COMPOSITION FOR REDUCING ABSORPTION OF DIETARY FAT

Abstract

This invention relates to compositions for reducing the absorption or bioavailability of lipids in the gastrointestinal tract, and to methods for the preparation and use of these compositions. The compositions comprise a source of dietary fibre together with a cyclodextrin. The compositions of the invention are suitable for use in the prevention and treatment of obesity or hyperlipidaemia.

Claims

1-38. (canceled)

39. A method of reducing weight gain, comprising the step of administering a an effective amount of a composition or healthcare preparation to a subject in need thereof, wherein the composition or healthcare preparation comprises (a) a dietary fibre preparation of plant or non-plant origin, and (b) at least one cyclodextrin.

40. A method of treatment of obesity, comprising the step of administering an effective amount of a composition or healthcare preparation to a subject in need of such treatment, wherein the composition or healthcare preparation comprises (a) a dietary fibre preparation of plant or non-plant origin, and (b) at least one cyclodextrin.

41. A method of treatment of hyperlipidaemia, comprising the step of administering a an effective amount of composition or healthcare preparation to a subject in need of such treatment, wherein the composition or healthcare preparation comprises (a) a dietary fibre preparation of plant or non-plant origin, and (b) at least one cyclodextrin.

42. The method of claim 39, wherein the dietary fibre preparation of plant or non-plant origin is chitosan.

43. The method of claim 39, wherein the composition or healthcare preparation comprises preparing a blend by subjecting the dietary fibre preparation and the at least one cyclodextrin to shear force.

44. The method of claim 39, wherein the composition or healthcare preparation comprises preparing a simple physical mixture of the dietary fibre preparation and the at least one cyclodextrin.

45. The method of claim 39, wherein the composition or healthcare preparation comprises a cyclodextrin selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, hydroxyalkyl-, alkyl-, sulphoallylether-, glycosylated-, maltosylated-, and partially acetylated alpha-, beta- and gamma-cyclodextrins, polymeric derivatives of alpha-, beta- and gamma-cyclodextrins, and combinations of two or more such cyclodextrins.

46. The method of claim 39, wherein the cyclodextrin is gamma-cyclodextrin.

47. The method of claim 39, wherein the weight ratio of the dietary fibre and cyclodextrin is between 95:5 and 60:40.

48. The method of claim 39, wherein the weight ratio of the dietary fibre and cyclodextrin is between 85:15 and 65:35.

49. The method of claim 40, wherein the composition or healthcare preparation further comprises a biologically-active agent suitable for treating obesity.

50. The method of claim 48, wherein the biologically-active agent is selected from the group consisting of absorption-altering, appetite-altering and metabolism-altering agent or combinations thereof.

51. The method of claim 41, wherein the composition or healthcare preparation further comprises a biologically-active agent suitable for treating hyperlipidaemia.

52. The method of claim 50, wherein the biologically-active agent is selected from the group consisting of cholesterol-lowering agents and combinations thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0085] FIG. 1 is a graph illustrating the effect of various compositions of cactus fibre and cyclodextrin on bioavailability of fat in a model emulsion.

[0086] FIG. 2 is a graph illustrating the anti-obesity effect of vehicle (spring water), first treatment group (cactus fibre & gamma-cyclodextrin preparation at 7.5:1.5) and second treatment group (cactus fibre).

[0087] FIG. 3 is a graph illustrating the fat indigestibility of vehicle (spring water), first treatment group (cactus fibre & gamma-cyclodextrin preparation at 7.5:1.5) and second treatment group (cactus fibre).

[0088] FIG. 4 is a bar chart illustrating the de-emulsification capacity of various plant and non-plant fibres.

DETAILED DESCRIPTION OF THE INVENTION

[0089] As described herein, a consumable healthcare preparation Is a dietary or health supplement which is adapted to be consumed by mouth, but is not in Itself a food.

[0090] An edible plant or product means one which can be ingested by humans or animals without causing any major adverse effects. Very minor and/or transient adverse effects may be tolerated.

[0091] Fibre is a polysaccharide constituent which is not degraded into absorbable units within the stomach or small intestine. Soluble fibres are more soluble in water relative to insoluble fibres. Dietary fibre is further classified as being either fermentable or non-fermentable, depending on whether or not undigested fibres are digested by microbes in the large bowel.

[0092] The expression bioavailability of fat refers to the degree and rate at which fats are absorbed into the body from the gut.

[0093] The expression absorption of dietary fat refers to the process by which the products of digestion of fats present in the diet pass through the gut mucosa into the blood or lymph. Dietary fat is predominantly neutral fat or triglyceride, and also includes phospholipids, sterols such as cholesterol, and many minor lipids, including fat-soluble vitamins. The small intestine also contains lipids from sloughed epithelial cells and cholesterol delivered in bile. In order for the triglyceride to be absorbed, large aggregates of dietary triglyceride, which are virtually insoluble in an aqueous environment, must be broken down physically and held in suspension; this process is called emulsification. Triglyceride molecules must also be enzymatically digested to yield monoglyceride and fatty acids, which can efficiently diffuse or be transported into the enterocytes. These two processes are mediated by bile salts and pancreatic lipase, both of which are mixed with chyme and act in the lumen of the small intestine. Bile salts are also necessary to solubilize other lipids, including cholesterol.

