PREPARATION METHOD OF A COLLOIDAL SYSTEM OF STABILISATION AND CONTROLLED RELEASE OF ROYAL JELLY COMPONENTS FOR VARIOUS USES

Abstract

A preparation method of a colloidal system of stabilisation and controlled release of royal jelly ingredients, wherein the system is characterized by the extraction of the components of the royal jelly and the simultaneous encapsulation in combinatorial liposome -cyclodextrin carriers for achieving controlled release, taking advantage of the special features of both carriers, namely: the ability of cyclodextrins to encapsulate polyphenols and their enhanced skin penetration ability as well as the ability of liposomes in encapsulating large amounts of components of various degrees of polarity, for local component transport and for controlled release rate.

Claims

1. A preparation method of a colloidal stabilization system and controlled release of royal jelly components, wherein, for the preparation of the release system of components of royal jelly: in the first stage the fresh royal jelly is kept in a temperature of 5-7° C. and contains 10-hydroxydecenoic acid (10-HDA) in a concentration of > 1.8% w/w, as determined by the method of Koshio et al. and is allowed to reach room temperature in the shortest possible time; in the second stage it is gradually dissolved, under stirring in a system of solvents which consists of deionized water, in which hydroxypropyl-β-cyclodextrin or β-cyclodextrin or γ-cyclodextrin in a concentration of 1% -22% w/w has been pre-dissolved and either vegetable 1, 3-propanediol or glycerol or vegetable butylene glycol in a ratio of 1, 3-propanediol (or glycerol or butylene glycol)/water: from 5/95 to 90/10, where the concentration of royal jelly in the solvent system ranges between 0.5% to 6.0% w/w; in the third stage the system of royal jelly/ cyclodextrin/ solvent system is subjected to stirring (1000-3000 rpm) for 30 to 90 minutes at a temperature of 20° C. to 26° C.; in the fourth stage, the system royal jelly/ cyclodextrin/ solvent is added to the liposomal system at a rate of 10 ml/sec at a concentration of 1.0-7.5% w/w, after the complete addition of which the pH of the system is set in the range 5.0-8.2 and either Disodium Ethylenediaminetetraacetic acid or Tetrasodium Glutamate Diacetate is added at a concentration of 0.1-0.2% w/w the system subjected to stirring at 4000 rpm for 4 hours; in the fifth step, the mixture is kept in a hermetically sealed container to rest for 24 hours at 5-7° C. and then, the colloidal system is cold filtered through an array cartridge filters of pore size 5 .Math.m-1 .Math.m-0.45 .Math.m and the pH is retested which if necessary is readjusted in the range 5.0-8.2; in the sixth stage, the system is allowed to reach room temperature and the mean hydrodynamic particles diameter is measured which should range between 220 nm to 650 nm with a polydispersity index ranging from 0.25 to 0.68, while in case the measured values are out of tolerances, a second filtration, at room temperature through an array of cartridge filters with pore size 0.45 .Math.m-0.2 .Math.m, follows; in the final stage, after finding that the values of mean hydrodynamic diameter and polydispersity index are within specifications, the total content of polyphenols and total content of 10-hydroxydecenoate acid are measured, while continuously the release rate of polyphenols and 10-hydroxydecenoic acid is measured in a buffer at pH 7.2 at 37° C. and the colloidal system is stored in a dark container at a temperature of 5-7° C., and is stable for 1 year.

2. A preparation method of a colloidal stabilization system and controlled release of royal jelly ingredients according to claim 1 wherein the requirement of deionized water quality is: <= 1 .Math.S/cm at 25° C.

3. A preparation method of a colloidal stabilization system and controlled release of royal jelly ingredients according to claim 1, wherein for the production of deionized water the water of the water supply network is introduced into a tank with a suitable pumping unit, passes through an automatic turbidity and activated carbon filter and then a scale inhibitor is dosed to remove its hardness and then it enters the main unit of the reverse osmosis unit, of 350 lt/h capacity with 70% recovery after having passed first, through a 1 micron cartridge filter and is then tanked in a stainless steel tank, from which it supplies the deionizer with a suitable system.

4. A preparation method of a colloidal stabilization system and controlled release of royal jelly ingredients according to claim 1, wherein, in a suitable container, the production of liposomal system, which consists of 50%-95% Phosphatidylcholine, 2%-10% Phosphatidylethanolamine, 0%-3% Lysophosphatidylcholine, 0% -3% Phosphatidylinositol, 0% -3% Phosphatidate acid, 0% -22% cholesterol, 0-17% bile salts, is performed, in solvent 1.3-propanediol or glycerol, in a concentration ranging from 20/80 to 80/20% w/w.

