METHOD FOR PREPARING A STABLE CONTROLLED-RELEASE PROPOLIS COLLOIDAL DISPERSION SYSTEM FOR VARIOUS USES

Abstract

Method for preparing a stable controlled-release propolis colloidal dispersion system, wherein propolis is micronized and then dispersed in the solvent system, which is under stirring and consists of deionized water and either natural 1,3-propanediol or glycerol, in which deionized water hydroxypropyl--cyclodextrin or -cyclodextrin has been predissolved, and as the propolis/cyclodextrin/solvents system is under intense stirring for 30-45 min, a liposomal suspension is added thereto, and stirring is performed for 1 to 4 hours under continuous measurement of the total content in polyphenols and of the encapsulation efficiency %, until the specific parameters reach the desired values so that the stirring process is ended and the colloidal system is filtrated through suitable filters, its pH is adjusted and the size of dispersed particles as well as the release rate of the polyphenols of propolis in a buffer solution at pH 7.2 and 37 C. are measured and the colloidal system is stored.

Claims

1. Method for preparing a stable controlled-release propolis colloidal dispersion system, characterized in that in order to produce the system, propolisafter being frozen for 24 hoursis micronized (<1 mm) and then dispersed at a rate of 1 kg/min in the solvent system, which is under stirring at 500-3000 rpm and consists of deionized water of <=1 S/cm at 25 C. and either natural 1,3-propanediol or glycerol at a mixture ratio 1,3-propanediol (or glycerol)/water from 15%/85% to 80%/20%, in which deionized water hydroxypropyl--cyclodextrin or -cyclodextrin has been predissolved to 2-10% w/w, and as the propolis/cyclodextrin/solvents system is under intense stirring (2000-3000 rpm) for 30-45 min and at a temperature of 20 C. to 38 C., a liposomal suspension is added thereto at a ratio of 0.3 to 3.5% w/w, and stirring is performed for 1 to 4 hours under continuous measurement of the total content in polyphenols by the Folin-Ciocalteu method and of the % encapsulation efficiency, until the specific parameters reach the desired values so that the stirring process is ended and the colloidal system is filtrated through suitable cartridge filters with a pore size of 0.45 m, its pH is adjusted at the range of 5.0-8.0 and the size of dispersed particles as well as the release rate of the polyphenols of propolis in a buffer solution at pH 7.2 and 37 C. are measured and the colloidal system is stored in a dark-coloured container at a temperature of 5-7 C., where it is kept stable for 2 years.

2. Method for preparing a stable controlled-release propolis dispersion colloidal system, according to claim 1, wherein the propolis used contains total polyphenols >1600 mg/l gallic acid after dissolution of 10% (w/w) propolis in ethanol and subsequent measurement in a spectrophotometer, its concentration ranging from 7.5% to 28% depending on its polyphenol content.

3. Method for preparing a stable controlled-release propolis dispersion colloidal system, according to claim 1, wherein the liposomal suspension added consists of large unilamellar liposomes, lipidic bilayers and natural 1,3-propanediol and is at a temperature higher than the phase transition temperature of its phospholipids

4. Method for preparing a stable controlled-release propolis dispersion colloidal system, according to claim 1, wherein the lipid composition of the liposomal suspension is: 50%-90% phosphatidylcholine 2%-10% phosphatidylethanolamine 1%-3% lysophosphatidylcholine 1%-3% phosphatidylinositol 1%-3% phosphatidic acid 0%-40% cholesterol and 0-20% cholate salts.

5. Method for preparing a stable controlled-release propolis dispersion colloidal system, according to claim 1, wherein the colloidal system is acceptable if its application on NHDF (primary human skin fibroblasts) leads to an increase in their vitality by at least 100% vs. control, which is represented by the increase in ATP (adenosine triphosphate) therein.

6. Method for preparing a stable controlled-release propolis dispersion colloidal system, according to claim 1, wherein the size of produced liposomes is in the range of 70 to 700 nm and their polydispersity index is less than 0.5 (<0.5), the cumulative release of the polyphenols at pH 7.2 and a temperature of 37 C. is 25-60% within 8 hours, while the system releases all encapsulated polyphenols within 24 hours.

Description

DISCLOSURE OF THE INVENTION

[0030] In order to make the present invention fully understood by those skilled in the art, a detailed description of the preparation method of the colloidal dispersion system of propolis in combinatorial liposome/cyclodextrin carriers is given.

