium salt of hydroxycitric acid, in a proportion so that the final composition comprises: a) 14 to 26% by weight of calcium, and, b) 24 to 40% by weight of potassium, or, 14 to 24% by weight of sodium, or a mixture of said potassium and sodium salts, calculated as a percentage of the total hydroxycitric acid content of said composition. The invention also provides a method for preparing a dietary supplement composition for reducing body weight containing hydroxycitric acid composition as stated above, wherein said. method comprises mixing the calcium salt of hydroxycitric acid, and at least one of the potassium salt of hydroxycitric acid or the sodium salt of hydroxycitric acid, wherein the said composition comprises at least 40% by weight of total hxdroxycitric acid. The invention further provides a method for preparing a food product for use in reducing body weight which comprises combining a prepared food product with a hydroxycitric acid composition as stated above, wherein said composition comprises 40% by weight of hydroxycitric acid together with 14 to 26% by weight of calcium, and 24 to 40% by weight of potassium or 14 to 24% by weight of sodium, or a mixture thereof, each calculated as a percentage of the total hydroxycitric acid content of said composition. -3- In another aspect of the invention, a method of manufacturing a composition for reducing body weight is provided which comprises at least approximately 40% by weight of total hydroxycitric acids which further comprises approximately 5 to 13% by weight of calcium, and approximately 9 to 20% by weight of potassium or approximately 5 to 12% by weight of sodium or a mixture thereof, calculated as a percentage of the total weight of said composition. Additional aspects of the invention include method of manufacturing dietary supplements and food products for use in reducing body weight which include the present compositions, and methods for reducing body weight by administering such compositions, dietary supplements and food products to mammals. - 4 - Detailed Description of tne lnvention The present invention provides method for manufacturing hydroxycitric acid compositions, dietary supplements and food products made therefrom and methods for their use in reducing body weight. In one aspect, the invention provides method for manufacturing a nydroxycitric acid composition for reducing body weight wherein the composition comprises approximately. 14 ro 16% by weight of calcium and approximately 24 to 40% by weigh of potassium or approximately 14 lo 24% by weight of sodium, or a mixture thereof, calculated as '¦* percentage of the total hydroxycitric acid content of said composition. Hydroxycitnc acrd has been known for years to be useful for inhibiting fatty acid synthesis. Crtraie is formed in the mitochondria by the citrate synthasc reaction. It is then metabolized via the citric acid cycle. Under certain metabolic conditions, some citrate is diverted to the cell cytosol where it is used for fatty acid synthesis, that is. for energy storage. The inhibition of fatty acid synthesis in biological systems by the use of bydroxycitric acid is believed to arise from the inhibition of the citrate cleavage enzyme cirrate-lyase by such compounds The cleavage of citrate is catalyzed by cinate-lyase and citrate is the major source of the acetyl group of acctyl coenzyme A which if utilized in the conversion of carbohydrates and various ammo acids to fat by non-ruminant mammals. Typically, hydroxycitric acid is utilized in the form of its phannaceutically acceptably uon-toxic basic salts Such salts include, for example, the alkali metars, e.g. sodium and potassium, the alkaline earth metals, e.g. calcium and magnesium, and complex salts, such as ammonium or substituted ammonium salts. In preparing various salts of hydroxycitric acid, it has been found that the pure potassium salt was highly sohiblc in water, but possessed high hygroscopiciry. an undesirable property. It was also determined that the pure calcium salt was moderately soluble in water, and possessed minimal hygroscopicity, a desirable property. - 5 - Surprisingly. It has been determined that a mixture of calcium salt with potassium or sodium salts produces a composition which is highly soluble (up to 25% weight to volume in water), but with minimal hygroscopicity. Furthermore, the composition according to the present invention displays minimal palatability concerns, as the caste of free hydxoxycitric acid is almost entirely climinated, as well as exceptional product application properties, including negligible odor, tasce and color While normally brown, the composition was found to be clear in solution ln addition, the composition was found to be largely free of the lactonc form ot hydroxycitric acid, and that in solution it did not equilibrate between the free and lactonc forms. Further