[0094] Cactus fibre refers to Cactus fibre powder produced from a cactus belonging to the family Opuntioideae. In one form of the invention, this powder is produced from Opuntia ficus-indica by the method described in US patent application No. US 20040126444.

[0095] Cyclodextrins (CD), sometimes called cyclic dextrins, are enzyme-modified starch derivatives, cyclic malto-oligomers made up of D-glucose units. (Szejtli, J.: Cyclodextrin Technology, Kluwer Ed. Dordrecht, Holland, 1988.) The industrially-produced, commercially-available cyclodextrins are called alpha-, beta- and gamma-cyclodextrins. They consist of six, seven or eight glucose units, linked via alpha-1,4-glycosidic bonds. Alpha-cyclodextrin is cyclohexaamylose, beta-cyclodextrin is cycloheptaamylose and gamma-cyclodextrin is cyclooctaamylose. All three classes of cyclodextrins are considered by regulatory authorities as falling into the category of products which are generally recognised as safe. As used herein, the term cyclodextrin also includes substituted alpha-, beta- and gamma-cyclodextrins, such as hydroxyalkyl-, alkyl-, sulphoallylether-, glycosylated-, maltosylated-, and partially acetylated-alpha-, beta- and gamma-cyclodextrins, their polymeric derivatives, and any combinations thereof. The cyclodextrin as used herein could further extend to the use of dextrin group which also includes maltodextrin and its derivatives thereof.

[0096] A Polymeric derivative of a cyclodextrin refers to a cross-linked cyclodextrin in which the monomers are covalently linked to each other via epichlorohydrin or other suitable bifunctional chemical reagent. The average molecular mass of the water-soluble cyclodextrin polymers is preferably between 1800-5000 Dalton.

[0097] A Blend as referred to in the Examples herein is a solid preparation formed by mixing dietary fibre and a cyclodextrin under conditions whereby the cyclodextrin is enabled to interact with the fibrous matrix of the dietary fibre, for example by subjecting the dietary fibre and cyclodextrin to shear force.

[0098] De-emulsifying ability, De-emulsifying effect or De-emulsifying efficiency means the removal of digestible micrometer-scale lipid droplets from a model chyme (emulsion) by inducing them to coalesce and thus transforming them into indigestible millimeter-scale droplets. The de-emulsifying ability of a composition or agent serves as a predictor of its ability to inhibit emulsification of lipids in the gut, and hence to inhibit lipid absorption. The de-emulsifying ability is also correlated with the fat-binding ability or fat elimination ability of the composition.

[0099] The term fat as used herein, unless otherwise specified, means oils, lipids, greasy materials, or combinations thereof.

[0100] Hyperlipidaemia is an elevation of lipids in the bloodstream. These lipids include cholesterol, cholesterol esters, phospholipids and triglycerides.

[0101] A bile salt is a natural surfactant which is essential for efficient digestion and absorption of fats for metabolism. Bile salts are bile acids conjugated to either glycine or taurine. In humans, the most important bile acids are cholic acid, deoxycholic acid, and chenodeoxycholic acid. Prior to secretion by the liver, they are conjugated with glycine or taurine. Bile salts act to some extent as detergents, helping to emulsify fats into a finely dispersed microemulsion. By increasing the interfacial surface area of fats in chyme (the semi-fluid mass of partly-digested food expelled by the stomach into the duodenum), bile salts assist the enzymatic action of lipases, and thus aids fatty acid absorption from the small intestine. If bile salts are absent from the duodenum, or if they are bound so that their action is blocked, not all fats are able to be digested, and the non-absorbed fat is excreted in the faeces.

[0102] In some embodiments the composition comprises a dietary fibre and a cyclodextrin in a weight ratio between 1:99 and 99:1. Thus the ratio may be 1:99, 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 11:89, 12:88, 13.87, 14:86, 15:85, 16:84 17:83, 18:82, 19:81, 20:80, 21:79, 22:78, 23:77, 24:76, 25:75, 26:74, 27:73, 28:72, 29:71, 30:70, 31:69, 32:68, 33:67, 34:66, 35:65, 36:64, 37:63, 38:62, 39:61, 40:60, 41:59, 42:58, 43:57, 44:56, 45:55, 46:54, 47:53, 48:52, 49:51, 50:50, 51:49, 52:48, 53:47, 54:46, 55:45, 56:44, 57:43, 58:42, 59:41, 60:40, 61:39, 62:38, 63:37, 64:36, 65:35, 68:34, 67:33, 68:32, 69:31, 70:30, 71:29, 72:28, 73:27, 74:26, 75:25, 76:24, 77:23, 78:22, 79:21, 8020, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 982, or 99:1.

[0103] In one embodiment the weight ratio of the dietary fibre and a cyclodextrin is between 95:5 and 60:40. In another embodiment the weight ratio of the dietary fibre and a cyclodextrin is between 85:15 and 65:35.

[0104] An in vitro model which simulates the in vivo bile-fat chyme (emulsion) was developed to evaluate the efficacy of the study materials.

[0105] The emulsion prepared with Na-deoxycholate was found to be the most suitable model chyme, since it distinguished changes which are of significance in the process of fat digestion, showing a clearer and consistent result. However, emulsions prepared from soy lecithin may also be used.

