Gel capsule containing sterol and solubilising agent

Abstract

The present invention relates to gel capsules comprising sterol and solubilizer for use as food supplement or as pharmaceutical.

Claims

1. A formulation for gel capsules comprising at least 61% by weight sterol ester and at least two solubilizers selected from the group consisting of polysorbates, lecithins, sodium stearoyl-2-lactylates, fatty acid esters of ascorbic acid, fatty acid esters of isoascorbic acid, and sugar esters with fatty acids.

2. The formulation according to claim 1, wherein the sterol ester has been obtained from sterols of plant origin (phytosterols) or the hydrogenated analogs thereof, the stanols.

3. The formulation according to claim 2, wherein the sterol ester has been obtained from sterols.

4. The formulation according to claim 1, wherein the sterol ester is obtained by esterification of fatty acids of natural origin.

5. The formulation according to claim 4, wherein the fatty acids are monounsaturated or polyunsaturated fatty acids.

6. The formulation according to claim 5, wherein the fatty acids comprise omega-3 fatty acids in an amount of at least 30 percent by weight, based on the fatty acid portion.

7. The formulation according to claim 1, wherein, at least one of the at least two solubilizers is selected from the group consisting of polysorbates, lecithins, sodium stearoyl-2-lactylates, and combinations thereof.

8. The formulation according to claim 7, comprising at least one wherein at least one of the at least two solubilizers is selected from the group consisting of fatty acid esters of ascorbic acid, fatty acid esters of isoascorbic acid, and sugar esters with fatty acids.

9. The formulation according to claim 1, wherein the at least two solubilizers are selected from the group consisting of ascorbyl fatty acid ester, lecithin, and polysorbate.

10. The formulation according to claim 1, wherein the formulation comprises water or oil, and wherein, the water or oil is present in an amount of not more than 15% by weight, based on the formulation.

11. A gel capsule comprising the formulation according to claim 1.

12. The gel capsule according to claim 11 in the form of a soft or hard capsule.

13. The gel capsule according to claim 11 for use as food supplement or as pharmaceutical.

14. A process for producing a formulation according to claim 1, comprising: (a) combining sterol ester and the at least two solubilizers to form a mixture, (b) heating the mixture to a temperature above the melting point of the sterol ester, and (c) mixing the heated mixture at 500 to 2500 rpm for 1 to 60 minutes to obtain a uniform mixture.

15. A process for producing a gel capsule according to claim 11, comprising: (a) combining sterol ester and the at least two solubilizers to form a mixture, (b) heating the mixture to a temperature above the melting point of the sterol ester, and (c) mixing the heated mixture at 500 to 2500 rpm for 1 to 60 minutes to obtain a uniform mixture; and (d) incorporating the uniform mixture into a gel capsule.

16. The formulation according to claim 1, wherein the sterol ester is obtained by esterification of fatty acids of plant or marine origin.

17. The formulation according to claim 8, comprising ascorbyl palmitate, lecithin and polysorbate 80.

18. The formulation according to claim 1, wherein the at least two solubilizers are lecithin and polysorbate.

19. The formulation according to claim 18, wherein the polysorbate is polysorbate 80.

Description

EXAMPLES

(1) Production of the Formulations

(2) Phytosterol ester and emulsifiers were combined and heated to 60? C. in order to avoid crystallization and/or, preferably and to reduce viscosity. Thereafter, mixing was carried out (Thinky mixer ARE-250 (Thinky Corporation, USA) at 2000 rpm for 1 min, or in an Ultra-Turrax tube drive (IKA, Germany) at 2000 rpm for about 30 min). The Thinky mixer was used when all input materials are present as liquid, whereas the Ultra-Turrax mixer was used when at least one of the input materials was present as semisolid or solid substance and could not easily dissolve in the liquid phytosterol ester phase or phytosterol ester phase liquefied at about 60? C. (e.g., protein, ascorbyl palmitate). The formulation was brought to room temperature (about 20 to 25C) in order to allow any possible foam formation during production to dissolve overnight (i.e., after about 8 to 12 hours), and examined for phase separation. If no phase separation was apparent, the application tests described below were carried out.

(3) All the results shown below have the numerical data in percent by weight.

(4) Application Tests

(5) Artificial Stomach Solution:

(6) 2 g of a 1 M hydrochloric acid were added to 900 ml of distilled water, the pH was adjusted to 1.6 with 1 M hydrochloric add, and distilled water was used to top up to 1 liter.

(7) Small Intestine Solution:

(8) 0.42 g of NaOH flakes, 3.95 g of NaH.sub.2PO.sub.4*H.sub.2O and 6.19 g of NaCl were dissolved in 900 ml of distilled water. The pH was adjusted to 6.5 with sodium hydroxide solution and the solution was topped up to 1 liter using distilled water.