5. A preparation method of a colloidal stabilization system and controlled release of royal jelly ingredients according to claim 1, wherein the total content of polyphenols in the system should be greater than 200 mg/L GAE.

6. A preparation method of colloidal stabilization system and controlled release of royal jelly ingredients according to claim 1, wherein the total content of 10-hydroxydecenoic acid 10-HDA in the system in should be greater than 0.04% w/w.

7. A preparation method of colloidal stabilization system and controlled release of royal jelly ingredients according to claim 1, wherein the release of polyphenols (cumulative release) at pH 7.2 and temperature 37° C. is 25 - 75% in 8 hours with total release of encapsulated polyphenols from the system at 24 hours.

8. A preparation method of colloidal stabilization system and controlled release of royal jelly ingredients according to claim 1, wherein the release of 10-hydroxydecenoate 10-HDA acid at pH 7.2 and 37° C. is 40% - 80% in 8 hours with total release of encapsulated 10-HDA-10-hydroxydecenoic acid from the system in 12 hours.

9. A stable colloidal dispersion system of royal jelly that releases in a controlled manner its components which is produced according to the method of claim 1, wherein its total content in polyphenols is greater than 200 mg/L GAE, its total content in 10-HDA 10-hydroxydecenoic acid is greater than 0.04% w/w, while the release, for polyphenols (cumulative release) at pH 7.2 and temperature 37° C. ranges from 25% to 75% in 8 hours with total release taking place in 24 hours, while for 10-HDA 10-hydroxydecenoic acid is 40% up to 80% in 8 hours with total release taking place in 12 hours.

Description

INVENTION DISCLOSURE

[0029] In order for our invention to be fully understood by those skilled in the art, we then proceed to the detailed description of the preparation method of the system of dispersion of fresh royal jelly in combinatorial liposome / cyclodextrin carriers.

[0030] In order for the system to be prepared, the fresh royal jelly, maintained at 5-7° C., is left to reach room temperature for the shortest time possible. Minimum requirement of the concentration of hydroxydecenoic acid (10-HDA) in the royal jelly is > 1.8% w/w, as determined by the method of Koshio et al.

[0031] The royal jelly is then gradually dissolved under stirring in a solvent system consisting of deionized water and either vegetable 1,3-propanediol or glycerol or vegetable butylene glycol in a ratio of 1,3-propanediol (or glycerol or butylene glycol) / water: 5/95 to 90/10. The concentration of royal jelly in the solvent system ranges between 0.5% to 6.0% w/w. Hydroxypropyl-β-cyclodextrin or β-cyclodextrin or γ-cyclodextrin is pre-dissolved in deionized water at a concentration of 1% -22% w/w.

[0032] The system royal jelly / cyclodextrin / solvent system is subjected to stirring (1000-3000 rpm) for 30-90 min and at a temperature of 20° C. to 26° C.

[0033] The quality requirement of deionized water is: <= 1 .Math.S/cm at 25° C., which meats the specification of European Pharmacopoeia for the preparation of parenteric pharmaceutical products. This quality requirement is necessary for the complete absence of charges in the final formulation that could cause the lipid membranes of the liposomes to agglomerate and eventually to a reduced product stability.

[0034] The preparation of deionized water to be used in our invention is as follows:

[0035] Water from the water supply system is introduced into the raw water tank (volume 2 m.sup.3 ), with a suitable pumping unit, it passes through an automatic turbidity filter to remove the turbidity and the solid particles and activated carbon to remove chlorine and organic charge and then a scale inhibitor is dosed to remove its hardness. Before its introduction to the main unit of reverse osmosis unit, it passes through a 1 micron cartridge filter.

[0036] Fully treated water for use in reverse osmosis is introduced in the reverse osmosis unit of 350 It/h capacity with 70% recovery. The water produced from the unit is tanked in a stainless steel tank, of 5 m.sup.3 volume. From this tank, water is supplied with a suitable pumping unit, to the deionizer and is driven on-line to the extraction tank via UV radiation. To avoid stagnant water in the network, water is under continuous recirculation with return to the tank.