desirable properties inctoded a balanced pH, and a low ( Thus the present compositions demonstrate a surprising, synergisric relationship between the calcium content and thc potassium (or sodium) content It has been determined that for a composition where the total hydroxycitric acid content exceed 40% by weight, a total of salts ot hydroxycitric acid which desirably comprise approximately 5 to 13% hy weight of calcium, preferably approximately 7 to 13%. and approximately 9 to 20% by weight of potassium, preferably approximately 14 to 18%. or approximately 5 to 1016 by weight of sodium, or mixtures of potassium and sodium As stated eatlicr. although sodium is acceptable, it is considered desirable to minimize the content of sodium, desirably to less than 1 % by weight. It is furtner considered desirable to provide a metnod ot manufacturing a compos it ion in wnicn the total hydroxycitric acid content is at least approximately 40% by weight, preferably at least approximately 50%, and desirably approximately 55-65%. Thus, in another aspect of the invent ion, a method for manufacturing a compos it ion for reducing body weight is provided .wnich comprises at least approximately 50% by weignt of total hydroxyc i t.r i c acids wnicn further comprise approximately 5 to 13% .by weight of calcium/ and approximately 9 to 20% by weignc of potassium or approximately "7 to 12% by weight of sodium, or a mixture thereof. - 6 - calculated as a percentage of the total weight of said composition Of the total amount, it is also considered desirable that the amount of hydroxycitric acid in the form of the lactone Dot exceed approximately 2% by weight. It is considered desirable to enrich the purity of free hydroxycitric acid from the Garcinia rind and prepare a calcium salt of the hydroxycitric acid. Generally, commercially available Garcinia rind comprises 25 to 30% moisture and 2 to 5% of sodium chloride. Garcinia rind contains 10 to 12% of free hydroxveitric acid. 12 ro 15% of the lactone form of hydroxyciiric acid and 2 to 3% citric acid on dry weight of die rind. A further aspect of the preparation of the salt of hydroxycitric acid is to mask the sour taste of byUroxycitric acid, minimize the percentage of hydroxycitric acid lactone and prepare a sodium free sail of hvdroxycicric acid. The process generally comprises washing the Garcinia rind, extracting hydroxycitric acid from die Garcinia rind, preparing an insoluble calcium salt of the hydroxveitric acid, dissociating the insoluble calcium salt.and thereafter preparing calcium and potassium salts of hydroxyciiric acid. The washing of the Garcinia rind is optional, as the sodium chloride can be reduced at other stages in the processing. The salt free water extract can be obtained from salted Garcinia rind by washing the Garcinia rind followed by hot extraction. The dilute water extract is filtered through a filtrate after adding clay to the extract and settled. The filtered dilute extract is then concentrated to 45% total solids under reduced pressure at elevated temperature. This concentrate contains 10 to 12% of free hydroxycitric acid by weight. 11 to 13% of hydroxycitric acid lactonc by weight and 2 to 3 % of citric acid by weight. The cootent of free hydroxycitric acid, hydroxycitric acid lactone, citric acid and non acid solu(cs can be determined by known techniques. The process of enriching free hydroxycitric acid from the rind is accomplished by preparing a water extract of the rind, converting the extract into a insoluble calcium citrate and removing non acidic impurities in the extract, such as pectin, sugar and color which will solubilizc, by washing the calcium hydroxycirrate. Thereafter, the calcium hydroxycitrate is dissociated with dilute phosphoric acid to form hydroxycicric - 7 - acid and calcium phosphate The calcium phosphate is then filtered out and the enriched hydroxycitric acid solution is converted to highly soluble calcium salt by first adjusting the pH of the hydroxycitric acid solution to 3.5 to 5 with calcium hydroxide suspension and second by the addition of potassium hydroxide solution to adjust the final pH to 8.0 to 9.0. This calcium salt solution is then filtered and concentrated under reduced pressure to approximate 50% total solids. The concentrate is then treated with 75% alcohol/acetone to crystalize white crystalline highly soluble calcium salt. There are numerous protocols available for preparing hydroxycitric acid extracts from Garcinu fruits. The rind of the Garcinia fruit which is commercially available typically consists of approximately 20% hydroxycitric acid, approximately 25% moisture and approximately 2.5% sodium chloride. It is considered desirable to eliminate as much sodium chloride from the rind as possible and since sodium chloride is freely soluble in water, this is easily accomplished. For example, one kilogram