(9) Testing in the Stomach Model:

(10) The phytosterol ester/emulsifier formulation was heated to about 60? C. in order to obtain a uniform liquid phase. Then, 100 ml of the stomach solution and 100 ml of the small intestine solution were each heated to 37-38? C., and 1 g of the formulation was added in each case. The systems were stirred at 200 rpm for one hour and then visually inspected.

(11) For the results, see Table 3.

(12) Caco2 Model

(13) The formulations were likewise tested in the so-called Caco2 model. This involves examining the effect of the administration of a formulation on the uptake of cholesterol into Caco2 cells. This is a measure of whether and how well the sterol esters in the formulation can lower the cholesterol uptake of the cells. The higher the prevention of the (i.e., the lower the) cholesterol uptake, the more effective a formulation.

(14) Principle Behind the Caco2 Testing:

(15) Cultivation of the Caco2 cells on a porous membrane; polarized structure of the cells, which take up nutrients (in this case: cholesterol) on the apical side and secrete them on the basal side.

(16) What is examined is the extent to which the transport of cholesterol is influenced by the simultaneous supplementation of phytosterols in combination with various vehicles.

(17) Testing steps: cultivation of the cells; production of the so-called mixed micelles; determination of the cytotoxicity and determination of the working concentration; measurement of the cholesterol concentration in the basal compartment (6 and 24 hours after the start of the supplementation) in 2 passes, triplicate measurement in each case.

(18) Cytotoxicity test: it was possible to use concentrations of 150 ?g/ml of all the test substances without impairment of the cells; working concentrations 50-100-150 ?g/ml were selected.

(19) For the formation of the micelles, the amount of cholesterol used for all treatment groups was kept constant. During the analysis of the apically applied solutions, it was established that the cholesterol content in the produced micelles varies (possibly caused by the intended mutual displacement of cholesterol and phytosterols during the micelle formation).

(20) The results of the cholesterol uptake or of the transport through were therefore corrected to the actual cholesterol content of the supplementation solutions.

(21) Sterol Ester Used:

(22) Vegapure 95 E, from BASF SE, comprises small amounts of various tocopherols and ascorbyl palmitates as antioxidant; proportion of sterol ester: at least 97% (area percentage); proportion of free sterol: up to 6% (area percentage).

(23) Solubilizers Used:

(24) Polysorbate 20: polyoxyethylene (20) sorbitan monolaurate, E432 Polysorbate 80: polyoxyethylene (20) sorbitan monooleate, E433, Tween 80 Lipoid P 75: lecithin fraction and phospholipids from soybeans, comprises about 75% phosphatidylcholine, 7% phosphatidylethanolamine Prefera SSL 6000: sodium stearoyl-2-lactylate having a degree of esterification of 100 to 140 Lametop P 65: FDA-specification DATEM (DATEM=diacetyl tartaric acid ester of mono- and diglycerides), E472e Phosal 40 IP: liquid composition comprising about 40 percent soybean phophatidylcholine and sunflower oil and also mixed tocopherols MCT: medium chain triglycerides, an oil comprising predominantly C8 to C10 fatty acids Lecico SUN FM 580: sunflower lecithin from Lecico; liquid, enzymatically modified sunflower lecithin, 56% AU CholestOff: commercially available comparative product from NatureMade, USA; comprising plant sterols and stanols.

(25) The following Table 1 (1a and 1b) shows tested formulations.

(26) TABLE-US-00001 TABLE 1a Compositions in gram Vegapure Propylene Sum No. Vehicle 95 E glycol MCT Water total 1 Polysorbate 20 7.5 31.5 0 0 0 39 2 Prefera SSL 6000 7.5 31.5 0 0 0 39 3 Polysorbate 80 + water 3.75 31.5 0 0 3.75 39 4 Polysorbate 80 7.5 31.5 0 0 0 39 5 Lametop P 65 + MCT 3.75 31.5 0 3.75 0 39 6 Lametop P 65 + propylene 3.75 31.5 3.75 0 0 39 glycol 7 Lametop P 65 7.5 31.5 0 0 0 39 8 Phosal 40 IP 7.5 31.5 0 0 0 39 9 Lipoid P 75 + water 0.63 31.5 0 0 6.87 39