[0031] In order to prepare a system, propolis is initially micronized (<1 mm) after being frozen for 24 hours. Any propolis may be used without previous treatment, as long as it contains total polyphenols >1600 mg/I gallic acid after dissolution of 10% (w/w) propolis in ethanol and subsequent measurement in a spectrophotometer by the Folin-Ciocalteau method. Thereafter, the micronized propolis is dispersed at a rate of 1 kg/min in the solvent system under stirring at 500-3000 rpm. The propolis concentration is in the range of 7.5 to 28% (w/w) depending on polyphenol content. The system of extraction solvents consists of deionized water and either 1,3-propanediol or glycerol at a ratio of 1,3 propanediol (or glycerol)/water: 15/85 to 80/20. In the deionized water, hydroxypropyl--cyclodextrin or -cyclodextrin has been predissolved at 2-10% w/w.

[0032] The quality specification of the deionized water is <=1 S/cm at 25 C., which meets the specification of European Pharmacopoeia for the preparation of parenteric pharmaceutical products. This quality is necessary for full absence of loads in the final formulation which would otherwise cause the lipid membranes of the liposomes to aggregate and finally lead to a decrease in the product stability.

[0033] The preparation of the deionized water used in our invention is as follows:

[0034] Tap water is introduced in the raw-water tank (volume 2 m.sup.3) by a suitable pumping system, passed through an automatic turbidity filter to remove turbidity and solid particles and activated carbon to remove chlorine and organic load and then an antiscalant is dosed to remove its hardness. Before its input in the central plant of the reverse-osmosis unit, it is passed through a 1-micron cartridge filter.

[0035] The fully treated water for use in reverse osmosis is introduced in the reverse-osmosis unit of a productivity of 350 It/h with a recovery of 70%. The water produced from the unit is kept in a stainless-steel 5-m.sup.3 tank. From this tank, water is supplied by a suitable pumping system to the deionizer and is directly supplied to the tank of extraction through U.V. radiation. In order to avoid stagnant water in the network, water is circulated continuously with return to the tank.

[0036] As the system of propolis/cyclodextrin/solvents is under intense stirring (2000-3000 rpm) for 30-45 min and at a temperature of 20 C. to 38 C., a liposomal suspension is added thereto at a proportion of 0.3 to 3.5% (w/w). If required, pH adjustment in the range of pH 5-8 is previously performed, depending on the propolis used. The liposomal suspension added consists of large unilamellar liposomes, lipidic bilayers and natural 1,3-propanediol.

[0037] The liposomal suspension is at a temperature higher than the phase transition temperature of its phospholipids and its lipidic composition is: [0038] 50%-90% phosphatidylcholine [0039] 2%-10% phosphatidylethanolamine [0040] 1%-3% lysophosphatidylcholine [0041] 1%-3% phosphatidylinositol [0042] 1%-3% phosphatidic acid [0043] 0%-40% cholesterol [0044] 0-20% cholate salts

[0045] Stirring is performed for 1 to 4 hours, and at the same time the following parameters are measured: [0046] Total polyphenol content by the Folin-Ciocalteau method [0047] % Encapsulation efficiency

[0048] When the above-mentioned parameters reach the desired values, the colloidal system is filtrated through suitable filters (cartridge filters) having a pore size of 0.45 pm and pH is adjusted to 5.0-8.0, as required, and stirring is ended.

[0049] The colloidal system is acceptable if its application on NHDF (primary human skin fibroblasts) leads to an increase in their vitality by at least 100% vs. control. The increase in the vitality of cells is represented by the increase in ATP (adenosine triphosphate) therein.

[0050] Thereafter, the size of dispersed particles and the release rate of the polyphenols of propolis is measured in a buffer solution at pH 7.2 at 37 C. and the colloid is stored in a dark-coloured container at a temperature of 5-7 C., where it is kept stable for 2 years.

[0051] The size of the liposomes produced by this method is in the range of 70 to 700 nm and the polydispersity index is less than 0.5 (<0.5), which is a requirement for their stability. The cumulative release of the polyphenols at pH 7.2 and at a temperature of 37 C. is 25-60% for 8 hours while the system releases practically all the encapsulated polyphenols within 24 hours.

