Patent Application
20200085744
The present invention relates to a method of producing a stable controlled-release propolis colloidal dispersion system and its use as an antioxidant, angiogenic, photoprotective, antimicrobial and immunostimulatory agent, alone or incorporated in cosmetic or pharmaceutical formulations, which is characterized by the fact that the components of propolis are extracted and at the same time encapsulated in combinatorial liposome-cyclodextrin carriers so that a controlled release rate of the components is obtained.
Until now, such a product or a method for the production thereof has not been known. In particular, propolis is a resinous substance which is produced by bees and presents many challenges in respect to its extraction and formulation. It exhibits antimicrobial, immunostimulatory and antioxidant activity and is employed in the preparation of functional foods and cosmetics as well as in traditional medicine. Its composition varies depending on the flora of the foraging region of the bees and the collection season. It contains ca. 50% resins, 30% waxes, 10% aromatic components, 5% pollen and 5% various other components. The bioactive components of propolis are polyphenols, terpenes, steroids, as well as sugars and aminoacids. Major polyphenols are flavonoids and phenolic acids. The complexity of the structure of propolis combined with the variable composition depending on the region and the collection season make both propolis and its extracts particularly difficult to formulate.
The extraction of propolis usually has a low yield in active component concentration. In order to increase the extraction yield, various extraction methods have been developed as well as methods of encapsulating its components in various carriers.
As regards extraction methods, either soaking in an extraction solvent for large time periods is performed, or higher extraction temperatures or organic solvents are used. These methods have the serious disadvantage that high temperatures, prolonged time and the use of high-energy techniques, such as microwave- or ultrasound-assisted extraction may lead to decomposition of the labile components of propolis, mainly of the polyphenols, and deterioration of the quality of the final extract.
Common solvents used for the extraction include various alcohols such as methanol and ethanol. Methanol presents the problem that -despite being an effective solvent for the active components of propolis- it is extremely toxic, therefore it is only used for research purposes. Ethanol is also an effective solvent for the active components of propolis, however it is involved in the rupture of the skin and local irritations, thus it is not indicated for topical administration.
Apart from the solubility of the components, the extraction of propolis presents other problems which have been addressed by the use of carriers for bioactive substances.
A limiting factor for the topical use of propolis extracts is the bioavailability of its components and the penetration depth in the skin. Polyphenols, which are responsible for the antioxidant and immunostimulatory action of propolis, depending on their physicochemical properties and in particular the lipophilicity thereof, accumulate upon topical delivery mainly on the corneal layer (Alonso et al., 2014, Abla et al., 2013), while a minor portion penetrates into the deeper layers of the skin.
Another serious problem is the protection of polyphenols against physical, chemical and biological decomposition due to environmental factors. As it is known in the literature, polyphenols oxidize easily and may leave black spots upon deposition on the skin, become malodorous -which limits their topical use- and lose their activity due to the decomposition (Munin and Edwards-Levy, 2011).
Finally, another problem is the duration of the contact of the polyphenols with the skin cells or mucosa, especially in cases when prolonged action against microorganisms and/or anti-oxidant protection and/or photoprotective and/or angiogenic action are required. As regards prolonged antimicrobial action, controlled-release systems of propolis (De Luca et al., 2014, Franca et al., 2014, Balata et al., 2014) have been described which either use ethanol as solvent -with the above-mentioned toxicity problems- or synthetic polymers. As regards controlled release of anti-oxidants, a system has been described which however relates to a final formulation and not a starting material which may be used alone or as a component of multiple final formulations for use on the skin (Zilius et al., 2013) as well as a nano-emulsion with photoprotective action on the skin which however contains—in addition to propolis—lycopene (Butnariu et al., 2011).
Also, until now, for the increase in solubility, bioavailability and protection of the components of propolis, carriers for bioactive substances such as cyclodextrins or liposomes have been used.
It is known from the literature that the formation of inclusion complexes of bioactive substances in cyclodextrins provides advantages, such as an increase in water solubility, protection against oxidation and protection against decomposition of the molecules, and therefore in the past complexes of polyphenols of propolis with cyclodextrins have been formed.
Encapsulation of polyphenols in liposomes has also been used previously (Tao et al., 2014, Yuan et al., 2013). In these cases, propolis was subjected to ethanol extraction, with all the above-mentioned problems, and then liposomal systems encapsulating polyphenols were formed. This is a multi-step procedure.
The present invention aims at eliminating the above disadvantages. In particular, the present invention allows for the first time simultaneous extraction and encapsulation of components of propolis in a combinatorial liposome-cyclodextrin system, which consists exclusively of natural and non-toxic ingredients and which presents controlled release of the encapsulated polyphenols of propolis.
More specifically the present invention exploits the particular properties of both carriers: the ability of the cyclodextrins to enclose polyphenols and enhance their permeability through the skin (Cutignelli et al., 2014) as well as the ability of the liposomes to encapsulate large quantities of components of various degrees of polarities, for topical transport of components and control of their release rate. At the same time, the extraction of the polyphenols and their encapsulation in the combinatorial system takes place in a single vessel.
Aim of the present invention is the preparation of a stable propolis colloidal dispersion system with a novel method where the components of propolis are extracted and at the same time encapsulated in combinatorial liposome-cyclodextrin carriers. The system, under suitable conditions, allows controlled release of the components while under suitable storage conditions it keeps the components encapsulated in the combinatorial system. The final preparation is suitable for antioxidant, photoprotective, antimicrobial, angiogenic and immunostimulatory use, either alone or after incorporation in cosmetic or pharmaceutical formulations.
The advantages and novel features of the method are the following:
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.
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-β-cyclodextrin or β-cyclodextrin has been predissolved at 2-10% w/w.
The quality specification of the deionized water is <=1 μS/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.
The preparation of the deionized water used in our invention is as follows:
Tap water is introduced in the raw-water tank (volume 2 m3) 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.
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-m3 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.
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.
The liposomal suspension is at a temperature higher than the phase transition temperature of its phospholipids and its lipidic composition is:
Stirring is performed for 1 to 4 hours, and at the same time the following parameters are measured:
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.
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.
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.
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.
In order to make the present invention fully understood, the following examples are presented:
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-β-cyclodextrin has been predissolved to a content of 6.45%.
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.
The final colloidal suspension after filtration presents the values given in table 2
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-β-cyclodextrin has been predissolved to a content of 4.45%.
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.
The starting material of propolis should contain total polyphenols >1600 mg/l gallic acid after dissolution of 10% w/w propolis in ethanol.
The final colloidal suspension after filtration presents the values given in table 4
The determination of the mean hydrodynamic diameter and Polydispersity index is effected by Dynamic Light Scattering.
An in vitro study of the release of the polyphenols of propolis was performed by means of dialysis sacks:
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
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).
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/cm2 was used. The UV lamp was used as means to form free radicals. The said experimental procedure includes the following steps:
With the specific experimental procedure, the previous results were confirmed.
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).
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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| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GR2015/000059 | 11/23/2015 | WO | 00 |