of raw Garcinia rind material is washed with approximately 2 liters of water in a stainless steel vessel. Thereafter, salt free Garcinia rind is extracted with 55 lo 60°C water on J continuing basis with approximately 15 liters of water. The spent rind is then tested tor hydroxycitric acid content and typically discarded. At this stage, recovery of total hydroxycitric acid is on the order of 90±2%. Next, approximately 2.5 kilograms of Fuller's Earth (a kaolin containing an aluminum magnesium silicate) is added to the solution obtained previously The mixture is stirred for one hour with continuous agitation at approximately 100 revolutions per miuute then allowed to settle far two hours. Thereafter, the material is filtered through a bed of a filter aid in a centrifuge. The filtrate is concentrated to 800 grams of approximately 45% total solids containing approximately 22% total hydroxycitric acid content. The yield at this step is approximately 97% of the extracted hydroxycitric acid. Next, the concentrated extract is filtered in a centrifuge to remove solids To the filtered extract is added calcium hydroxide (100 grams in 500 ml of water). The mixture is stirred for approximately four hours maintaining the pH of the solution at _ 8 - approximately 8.5. Thereafter, the mixture is filtered through the centrifuge and the supernatant is. discarded. The moist pellet is washed continuously with S liters of water until the water is colorless and no solid arc extracted from the filtrate. The yield of hydroxycitric acid at this phase is approximately 96.5%. In the next step, the wet pellet of calcium hydroxycitrate obtained previously is treated with 500 ml of 2N phosphoric acid solution to convert the calcium hydroxycicrate to hydroxycitric acid and calcium phosphate. Calcium phosphate precipitate is removed by centrifugal ion and washed with 2 liters of water. The filtrate contains approximately 165 grams of hydroxycitric acid with total solids of approximately 6 8% and the hydroxycitric acid yield is approximately 91.6%. Finally, the hydroxycitric acid solution obtained previously is treated with calcium hydroxide (61.7 grams in 600 ml of water to adjust to.pH 4.5). This solution is treated with neutral charcoal (60 grams at 75 °C for two hours under agitation, cooled and filtered). To the filtrate is added potassium hydroxide (52 grams in 50 ml of warer). which adjusts the pH 10 between 8.0 and 8.5. This salt solution is concentrated to 555 grams under reduced pressure to provide 50% total solids. The concentrate is treated with 75% acetone to obtain pure crystalline highly soluble calcium salt of hydroxycitric acid. The amount of (-) hydroxycitric acid can be estimated by high pressure liquid chromatography (HPLC), gcneralIy as follows. - 9 - Estimation of (-) HCA by HPLC: HPLC System: SHIMADZU or equivalent LC 10AT Pump or equivalent SPD 10 Detector or equivalent CR I0A Software or equivalent Column: ALLTIMA C12(5 ) (4.6x250 mm) Wave Length: 210nm How rate. lmL/min Volume of Injection: 20 1 Temperature: 25°±2X. Mobile Phase: 0.05M sodium sulphate solution in water (pH adjusted to approximately 2.3 with cone. H2SO4 Standards: 1) Ethylenediaminc Salt of (-) HCA. 2) (-) HCA Lacionc Standards Preparation Weigh accurately about 50mg of each standard into two different 25mL volumetric flasks. Dissolve in water and make up to volume with water. Filter unough 0.22 membrane filter and inject the standard solutions separately. Sample preparation: Weigh accurately about 50mg of sample in a 25mL volumetric flask. Dissolve it in water and make up ihe volume with water. Filler through 0.22 membrane filter and inject the solution. Retention Times: foT free (-) HCA - 5 min. (-) HCA Lactonc - 4.1 min. Calculations: % of Free (-) HCA = Sample Area x Standard Cone x Purity of Standard Standard Area x Sample Cone. % of (-) HCA Lactonc = Sample l^ctmic Area x Lactone Standard CONE. X Purity of Lactonc lactonc Standard Area x Sample Cone. - 10 - REAGENTS Unless otherwise stated all chemicals used arc reagent grade. o All glass washed with double distilled water. o Dilute ammonia solution. 25% w/v. o Ammonium chloride. o EDTA o Mordant Black T mixture o Sodium sulphate, analytical grade, or equivalent o Sulphuric acid, analytical grade or equivalent WATER SOLUBLE EXTRACTIVES: As per USP XX. pH : As per USP (1 % Solution) MOISTURE CONTENT : As per USP (K.F. rrtrimetCT) CALCIUM: Weigh accurately about 10mg of sample into a l00mL conical flask, dissolve it in 50mL of water. Add 2mL of ammonia - ammonium chloride (pH 9.2) buffer. Then titrate with 0.01M EDTA solutiou. Using Mordant Black-T Mixture as indicator. End point is blue. % of Calcium = Titrc value x Molaritv of EDTA x 0.4 x 100 0.01 x Weight of the sample in mg. ESTIMATION OF SODIUM AND POTASSIUM BY FLAME PHOTOMETER: Place 100mg of sample in a Silica crucible, and