(27) TABLE-US-00002 TABLE 1b Compositions (continuation of Table 1a) Percent by weight Vegapure Propylene Sum No. Vehicle Vehicle 95 E glycol MCT Water total 1 Polysorbate 20 19.23% 80.77% 0.00% 0.00% 0.00% 100.00% 2 Prefera SSL 6000 19.23% 80.77% 0.00% 0.00% 0.00% 100.00% 3 Polysorbate 80 + water 9.62% 80.77% 0.00% 0.00% 9.62% 100.00% 4 Polysorbate 80 19.23% 80.77% 0.00% 0.00% 0.00% 100.00% 5 Lametop P 65 + MCT 9.62% 80.77% 0.00% 9.62% 0.00% 100.00% 6 Lametop P 65 + propylene 9.62% 80.77% 9.62% 0.00% 0.00% 100.00% glycol 7 Lametop P 65 19.23% 80.77% 0.00% 0.00% 0.00% 100.00% 8 Phosal 40 IP 19.23% 80.77% 0.00% 0.00% 0.00% 100.00% 9 Lipoid P 75 + water 1.62% 80.77% 0.00% 0.00% 17.62% 100.00%
Results of the Tests: Table 1 (1a and b): tested formulationscomposition FIG. 1: Cholesterol content of the basal recipient medium after 6 hours (in % of the applied starting concentration) FIG. 2: Cholesterol content of the basal recipient medium after 24 hours (in % of the applied starting concentration) FIG. 3: Infrared spectra of the micelle solutions of cholesterol (CHOL) alone and in combination with the tested formulations FIG. 4: PCA analysis of the micelle solutions of cholesterol (CHOL) alone and in combination with various test substances (C1 to C10) FIG. 5: Results relating to Table 4

(28) Further results from the Caco2 model (figure relating to Table 3) Table 2: Influencing of the transport of cholesterol by the phytosterol-vehicle combination in comparison with Vegapure 95E as individual substance Table 3: Further results from the Caco2 model Table 4: Results of the testing in the stomach model (Sterol ester=Vegapure 95 E from BASF)

(29) TABLE-US-00003 TABLE 2 Influencing of the transport of cholesterol by the phytosterol-vehicle combination in comparison with Vegapure 95E as individual substance After 6 hours After 24 hours Cholesterol in % less Cholesterol in % less the recipient cholesterol the recipient cholesterol Formulation Dosage of compartment than Vegapure compartment than Vegapure comprising as the test (% of the 95E without (% of the 95E without solubilizers substance amount used) vehicle amount used) vehicle Cholesterol control 78.72 76.11 No solubilizer (only 50 ?g/ml 70.85 67.62 Vegapure 95 E) 100 ?g/ml 70.79 63.54 150 ?g/ml 66.32 59.05 Prefera SSL 6000 50 ?g/ml 54.46 23% 56.96 16% 100 ?g/ml 54.46 23% 59.97 6% 150 ?g/ml 45.10 32% 55.46 6% Polysorbate 80 50 ?g/ml 59.23 16% 56.77 16% 100 ?g/ml 66.56 6% 60.32 5% 150 ?g/ml 65.02 2% 62.11 n.a. Lametop P65 + MCT 50 ?g/ml 69.41 2% 72.51 n.a. 100 ?g/ml 53.04 25% 63.92 n.a. 150 ?g/ml 50.74 23% 55.64 6% Phosal 40 IP 50 ?g/ml 57.76 18% 71.85 n.a. 100 ?g/ml 53.88 24% 65.75 n.a. 150 ?g/ml 49.67 25% 58.78 0%
Measurement of the Displacement of Cholesterol by Phytosterol During the Micelle Formation

(30) Cholesterol and phytosterols are poorly water-soluble and are transported in the body together with fatty acids and bile salts in the mixed micelles.

(31) The displacement of cholesterol from the micelles is seen as an important mechanism of action of the phytosterols for the lowering of cholesterol.

(32) The goal of the measurement is to determine the influence of the test substances on the cholesterol concentration in the micelle solution obtained in order to thus obtain an indication of the possible displacement of the cholesterol from the micelles.

(33) The test substances were used in 5 different concentrations together with a defined cholesterol concentration for in vitro micelle formation.

(34) The micelle emulsions obtained were examined by infrared spectroscopy using the SpeCCs analyzer (Cetics Healthcare).

(35) At first glance, the infrared spectra (FIG. 3) differ only slightly owing to the similarity in the chemical structure of cholesterol and phytosterols. Further evaluation is done statistically using principal-component analysis (PC), by means of which it is possible to determine the similarity of samples.

(36) PCA analysis of the micelle solutions of cholesterol (CHOL) alone and in combination with various test substances (C1 to C10) (FIG. 4)

(37) Result: The formulation comprising Lipoid P75+water differs most distinctly from the pure cholesterol micelles, followed by Vegapure 95 E, polysorbate 80, Lametop P65+MCT and Prefera SSL 6000, and this can be seen as a measure of the displacement of cholesterol from the micelles.