[0106] The model fat-Na-deoxycholate emulsions showed suitable stability for, relatively rapid comparison laboratory tests. The changes in stability were recorded when these emulsions were exposed to the effect of cactus powder, apple fibre, oat fibre, shark cartilage, chondroitin sulphate, chitosan, cyclodextrins, or combinations of each of these fibres with cyclodextrin. Phase separation studies were then developed to enable the visualization and quantification of de-emulsification process over time.

[0107] An exponential increased de-emulsification effect was demonstrated for certain fibre/cyclodextrin combinations in the experimental fat-bile salt emulsion model using a blended composition according to the invention. The degree of de-emulsification of the model fat-bile salt emulsion caused by the composition serves as a predictor of the ability of compositions of the invention to inhibit emulsification of lipids in vivo by bile salts, and consequently to inhibit dietary fat absorption.

[0108] Without wishing to be limited by any proposed mechanism for the observed increased effects, we believe that certain dietary fibres undergo a physical interaction when blended with a cyclodextrin, which substantially increases their fat-binding or fat elimination ability, and that the structural alterations taking place during the physical interaction together with the properties of the native ingredients result in an increased effect in reducing dietary fat absorption. Thus, in addition to the use of a much lower dose of a cyclodextrin than has been proposed in the prior art, the physical interaction between certain dietary fibres and cyclodextrin has not been previously disclosed or suggested.

[0109] We have found that the de-emulsification of fat by dietary fibres of plant and non-plant origin, and in particular cactus fibre, apple fibre and chitosan, in an in vitro experimental fat-bile salt emulsion model can be improved by prior processing with a cyclodextrin. We have also demonstrated in this model that a low dosage of cyclodextrin in a blend with certain plant fibres, including cactus fibre or apple fibre, exerts higher efficacy than that of prior art compositions which use a much higher dose of cyclodextrin, thereby lowering the cost of prevention or treatment.

[0110] In the in vitro studies with the model emulsion, the Cactus fibre alone was found to be an effective de-emulsifying agent in that it resulted in agglomeration of micro-scale droplets of the emulsion, eventually causing phase separation of, the fat and water in the emulsion. It was also demonstrated that cyclodextrin alone does not produce a do-emulsification effect in the model emulsion. This phenomenon is likely to be attributable to the affinity of surfactants to the cactus fibres. The extent of de-emulsification elicited by the test materials was used as a predictor of the ability of the materials to reduce fat bioavailability in vivo. While we believe that the specific class or identity of the cyclodextrin in the aforementioned experiments is not important, gamma-cyclodextrin was found to be the most efficient.

[0111] In a series of further studies, it was demonstrated that while both the simple physical mixture and the prior processed blend of the Cactus fibre with cyclodextrins resulted in Increased de-emulsification ability compared to effect of the cactus fibre alone, an exponential increase in de-emulsification effect was observed only with the emulsion treated with a Cactus fibre-cyclodextrin blend. Similar exponential increase in de-emulsification was demonstrated when apple fibre was blended with cyclodextrin. Of the cyclodextrins tested, the most significant synergism was observed with the prior-processed blend of gamma cyclodextrin and Cactus fibre, as illustrated in FIG. 1.

[0112] The results of the in vitro tests suggest that a composition comprising Cactus fibre and gamma-cyclodextrin in a solid blend formula is particularly suitable for effective weight management. The efficacy of the composition may readily be tested in vivo, using methods well known in the art.

[0113] In a particular embodiment of the present invention, the composition of Cactus fibre and a cyclodextrin may further comprise a biologically-active agent suitable for treating obesity and hyperlipidaemia. The biologically-active agent may be selected from the group consisting of absorption-altering agents, including pharmaceutically-active agents such as orlistat and cetilistat, or naturally-derived ingredients such as white kidney bean extract; appetite-altering agents, including pharmaceutically-active agents such as sibutramine, phentermine, diethylpropion, rimonabant and benzphetamine, or naturally-derived ingredients such as potato starch and glucomannan; metabolism-altering agents such as moxonidine or naturally-derived ingredients such as extracts of green tea, Citrus aurantium, or Garcinia cambogia; cholesterol-lowering agents, including pharmaceutically-active agents, such as statins (e.g. atorvastatin, simvastatin, lovastatin, pravastatin, rosuvastatin etc.), fibrates (e.g. gemfibrozil, bezafibrate, fenofibrate or ciprofibrate), vitamin B3 (niacin), bile acid sequestrants (e.g. colestipol, cholestyramine) or naturally-derived ingredients such as plant sterol compounds (e.g. sitosterol, stigmasterol, campesterol), or any combination thereof.

[0114] In another particular embodiment of the present invention, the composition of Cactus fibre and cyclodextrins may further comprise a nutrient ingredient selected from the group consisting of vitamins and minerals, and any combination thereof. The vitamin may be any of a variety of vitamins or related nutrients, non-limiting examples of which include vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B12, carotenoids (e.g., beta-carotene, zeaxanthin, lutein, lycopene), niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol, salts and derivatives thereof, and combinations thereof. The mineral may be any of a variety of minerals, non-limiting examples of which include calcium, phosphorus, magnesium, iron, zinc, manganese, copper, iodine, sodium, potassium, molybdenum, selenium, chromium, chloride, and combinations thereof.

[0115] The invention will now be described in detail by way of reference only to the following non-limiting examples and drawings. All compounds and materials used in the examples are commercially available.