[0052] In order to make the present invention fully understood, the following examples are presented:

Example 1

[0053] Propolis which was micronized (<1 mm) after deep freezing for 24 hours is dispersed in the solvent system consisting of water and natural 1,3-propanediol at a ratio 1,3-propanediol/water: 45/55. In the aqueous phase hydroxypropyl--cyclodextrin has been predissolved to a content of 6.45%.

[0054] As the system is under intense stirring (3000 rpm) and at a temperature of 20 C., a liposomal suspension is added thereto at a proportion of 3.5% (w/w). The end point of the extraction is determined by the values of Total polyphenols and Encapsulation efficiency % determined, and is confirmed by the value of ATP increase vs control in NHDF % as shown in the Table 1.

TABLE-US-00001 TABLE 1 Parameter Value Total polyphenols* >1600 80 (mg/l gallic acid) Encapsulation efficiency % >95 ATP increase vs control in NHDF % >100 *The starting material of propolis should contain total polyphenols >1600 mg/l gallic acid after dissolution of 10%(w/w) propolis in ethanol.

[0055] The final colloidal suspension after filtration presents the values given in table 2

TABLE-US-00002 TABLE 2 Parameter Value Mean hydrodynamic particle diameter 100-250 nm Polydispersity index <0.5 Cumulative release in 8 hours % 25-60

Example 2

[0056] Propolis which was micronized (<1 mm) after deep freezing for 24 hours is dispersed in the solvent system consisting of water and natural 1,3-propanediol at a ratio 1,3-propanediol/water: 25/75. In the aqueous phase hydroxypropyl--cyclodextrin has been predissolved to a content of 4.45%.

[0057] As the system is under intense stirring (3000 rpm) and at a temperature of 35 C., a liposomal suspension is added thereto at a proportion of 1.5% (w/w). The end point of the extraction is determined by the values of Total polyphenols and Encapsulation efficiency % determined, and is confirmed by the value of ATP increase vs control in NHDF % as shown in the Table 3.

After completion of the extraction, propolis is filtrated to remove insoluble components.

TABLE-US-00003 TABLE 3 Parameter Value Total polyphenols* >1600 80 (mg/l gallic acid) Encapsulation efficiency % >95 ATP increase vs control in NHDF % >100

[0058] The starting material of propolis should contain total polyphenols >1600 mg/l gallic acid after dissolution of 10% w/w propolis in ethanol.

[0059] The final colloidal suspension after filtration presents the values given in table 4

TABLE-US-00004 TABLE 4 Parameter Value Mean hydrodynamic particle diameter 100-350 nm Polydispersity index <0.5 Cumulative release in 8 hours % 25-60

[0060] The determination of the mean hydrodynamic diameter and Polydispersity index is effected by Dynamic Light Scattering.

[0061] An in vitro study of the release of the polyphenols of propolis was performed by means of dialysis sacks:

[0062] A specific quantity of the colloid is placed in the dialysis sacks of a MWCO=1000. The sack is placed in distilled water having pH=7.2 and a temperature of 37 C. under mild stirring. On specific time points, samples are taken and their polyphenol concentration is measured, while the water quantity removed is replaced by distilled water having pH=7.2 and a temperature of 37 C. to maintain the tank conditions.

The prepared colloidal system of propolis dispersion in combinatorial liposome/cyclodextrin carriers presents advantageous properties in various uses, including for example antioxidant, angiogenic, photoprotective, antimicrobial and immunostimulatory properties.
In particular, it was shown that the prepared colloidal system of propolis did not show cytotoxicity on skin fibroblasts at any concentration. On the contrary, 100% increase in the

[0063] ATP levels of fibroblasts with the colloidal system of propolis (0.01%, 0.1%, 1%) compared to the ATP levels of the untreated fibroblasts (p<0.05)(barchart 1). Also the colloidal system of propolis of the present invention at a concentration of 1%, protects the cells against photo-oxidative stress. In particular, after the relevant experimental procedure, an increase in the vitality of the cells (ATP increase) with the colloidal system of 1% propolis under photo-oxidative stress conditions (UVA radiation) in relation to untreated cells was observed. This is linked to the antioxidant capacity of the colloidal system of 1% propolis as well as to its protective role against premature aging of the cells (photo-aging).