(38) TABLE-US-00004 TABLE 3 Caco2 model preliminary results - influencing of the transport of cholesterol by combinations of phytosterol esters and emulsifiers Cholesterol Cholesterol Dosage of concentration in concentration in the test the recipient the recipient substance medium in ?g/ml medium in ?g/ml Formulations tested (?g/ml) (after 6 hours) (after 24 hours) Experiment 88; Vegapure 95 E 150 69.9 64.9 (without emulsifier) Experiment 88; Vegapure 95 E 100 65.0 64.7 (without emulsifier) Experiment 88; Vegapure 95 E 50 68.2 64.4 (without emulsifier) Experiment 89: Vegapure 95 E with 150 63.2 63.3 Lipoid 75 and sunflower oil Experiment 89: Vegapure 95 E with 100 65.7 61.0 Lipoid 75 and sunflower oil Experiment 89: Vegapure 95 E with 50 62.7 59.4 Lipoid 75 and sunflower oil Experiment 100: Vegapure 95 E and 150 61.3 57.4 Lecico SUN FM 580 and Tween 80 Experiment 100: Vegapure 95 E and 100 60.6 60.1 Lecico SUN FM 580 and Tween 80 Experiment 100: Vegapure 95 E and 50 57.2 58.6 Lecico SUN FM 580 and Tween 80 CholestOff (commercially available 150 59.42 60.4 comparative product) CholestOff (commercially available 100 56.93 58.3 comparative product) CholestOff (commercially available 50 60.66 60.5 comparative product)

(39) The results in Table 3 show that the transport of cholesterol can be influenced more greatly when using a combination of phytosterol ester and emulsifier(s) than by the phytosterol ester alone.

(40) Table 4 shows the assessment of the testing of the formulations from Table 3.

(41) FIG. 5 shows selected results in the Caco2 test of formulations from Table 3.

(42) Evaluation relating to Table 4: Visual assessment scale

(43) 1=Homogeneous, no oil on the surface

(44) 2=Largely homogeneous with small to moderate oil droplets on the surface

(45) 3=Moderate turbidity, noticeable oil layer on the surface

(46) 4=Slightly whitish-turbid appearance, thick oil layer on the surface

(47) 5=No emulsification effect, i.e., virtually complete phase separation

(48) TABLE-US-00005 TABLE 4 Results of the testing in the stomach model (Sterol ester = Vegapure 95 E from BASF); see also FIG. 5 Input Input Input Sterol ester material 1 material 2 material 3 Experiment concentration Input conc. Input conc. Input conc. number (% by weight) material 1 (% by weight) material 2 (% by weight) material 3 (% by weight) 88 100 Vegapure 95E 100 89 80 P75 1.6 Sunflower oil 17.6 90 80 Lecico SUN FM 20 580 91 85 Lecico SUN FM 15 580 92 90 Lecico SUN FM 10 580 93 80 Tween 80 20 94 98 Tween 80 2 95 90 Ascorbyl 10 palmitate 96 98 Ascorbyl 2 palmitate 97 80 Lecico SUN 20 400 98 80 Lecico SUN FM 19 Tween 80 1 580 99 80 Lecico SUN FM 18 Tween 80 2 580 100 80 Lecico SUN FM 16 Tween 80 4 580 101 80 Lecico SUN FM 18 Ascorbyl 2 580 palmitate 102 80 Lecico SUN FM 16 Ascorbyl 4 580 palmitate 103 80 Lecico SUN FM 12 Ascorbyl 8 580 palmitate 104 80 Lecico SUN FM 8 Ascorbyl 12 580 palmitate 105 85 Lecico SUN FM 13.5 Ascorbyl 1.5 580 palmitate 106 90 Lecico SUN FM 9 Ascorbyl 1 580 palmitate 107 80 Lecico SUN FM 16 Ascorbyl 2 Tween 80 2 580 palmitate 108 80 Lecico SUN FM 19 Systerna 1 580 SP70 109 80 Lecico SUN FM 18 Systerna 2 580 SP70 Stomach Stomach Intestine Intestine solution: solution: solution: solution: Experiment turbidity visual turbidity visual number (NTU) assessment (NTU) assessment 88 0.3 5 1 5 89 485 2 317 3 90 266 3 1000 2 91 59 4 763 3 92 71 4 492 4 93 63 4 111 4 94 102 3 27 4 95 9.2 4 1000 2 96 1.1 5 90 4 97 31 4 814 2 98 554 2 1000 1 99 1000 1 1000 1 100 769 2 1000 1 101 25 4 922 2 102 237 2 46 5 103 516 2 50 5 104 1000 3 1000 1 105 43 4 679 2 106 18 5 379 3 107 383 2 1000 2 108 375 2 830 2 109 51 4 539 3