EXAMPLES

Cactus Powder:

[0116] The cactus powder used in the experiments described herein is produced from Opuntia ficus-indica by the method described in US patent application (publication number US20040126444). The fibre complex contains two kinds of fibre, in approximately equal proportions: Insoluble fibre and soluble fibre. The insoluble fibre is a polymer, insoluble in water at neutral pH and ambient temperature. The soluble fibre is a soluble polysaccharide dietary fibre containing pectin, gum and mucilage, which form a fluid gel in the stomach. The increase of pH from stomach to small intestine does not affect the gel's stability.

[0117] The pectin component of Opuntia ficus-indica fibre is both low-methoxy (ie degree of methoxylation <50%) and high-methoxy (ie degree of methoxylation >50%); the relative proportions of low-methoxy and high-methoxy depend on the part of the cactus (Goycoolea F. M. and A. Cardenas: Pectins from Opuntia spp: a short review J. PACD 2003 17-19). The fibre complex from Opuntia ficus-indica contains dietary fibre (about 45%), soluble sugars, proteins, lipids, vitamins minerals (mainly calcium and phosphorus) and amino adds. The cactus powder may optionally comprise granulated gum acacia excipient in the range of 5 to 35% as a granulation aid.

[0118] Apple fibre, oat fibre, cartilage, chondroitin and chitosan tested herein for de-emulsifying effects were obtained from the following sources with the described specifications:

TABLE-US-00001 Test Material Source Specification Apple Fibre Plant >50% dietary fibre Oat Fibre Plant >90% dietary fibre Cartilage Animal (Shark) 40% Protein Chondroitin Animal (Shark) >90% Chondroitin sulphate Chitosan Animal (Crustaceans) >70% Deacetylation degree

Surfactant

[0119] Sodium deoxycholate: product of Sigma-Aldrich (>97%):

[0120] Sodium dodecyl sulphate (SDS): product of Merck (>99.0%);

[0121] Soy bean lecithin: commercial food supplement (96%) (BiYo-Product Ltd., Hungary)

Cyclodextrins:

[0122] alpha-cyclodextrin (CYL-2322);

[0123] beta-cyclodextrin (CYL-2518/2);

[0124] gamma-cyclodextrin (CYL-2323):

[0125] each a product of CycloLab Ltd., Hungary

Other Reagents

[0126] Sunflower oil: commercial food grade product Vnusz brand (product of Bunge Co., Hungary)

[0127] Sudan red, an oil-soluble dye (product of Reanal, Hungary)

All other reagents used were of analytical grade commercial products, and distilled water was used in all experiments.

Example 1: Model Chyme (Fat-Na Deoxycholate Emulsion)

[0128] The model emulsion was composed of, [0129] 48 g sunflower oil (saturated with Sudan red) [0130] 350 g distilled water [0131] 0.58 g sodium deoxycholate
and was prepared by homogenization in an Ultra-turrax homogenizer for 5 min at 9000 RPM.

Example 2 (a): Preparation of Gamma-Cyclodextrin and Cactus Fibre Blend

[0132] 600 g dried plant fibre composition originating from species Opuntia ficus indica was triturated with 300 g of previously amorphized gamma-Cyclodextrin powder. After addition of 200 ml water, the mixture was treated by means of shear force mechanical activation by co-kneading by a twin-screw kneader, at ambient temperature for 30 minutes until a homogenous blend was obtained, followed by extrudation. The water content of the blend was removed in vacuo at 40 C. to below 5.0 w %. The dried blend was granulated and sieved to break any lumps into powder; the particle size is not critical.

[0133] (b) In a similar manner were prepared blends of either alpha- or beta-Cyclodextrin with cactus fibre.

[0134] (c) Similarly prepared was a blend of gamma-Cyclodextrin and Cactus fibre in a ratio of 1.5:7.5 using 150 g gamma-Cyclodextrin and 750 g cactus fibre.

[0135] (d) Similarly prepared were blends of gamma-Cyclodextrin and other plant fibres and non-plant fibres, including apple fibre, oat fibre, shark cartilage, chondroitin sulphate and chitosan

Example 3 (a): Preparation of Physical Mixture of Gamma-Cyclodextrin and Cactus Fibre

[0136] 600 g dried plant fibre composition originating from the species Opuntia ficus-indica was triturated with 300 g of previously amorphized gamma-cyclodextrin powder. The physical powder mixture was homogenized by dry blending and used without further treatment.

[0137] (b) In a similar manner were prepared physical mixtures of either alpha- or beta-Cyclodextrin with cactus fibre.

[0138] (c) Similarly prepared was a physical mixture of gamma-Cyclodextrin and Cactus fibre in a ratio of 1.5:7.5 using 150 g gamma-Cyclodextrin and 750 g cactus fibre.

[0139] (d) Similarly prepared were physical mixtures of gamma-Cyclodextrin and other plant fibres and non-plant fibres, including apple fibre, oat fibre, shark cartilage, chondroitin sulphate and chitosan

Example 4: In Vitro Efficacy of Study Materials with Model Emulsion

[0140] The test materials prepared according to Examples 2(a), 2(b), 2(d), 3(a) 3(b) and 3(d) were each added to 50 g of emulsion prepared as in Example 1, according to the schedule set out in Tables 1 and 2. The de-emulsifying effect of a test material is a measure of its ability to reduce the bioavailability of dietary fat.