[0037] The preparation of the liposomal system takes place in a separate container. The liposomal system consists of 50%-95% Phosphatidylcholine, 2%-10% Phosphatidylethanolamine, 0%-3% Lysophosphatidylcholine, 0%-3% Phosphatidylinositol, 0%-3% Phosphatidic acid, 0%-22% cholesterol and 0-17% bile salts, in solvent 1,3-propanediol or glycerol. The ratio of the above components to 1,3-propanediol or glycerol range from 20/80 to 80/20% w/w. Then, the system royal jelly / cyclodextrin / solvent system is added into the liposomal system at a rate of 10 ml/sec in a ratio of 1.0-7.5% w/w. After complete addition, the system pH is adjusted in the range between 5.0-8.2 and either Disodium Ethylenediaminetetraacetic acid or Tetrasodium Glutamate Diacetate is added at a concentration of 0.1-0.2% w/w. The system is stirred at 4000 rpm for 4 hours.

[0038] The mixture is then left in a hermetically sealed container at 5-7° C. for 24 hours. The colloidal system is then subjected to cold filtration through an array cartridge filters of pore size 5 .Math.m-1 .Math.m-0.45 .Math.m and the pH is retested which if necessary is readjusted in the range 5.0-8.2.

[0039] The system is then allowed to reach room temperature and the mean hydrodynamic particles diameter is measured which should range between 220 nm to 650 nm with a polydispersity index ranging from 0.25 to 0.68. In case the measured values are out of limits, a second filtration, at room temperature, through an array of cartridge filters with pore size 0.45 .Math.m-0.2 .Math.m follows.

[0040] Once the values of mean hydrodynamic diameter and polydispersity index are within the specifications the total content of polyphenols and total 10-HDA content in the system are assessed.

[0041] Consequently the release rate of polyphenols and 10-HDA is determined in a buffer at pH 7.2 at 37° C. and the colloidal system is stored in a dark container at a temperature of 5-7° C., where it remains stable for 1 year.

[0042] Based on the method described above in order for the invented system to be suitable for its intended use, the total content in polyphenols should be greater than 200 mg/L GAE while the total content in 10-HDA should be greater than 0.04% w/w.

[0043] The release of polyphenols (cumulative release) at a pH of 7.2 and at temperature of 37° C. is 25%-75% in 8 hours while the system releases practically all encapsulated polyphenols in 24 hours. The respective values for 10-HDA are 40%-80% in 8 hours while the system releases practically the total amount of encapsulated 10-HDA in 12 hours.

[0044] In order to make the present invention, fully understood, we proceed to following examples:

Example 1

[0045] RJ after reaching ambient temperature is dispersed in a concentration of 2% to a solvent system comprised of water and vegetable 1,3-propanediol in a ratio of 1,3-propanediol/water: 50/50. In the aqueous phase hydroxypropyl-β-cyclodextrin is pre-dissolved at a concentration of 9%.

[0046] As the system is under intense stirring (3000 rpm) and at temperature of 20° C. the liposomal suspension is added at a per weight concentration of 3.0%. The procedure follows as described in the disclosure of the invention.

[0047] The final colloidal suspension after the filtration exhibit the values shown in Table 1.

TABLE-US-00001 Parameter Value Mean particle hydrodynamic diameter 120-250 nm Polydispersity index <0.68 10-HDA 0.05% w/w Total polyphenols (gallic acid equivalents) 350 mq/L Cummulative release of 10- HDA in 8 hours% 65-75 Cummulative release of polyphenols in 8 hours% 42-60

Example 2

[0048] RJ after reaching ambient temperature is dispersed in a concentration of 5% to a solvent system comprised of water and vegetable 1,3-propanediol in a ratio of 1,3-propanediol/water: 75/25. In the aqueous phase, hydroxypropyl-β-cyclodextrin is pre-dissolved at a concentration of 12%.

[0049] As the system is under intense stirring (3000 rpm) and at temperature of 20° C. the liposomal suspension is added at a per weight concentration of 3.5%. The procedure follows as described in the disclosure of the invention.

[0050] The final colloidal suspension after the filtration exhibit the values shown in Table 1.

TABLE-US-00002 Parameter Value Mean particle hydrodynamic diameter 120-250 nm Polydispersity index <0.68 10-HDA 0.08% w/w Total polyphenols (gallic acid equivalents) 430 mq/L Cummulative release of 10- HDA in 8 hours% 60-75 Cummulative release of polyphenols in 8 hours% 40-65

[0051] Determination of mean hydrodynamic diameter and Polydispersity index is performed via Dynamic Light Scattering.

REFERENCES CITED IN THE DESCRIPTION

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