reduce to ash in a muffle furnace at 400"C Transfer ash into a 50mL volumetric flask, add I drop cone. HC1 and water to dissolve and make up to volume with water. Calibrate the flame photometer with 100, 50 and 10ppm standard sodium and potassium solutions. Now place the sample solution in the flame photometer. Note the ppm reading corresponding to Sodium and Potassium. % of corresponding Ion = ppm reading corresponding Ion x 50 x 100 1000 x Weight of sample in mg. - 11 - By inhibiting the synthesis of fatty acids, hydroxycitric acid is useful for the reduction of body weight in mammals. These useful compositions can be provided in the form of conventional pharmaceutical preparations or dietary supplements, for example, they can be mixed with conventional organic or inorganic inert pharmaceutical carriers or dietary supplements saleable for oral or parenteral administration, such as, for example, water, gelatin, lactose, starch, magnesium stcarate, talc, vegetable oil, gums or the like. They can be administered in conventional form, e.g.. solid forms, for example powders, tablets, capsules, suppositories or the like; or in liquid,forms, for example, suspensions or emulsions. In addition, such compositions can be formulated as a part of a processed food product for example in a form of a bar, baked good, beverage and the like. Moreover, the pharmaceutical compositions and dietary supplements can be subject ro conventional pharmaceutical or dietary supplements expedients, such as sterilization, and can contain conventional pharmaceutical or dietary supplements excipients, such as preservatives, stabilizing agents, emulsifying agents, salts for the adjustment of osmotic pressure or buffers, and the like. The compositions can also contain other therapcutically active materials. A suitable dosage unit will typically contain from about 15 to about 3000mg of hydroxycitric acid, administered up to three times per day. Suitable parenteraj dosage regimens in mammals can comprise from about Img per kilogram of body weight to about 50mg per kilogram of body weight per day. However, for any particular subject, the specific dosage regimen should be adjusted according to individual need and the professional judgment of the person administering or supervising the administration of the aforesaid compounds. Additional aspects of the invention include food method for manufacturing product and the like for use in reducing body weight which include the present compositions, and methods for reducing body weight by administering such compositions and dietary supplements and food products. When provided for oral administrarion as a proccessed food product, such as a beverage or a snack bar. the hydroxycitric acid content will desirably - 12 - comprise approximately 0.001 to 25%, preferably 0.05 to 5% by weight of the total weight of the food product. Preparation of processed food products to include hydroxycicric acid compositions of the present invention involves, for example, diluting a concentrate of the composition containing at least approximately 40% hydroxycitnc acid in water, adding supplements, blending, heating and/or periodic agitation. For both snack bars and beverages, it is desirable to pre-pastcurize the concentrate in a highly diluted ratio with purified water. For example, approximately lmL of the concentrate can be added to 12 fluid ounces of water. These figures will however vary depending upon the types of products desired, ranging from 1 to 25% for a beverage and 1 to 40% for a snack bar. After the blending step, the temperature of the vessel which the preprocessing step takes place is increased, frequently using steam as in the case of beverage manufacture or low heat as in the case of snack bars and baked goods. Before heat is applied, 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 bomogeneosity, the liquid is pumped by a stainless steel pipeline into a bottling facility. High temperature steam is applied from the outside to the pipes which in turn maintains, the temperature of the liquid during its transit. The pipes are placed so as to facilitate the bottling of the liquid beverage in an efficient manner. In the preparation of, for example, snack bars, the preprocessing follows approximately the same protocol as for beverages. The environment for the development of this product is typically an industrial kitchen with the use of large cooking pots. The diluted hydroxycitric acid composition is blended with water, covered and heated, bringing it to a boil for a certain period of time. This boiling also provides agitation to insure thorough mixing. Thereafter, snack bars, baked goods or other processed food products are produced in accordance with the techniques well known in the art. - 13 - Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will he apparent to those of ordinary skill in the art in light of the disclosnrc that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. -14- We claim : 1. A method for preparing a hydroxycitric acid composition for reducing body weight wherein said method comprises mixing the calcium salt of hydroxycitric acid with at least one other salt of hydroxycitric acid selected from the potassium salt of hydroxycitric acid and the sodium salt of hydroxycitric acid, in a proportion so that the final composition comprises: a) 14 to 26% by weight of calcium, and, b) 24 to 40% by weight of potassium or, 14 to 24% by weight of sodium, or a mixture of said potassium and sodium salts, calculated as a percentage of the total hydroxycitric acid content of said composition. 