TABLE-US-00002 TABLE 1 Test materials for evaluation of the de-emulsifying effects of Cactus fibre/cyclodextrin in the model emulsion system Cactus Alpha- Beta- Gamma- Test Sample fibre CD CD CD Emulsion + Cactus fibre 0.6 g Emulsion + Alpha-CD 0.3 g Emulsion + Beta-CD 0.3 g Emulsion + Alpha-CD + 0.15 g 0.15 g Beta-CD (Physical mixture) Emulsion + Cactus fibre + 0.6 g 0.3 g Alpha-CD (Physical mixture) Emulsion + Cactus fibre + 0.6 g 0.3 g Beta-CD (Physical mixture) Emulsion + Cactus fibre + 0.6 g 0.15 g 0.15 g Alpha-CD + Beta-CD (Physical mixture) Emulsion + Cactus fibre + 0.6 g 0.3 g Gamma-CD (Physical mixture) Emulsion + Cactus fibre + 0.6 g 0.3 g Alpha-CD (Blend) Emulsion + Cactus fibre + 0.6 g 0.3 g Beta-CD (Blend) Emulsion + Cactus fibre + 0.6 g 0.3 g Gamma-CD (Blend)

Phase Separation StudyVolumetric Test

[0141] 50 g of stock emulsion was portioned on to the previously-weighed solid additives while the stock emulsion was continuously stirred gently. 50 ml of the homogenized samples were filled into a graduated cylinder so that the rate and the extent of phase separation could be observed were recorded, and the volume of the separated (upper) oil-rich emulsion phase and the quantity of pure oil layers which eventually appeared were a measure of the de-emulsifying efficiency of the additives.

[0142] During phase separation, microdroplets of oil in the emulsion coalesced into bigger oil droplets, forming a layer of oil-rich emulsion, which floated on the upper portion of the emulsion. This was indicated by the more intense Sudan red colour of the oil. A more compact oil-rich phase indicated a stronger de-emulsification capability of the test materials. After 1 hour of standing, the test samples were examined and the volume of the oil-rich phase measured. The results are shown in Table 3.

TABLE-US-00003 TABLE 2 Test materials for evaluation of the de-emulsifying effects of various plant/gamma-CD and non-plant/gamma CD in the model emulsion system Cactus Gamma Test Sample Fibre CD Emulsion + Cactus fibre 0.6 g 0 g Emulsion + Cactus fibre + Gamma CD 0.5 g 0.1 g (Physical mixture) Emulsion + Cactus fibre + Gamma CD (Blend) 0.5 g 0.1 g Emulsion + Apple fibre 0.6 g 0 g Emulsion + Apple fibre + Gamma CD 0.5 g 0.1 g (Physical mixture) Emulsion + Apple fibre + Gamma CD (Blend) 0.5 g 0.1 g Emulsion + Oat fibre 0.6 g 0 g Emulsion + Oat fibre + Gamma CD (Physical 0.5 g 0.1 g mixture) Emulsion + Oat fibre + Gamma CD (Blend) 0.5 g 0.1 g Emulsion + Cartilage 0.6 g 0 g Emulsion + Cartilage + Gamma CD (Physical 0.5 g 0.1 g mixture) Emulsion + Cartilage + Gamma CD (Blend) 0.5 g 0.1 g Emulsion + Chondroitin 0.6 g 0 g Emulsion + Chondroitin + Gamma CD 0.5 g 0.1 g (Physical mixture) Emulsion + Chondroitin + Gamma CD (Blend) 0.5 g 0.1 g Emulsion + Chitosan 0.6 g 0 g Emulsion + Chitosan + Gamma CD (Physical 0.5 g 0.1 g mixture) Emulsion + Chitosan + Gamma CD (Blend) 0.5 g 0.1 g

Phase Separation StudyVolumetric Test

[0143] 50 g of stock emulsion was portioned on to the previously-weighed solid additives and stirred continuously to form a homogenized sample. The homogenized samples were filled into a graduated cylinder so that the rate and the extent of phase separation could be observed and recorded.

[0144] During phase separation, microdroplets of oil in the emulsion coalesced into bigger oil droplets, forming a layer of oil-rich emulsion, which floated on the upper portion of the emulsion. This was indicated by the more intense Sudan red colour of the oil. A more compact oil-rich phase indicated a stronger de-emulsification capability of the test materials. After 24 hour of standing, the test samples were examined and the volume of a more compact oil-rich phase were measured. The results are shown in Table 4.

TABLE-US-00004 TABLE 3 Results of de-emulsification test Volume % Increase in De- of Oil-rich emulsification Phase (D.sub.x) vs Test Samples (V.sub.x) Reference* Emulsion + Cactus fibre 17 ml Reference Emulsion + Alpha-CD 0 ml No de- emulsification Emulsion + Beta-CD 0 ml No de- emulsification Emulsion + Alpha-CD + Beta-CD 0 ml No de- (Physical mixture) emulsification Emulsion + Cactus fibre + Alpha-CD 15 ml 12 (Physical mixture) Emulsion + Cactus fibre + Beta-CD 14 ml 18 (Physical mixture) Emulsion + Cactus fibre + Alpha-CD + 12 ml 29 Beta-CD (Physical mixture) Emulsion + Cactus fibre + Gamma-CD 13 ml 24 (Physical mixture) Emulsion + Cactus fibre + Alpha-CD 11 ml 35 (Blend) Emulsion + Cactus fibre + Beta-CD 8 ml 53 (Blend) Emulsion + Cactus fibre + Gamma-CD 6 ml 65 (Blend) *calculated according to the formula D.sub.x = (V.sub.x V.sub.1 + V.sub.1 100 where x = test sample; V.sub.1 = volume of oil rich phase for the Reference (test sample 1; Emulsion + Cactus fibre).