[0064] In particular, ultraviolet radiation is a major cause of oxidative stress for the skin. Apart from the generation of free radicals, ultraviolet radiation affects the defense enzymes of the skin against oxidation, making the skin more vulnerable to permanent cellular damage (acceleration of skin aging). When the skin is exposed to ultraviolet light for a long time without protection with sunscreen filters, skin protection depends exclusively on the endogeneous antioxidant defense systems. In our experimental procedure, photooxidative stress was simulated. Therefore, a UV lamp with a wavelength of 365 nm and a radiation dose of 5 J/cm.sup.2 was used. The UV lamp was used as means to form free radicals. The said experimental procedure includes the following steps: [0065] Incubation of the colloidal system of 1% propolis on the NHDF cells for 48 hours [0066] Removal of cell medium and washing with phosphate/salt (PBS) buffer solution to avoid intramolecular reactions [0067] Addition of phosphate/salt (PBS) buffer solution and activation of the UV lamp at a wavelength of 365 nm for 20 minutes, [0068] Addition of cell medium [0069] Measurement of the viability of the cells based on ATP.

[0070] With the specific experimental procedure, the previous results were confirmed. [0071] The colloidal system of 1% propolis causes advantageous on the transcriptomic profile of skin fibroblasts, so that it has an antimicrobial and immunostimulatory action and influences angiogenesis. [0072] Last but not least, we evaluated for potential skin irritancy of colloidal system of propolis (1%) as well as cosmetic formulation containing of this extract. For this reason we use the reconstructed human epidermal model EpiDerm. This model has been manufactured by MatTek Corporation (Ashland, Mass., USA) since 1993. The model consists of human-derived epidermal keratinocytes, which have been cultured to form a multi-layered, highly differentiated model of human epidermis. The tissue exhibits organized basal, spinous, granular layers, and multi-layered stratum corneum containing intercellular lamellar lipidis layers arranged in patterns analogous to those found in vivo. Because it closely mimics the human epidermis, it has proven its scientific relevancy in studies where dermal exposure and toxicity is anticipated (Kandrov H et al., 2009). The parameters measured for skin irritancy assessments of colloidal system of propolis (1%) were the percent MTT cell viability relative to the corresponding negative as well as positive control. According to the MTT assay colloidal system of propolis was proven non skin irritant, showing a cell viability >50% and confirming the aforementioned results on cell viability. Furthermore, for skin irritancy assessments of colloidal system of propolis based cosmetic formulation we examined the percentage cell viability in the MTT reduction assay with ET.sub.50 determination after topical application of the product for different exposure times (2 hr, 5 hr and 18 hr). The colloidal system of propolis-based cosmetic formulation was proven mild for skin after 11 h of exposure (cell viability>50%). [0073] For the experimental confirmation of the above, real-time polymerase chain reaction (RT-PCR) (Giulietti A et al., 2001) was employed. For the quantification of the results, two endogenous reference genes were employed, b-actin ACTB and GAPDH. The normalization to the endogenous reference genes is required in order to correct possible differences between samples due to a different concentration of the initial substrate (cDNA) or to differences in the yield of the amplification reaction (Livak K J et al., 2001). [0074] From the transcriptomic study it was concluded that the fibroblasts with the colloidal system of 1% propolis presented increased levels on the transcripts of gene endothelial vascular growth factor (VEGFA) in relation to untreated fibroblasts (barchart 2). The vascular growth factor (VEGFA) has been shown to have a major contribution in angiogenesis, increasing the number of capillaries in a network (Moens S et al., 2014).

[0075] Also, the colloidal system of 1% propolis has a strong antimicrobial and immunostimulatory action. In order to confirm this, transcripts of genes interleukin-4 (IL4) and integrin B2 (ITGB2) were checked. In particular, an increase in the levels of transcript of gene IL4 in fibroblasts was observed with the colloidal system of 1% propolis in relation to untreated fibroblasts (barchart 3). The increased levels of transcripts of gene IL4 correlates with the activation of the defense pathways against pathogenic microbes on skin fibroblasts (Niebuhr M et al., 2010).

[0076] Finally, by the use of the colloidal system of 1% propolis, an increase on the levels of transcripts of gene ITGB2 in fibroblasts, in relation to untreated fibroblasts, is observed, which demonstrates that its use increases the immune response of the organism, since increased levels of transcripts of gene ITGB2 have been correlated with an immune response (Striz I et al., 1992(barchart 4))

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