2. A method as claimed in claim 1, wherein the total sodium in the composition comprises less than 2% by weight. 3. A method as claimed in claim 1, wherein the calcium comprises 18 to 26% by weight. 4. A method as claimed in claim 1, wherein the potassium comprises 28 to 36% by weight. 5. A method as claimed in claim 1, wherein the lactone forms of hydroxycitric acid comprise less than 4 % by weight of the total hydroxycitric acid content of the composition. 6. A method for preparing a dietary supplement composition for reducing body weight containing hydroxycitric acid composition as claimed in claim I, wherein said method comprises mixing the calcium salt of hydroxycitric acid, and at least one of the potassium salt of hydroxycitric acid or the sodium salt of hydroxycitric acid, wherein the said composition comprises at least 40% by weight of total hydroxycitric acid -15- 7. A method as claimed in claim 6, wherein the hydroxycitric acid composition comprises 55-65 % by weight. 8. A method as claimed in claim 7, wherein the calcium comprises 9 to 13 % by weight. 9. A method as claimed in claim 7, wherein the potassium comprises 14 to 18 % by weight. 10. A method as claimed in claim 6, wherein the lactone forms of hydroxycitric acid comprise less than 2% by weight of the total weight of the composition. 11. A method as claimed in claim 6, wherein the sodium comprises less than I % by weight. 12. A method for preparing a food product for use in reducing body, weight which comprises combining a prepared food product with a hydroxycitric acid composition as claimed in claim 1, wherein said composition comprises 40% by weight of hydroxycitric-acid together with 14 to 26% by weight of calcium, and 24 to 40% by weight of potassium or 14 to 24% by weight of sodium, or a mixture thereof, each calculated as a percentage of the total hydroxycitric acid content of said composition. 13. A method as claimed in claim 12, wherein the hydroxycitric acid composition comprises 55-65 % by weight. 14. A method as claimed in claim 13, wherein the calcium comprises 18 to 26% by weight. 15. A method as claimed in claim 13, wherein the potassium comprises 28 to 36% by weight. -16- 16. A method as claimed in claim 12, wherein the sodium comprises less than 2% by weight. 17. A method as claimed in claim 12, wherein the lactonc forms of hydroxycitric acid comprise less than 4% by weight of the total hydroxycitric acid content of the composition. 18. A method as claimed in claim 12, wherein said hydroxycitric acid composition comprises 0.001 to 25% by weight of the total weight of said prepared food product. 9. A method for preparing hydroxycitric acid composition substantially as herein described. 0. A method for preparing a dietary supplement substantially as herein described. 1. A method for preparing a food product substantially as herein described. There is disclosed a method for manufacturing a hydroxycitric acid composition for reducing body weight, said method comprising formulating a composition by mixing calcium salt of hydroxycitric acid having approximately 14 - 26% by weight of calcium and at least one of the potassium salt and sodium salt of said hydroxycitric acid, in proportions 24-40% by weight and 14-24% by weight respectively, all calculated as a percentage of the total hydroxycitric acid content of said composition.

Documents:

00268-kol-2003-abstract.pdf

00268-kol-2003-assignment.pdf

00268-kol-2003-claims.pdf

00268-kol-2003-correspondence.pdf

00268-kol-2003-description(complete).pdf

00268-kol-2003-form-1.pdf

00268-kol-2003-form-18.pdf

00268-kol-2003-form-2.pdf

00268-kol-2003-form-3.pdf

00268-kol-2003-form-5.pdf

00268-kol-2003-g.p.a.pdf

00268-kol-2003-letters patent.pdf

00268-kol-2003-priority document.pdf

00268-kol-2003-reply f.e.r.pdf

268-KOL-2003-07-03-2012-CORRESPONDENCE.pdf

268-KOL-2003-CORRESPONDENCE.pdf

268-KOL-2003-FORM 27.pdf

268-KOL-2003-FORM-27.pdf

268-kol-2003-granted-abstract.pdf

268-kol-2003-granted-claims.pdf

268-kol-2003-granted-description (complete).pdf

268-kol-2003-granted-form 2.pdf

268-kol-2003-granted-specification.pdf

268-kol-2003-priority document.pdf