[0145] The results of this phase separation study are illustrated in FIG. 1 (Cactus fibre), and show that the method of pre-treatment (physical mixing vs. Blending) has a very significant effect on reduction of fat bioavailability, with the Blends having a much greater effect when Cactus fibre is used. The differences in cavity sizes of the cyclodextrins may also play a role in fat removal, since there was a difference in effect between alpha-, beta- and gamma-cyclodextrin.

[0146] Therefore, it has been demonstrated that gamma-cyclodextrin in a cactus-fibre blended composition is superior to the other cyclodextrins as an inhibitor of fat absorption.

[0147] It will be appreciated that the same method can be used to test Blends and mixtures in which the cyclodextrin is replaced by maltodextrin or other suitable starch derivatives, and the Cactus fibre is replaced by dietary fibre selected from the group consisting of soluble dietary fibres, such as gum acacia, guar gum, citrus pectin, low- and high-methoxy pectin, oat and barley beta-glucans, carrageenan, psyllium and combinations thereof, and insoluble dietary fibres, such as oat hull fibre, pea hull fibre, soy hull fibre, soy cotyledon fibre, sugar-beet fibre, cellulose, corn bran, and combinations thereof.

TABLE-US-00005 TABLE 4 Results of de-emulsification test Volume of Oil- rich Phase Test samples (V.sub.x) % De-emulsification, Dx* Emulsion + Cactus fibre 0 ml 0 Emulsion + Cactus fibre + Gamma 0 ml 0 CD (Physical mixture) Emulsion + Cactus fibre + Gamma 5 ml 50 CD (Blend) Emulsion + Apple fibre 0 ml 0 Emulsion + Apple fibre + Gamma 0 ml 0 CD (Physical mixture) Emulsion + Apple fibre + Gamma 1 ml 10 CD (Blend) Emulsion + Oat fibre 1 ml 10 Emulsion + Oat fibre + Gamma 1 ml 10 CD (Physical mixture) Emulsion + Oat fibre + Gamma 1.5 ml 15 CD (Blend) Emulsion + Cartilage 0 ml 0 Emulsion + Cartilage + Gamma 0 ml 0 CD (Physical mixture) Emulsion + Cartilage + Gamma 0 ml 0 CD (Blend) Emulsion + Chondroitin 2 ml 20 Emulsion + Chondroitin + Gamma 2.5 ml 25 CD (Physical mixture) Emulsion + Chondroitin + Gamma 1 ml 10 CD (Blend) Emulsion + Chitosan 2.5 ml 25 Emulsion + Chitosan + Gamma 7.5 ml 75 CD (Physical mixture) Emulsion + Chitosan + Gamma 3.5 ml 35 CD (Blend) *calculated according to the formula Dx = (Vx/R) 100 wherein R is 10 ml of compact oil in the emulsion/Negative Control

[0148] The results of this phase separation study are illustrated in FIG. 4 and show (1) that certain fibres such as cartilage have no de-emulsification capacity with or without gamma-cyclodextrin and (2) that the method of pre-treatment (physical mixing vs. Blending) can have a very significant effect on reduction of fat bioavailability, with the Blends having a much greater effect when Cactus fibre is used whereas physical mixtures have a greater effect when chitosan is used.

Example 5: Gas Chromatographic Study of Fat-Binding Efficiency

[0149] It was observed that the model emulsions containing cactus powder/cyclodextrin Blends had a fibrous sediment which sank to the bottom of the graduated cylinders. This was not observed when cyclodextrins were added to the cactus powder as a physical mixture. This phenomenon suggests that during the pre-treatment of the cactus powder by cyclodextrins (Blending), the cactus fibres undergo significant physical changes. Aggregated hydrophobic fibre moieties may become unfolded or swollen due to this processing, making additional regions available for triggering de-emulsification. Cyclodextrin may be extracted from the fibrous matrix by water when it comes into contact with the emulsion. Meanwhile the fibres may aggregate again, forming a separate hydrogel phase. It was very noticeable that the volume of the sediment was greatest when the Blend prepared with gamma-cyclodextrin, which showed the most efficient de-emulsifying effect, was added to the model emulsions.

[0150] We have developed a gas chromatographic method in order to determine the amount of oil bound to the cactus fibres. Aliquots were taken from the sedimented fibrous layers of a model emulsion containing soybean lecithin as emulsifier. Blends and physical mixtures of cactus fibre and cyclodextrin, prepared as in Examples 2(a), 2(b), 3(a) and 3(b) were each added to a stock emulsion of the following composition, prepared in the same way as described in Example 1:

[0151] 48 g sunflower oil (saturated with Sudan red)

[0152] 350 g distilled water

[0153] 0.73 g soybean lecithin

[0154] The sample preparation method was adapted from European Pharmacopoeia 2.4.22. 0.6-3.0 g samples comprising dissolved, bound or emulsified triglycerides (oil) were weighed exactly into test tubes. 2.00 ml hexane was added to the samples, and the mixture was vigorously shaken to extract the triglycerides into the organic phase for 30 seconds. When complete phase separation was attained, 1.00 ml of the upper (organic) phase was pipetted into test tubes. Hexane was removed by evaporation in a nitrogen stream. The remaining oil was transesterified by adding 1.0 ml methanol and 25 l solution of potassium hydroxide in methanol (c=60 g/l). The mixtures were gently boiled for 10 min under a nitrogen atmosphere. The reaction mixture was cooled and the transesterified fatty adds were extracted by 0.80 ml hexane. The solution obtained analysed by gas chromatography, using a Shimadzu GC-17A gas chromatograph with a Supelcowax 10 column (30 m0.32 mm1.0 m) Shimadzu AOC-5000 auto injector and a flame ionization detector, under standard conditions (Temperature program: injector temperature: 260 C., detector temperature: 260 C.; 170 C. initially, increased to 230 C. at 3 C./min and held for 15 min, increased to 260 C. at 15 C./min and held for 33 min) with split ratio 11:1. Shimadzu Class-VP 7.4 Version software was used.

[0155] As a reference sample, sunflower oil saturated with Sudan red dye was also transesterified and analysed. The numerical data showing the distribution of oil in the different separated phases are summarized in Table 5.

TABLE-US-00006 TABLE 5 Oil distribution amongst the separated phases in 50 ml of lecithin- containing model emulsions after a 24 hour storage period V.sub.oil in V.sub.oil in V.sub.oil in V.sub.oil in oil emulsion aqueous fibrous phase phase phase phase Sample (ml) (ml) (ml) (ml) Cactus powder 0.5 5.7 Not Not detected separated Cactus powder + 2.0 4.2 <0.001 Not alpha-CD separated (physical mixture) Cactus powder + 1.5 4.7 0.009 Not beta-CD separated (physical mixture) Cactus powder + 1.5 4.7 Not Not gamma-CD detected separated (physical mixture) Cactus powder + 5.9 0.31 Not 0.018 alpha-CD (Blend) detected Cactus powder + 5.6 0.61 Not 0.034 beta-CD (Blend) detected Cactus powder + 5.2 1.0 0.008 0.052 gamma-CD (Blend)

[0156] Oil was removed virtually completely from the aqueous phases (theoretically oil could be present in the form of micelles or phospholipid bilayers in the aqueous medium). The quantity of oil bound by the separated fibres was negligible. The highest amount was detected in the sample comprising cactus powder/gamma-cyclodextrin Blend; nevertheless this amount is only about 1% of the total initial amount of fat.

[0157] The Blends clearly showed higher de-emulsifying ability than physical mixtures. In this example cactus powder/alpha-cyclodextrin Blend removed the highest amount of fat from the emulsion state, but we have found that under these conditions the gamma-cyclodextrin-based Blend provided the highest rate of de-emulsification.

[0158] This gas chromatographic method also enabled the detection of eventual changes in the layer-by-layer distribution profile in terms of the acyl chain length of fatty acids. Preferential enrichment was not observed, ie. Oil was present in all layers in identical chemical compositions, regardless of the presence of very different quantities. Thus our results provide further support for the suggestion that the mechanism of the fat removal brought about by the cactus fibres is not likely to result from mere sorption of triglycerides into the formed fibrous phase.

Example 6: In Vivo Study of Efficacy in Animals

[0159] An in vivo study was performed on female Sprague-Dawley rats, with a study duration of 5 weeks. Groups of rats (6 rats per group) were housed individually in the polycarbonate cages, and were fed with the high fat diet (Harlan, TD 06414) throughout the study duration. The control group was treated orally with vehicle (spring water), while the first treatment group was treated orally with a daily dose of 200 mg/kg/day of the test composition prepared according to Example 2(c). Meanwhile, the second treatment group was treated orally with a daily dose of 200 mg/kg/day of fibre preparation (Cactus Fibre) only. Parameters such as body weight, absolute food intake, faeces consistency and animal behaviour (morbidity and mortality) were evaluated daily. Faecal fat was evaluated once on day 15.

[0160] The body weight of individual rats was measured daily, using a calibrated electronic weighing scale. The anti-obesity effect was evaluated based on the body weight different between the last treatment day (day 36) and baseline (day1). The numerical data showing the anti-obesity effect is summarized in Table 6.

[0161] The absolute food intake was assessed with reference to food intake and spillage.

[0162] Faeces consistency, morbidity and mortality were monitored by daily observation.

[0163] Faeces sampling was performed on day 15 by placing the rats in the metabolic cages for 24 hours. Faecal analysis was then performed to determine the faecal fat content. The fat indigestibility (%) was determined by the ratio of total faecal fat excreted (mg) and total dietary fat intake (mg). The numerical data showing the fat indigestibility (%) is summarized in Table 7.

TABLE-US-00007 TABLE 6 Anti-obesity effect of first treatment group (cactus fibre & gamma-cyclodextrin preparation at 7.5:1.5) and second treatment group. (cactus fibre) Cactus Fibre & gamma- Cyclodextrin preparation (7.5:1.5) Cactus Fibre Anti-Obesity Effect (g) 200 mg/kg/day 200 mg/kg/day Mean Body Weight 49.83* 26.00 difference between treatment group and vehicle Standard Deviation 14.59 10.51 *Significant difference (P < 0.05) compared with Cactus Fibre alone

TABLE-US-00008 TABLE 7 Fat indigestibility of first treatment group (cactus fibre & gamma-cyclodextrin preparation at 7.5:1.5) and second treatment group (cactus fibre) Cactus Fibre & gamma- Cyclodextrin preparation (7.5:1.5) Cactus Fibre Fat Indigestibility (%) 200 mg/kg/day 200 mg/kg/day Mean Fat Indigestibility 0.60* 0.16 difference between treatment group and vehicle Standard Deviation 0.21 0.19 *Significant difference (P < 0.05) compared with Cactus Fibre alone

Mean body weight (MBW) difference between treatment group & vehicle (g)=[MBW of treatment group on day 36MBW of treatment group on day 1][MBW of vehicle on day 36MBW of vehicle on day 1].

Fat Indigestibility (%)=[Total faecal fat excreted/Total dietary fat intake]100%.

Fat Indigestibility difference between treatment group & vehicle (%)Fat indigestibility of treatment groupFat Indigestibility of vehicle.

Example 7: Tablet Formulation I

[0164]

TABLE-US-00009 TABLE 8 Composition of tablet formulation comprising cactus powder and cyclodextrin Ingredient Quantity per tablet 1. Cactaceae-based composition comprising 500.0 mg cactus fibre/cyclodextrin Blend 2. Microcrystalline cellulose 142.0 mg 3. Calcium hydrogen phosphate dihydrate 20.0 mg 4. Silica (silicon dioxide) 6.5 mg 5. Povidone (Kollidon CL) 24.0 mg 6. Magnesium stearate 7.5 mg

[0165] Ingredients are dispensed according to the above formulation for a batch size of 2000 tablets. Ingredients 1 to 5 are sieved through a sieve of mesh size #20 (850 microns), and blended homogeneously in a laboratory-scale drum blender. Ingredient 6 through a sieve of mesh size #30 (600 microns) into the powder mixture, and further blended for 1 minute. The mixture is compressed into oblong tablets of 700 mg each using a rotary tablet press. The tablet hardness is approximately 200 kN, and disintegration time <60 minutes

Example 8: Tablet Formulation II

[0166]

TABLE-US-00010 TABLE 9 Composition of tablet formulation comprising cactus powder and cyclodextrin Quantity per tablet Ingredients (A) 1. Cactaceae-based composition comprising 500 mg cactus fibre/cyclodextrin Blend 2. Sodium selenite 2 mg 3. Chromium chloride hexahydrate 24 mg 4. Microcrystalline cellulose 122 mg 5. Calcium hydrogen phosphate 20 mg 6. Sodium starch glycolate 25 mg 7. Magnesium stearate 7 mg Ingredients (B) 1. Calcium carbonate 250 mg 2. Zinc gluconate 8 mg 3. Vitamin A acetate 500000 IE/g 1 mg 4. Vitamin E acetate 50% 10 mg 5. Vitamin D3 100000 IE/g 1 mg 6. Coenzyme Q10 5 mg 7. Kolidon 30 10 mg 8. Hydroxypropylmethylcellulose 60 mg 9. Cellulose 10 mg 10. Magnesium stearate 5 mg Ingredients (C) Opadry (II) white Y-22-7719 25 mg

[0167] Ingredients are dispensed according to the above formulation for a batch size of 2000 tablets.

[0168] To prepare granulate (A), ingredients (A) 1 to 6 are sieved through a sieve of mesh size #20 (850 microns), and blended homogeneously in a laboratory-scale drum blender. Ingredient (A) 7 is then sieved through a sieve of mesh size #30 (600 microns) into the powder mixture, and further blended for 1 minute.

[0169] To prepare granulate (B), ingredients (B) 1 to 9 are sieved through a sieve of mesh size #20, and blended homogenously in a laboratory-scale drum blender. Ingredient (B) 10 is then sieved through a sieve of mesh size #30 into the powder mixture, and further blended for 1 minute.

[0170] The granulates (A) and (B) are compressed into double-layer oblong tablets of 1060 mg each with a rotary tablet press. The hardness is approximately 200 kN.

[0171] The tablet may be film-coated in white to improve appearance with a commercial coating formulation such as Opadry (II) White Y-22-7719 (Colorcon, Inc.). Opadry (II) White Y-22-7719 colouring agent consists of titanium dioxide, polydextrose, hypromellose, triacetin and polyethylene glycol 8000. To prepare the coating suspension, Opadry (II) White Y-22-7719 is dispersed in water with a homogenizer at a concentration of 15% w/w, stirred for 45 minutes, and filtered to form a coating suspension. The tablets are coated with the suspension in a perforated bed coater until a weight gain of 25 mg per tablet is achieved.

[0172] Tablet formulations for other plant fibres or non-plant fibres (e.g. chitosan) with cyclodextrin may be prepared in a similar manner to methods described in Examples 7 and 8 above. In some cases, such as chitosan-cyclodextrin combinations (e.g. chitosan-gamma cyclodextrin), it may be preferable to include in the tablet a physical mixture of the fibre and cyclodextrin.

[0173] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.