Process for production of combination delivery system of grape leaves and propolis polyphenols for multiple uses

Abstract

A process for the production of a combination delivery system of grape leaves and propolis polyphenols, using grape leaves comprising Syrtiko / Therii / Aidani in a ratio of 25-50% / 25-50% / 25-50%, the grape leaves being cut into lt; the present invention relates to a method for extracting propolis, comprising the following steps: (1) mixing a portion having a particle size of 0.5 mm and a particle size of 0.8 mm until being completely homogenized, then dispersing at a rate of 1 kg / min in a container containing an extraction solvent system stirred at 500-3000 rpm and having a ratio of propolis of 75-25% to 50-50%, without pre-treatment, as long as 10% by weight of propolis is dissolved in ethanol, which contains total polyphenols of > 1800 mg / l gallic acid equivalent, and which is pulverized into 1t; the present invention relates to a method for preparing liposomes, in which particles having a particle size of 1 mm are dispersed at a rate of 1 kg / min in another container containing an extraction solvent system stirred at 500-3000 rpm after being cooled at a temperature of-20 DEG C for 24 hours and then placed together in a container in which a liposomal system has been produced.

Description

Technical Field

[0001] The present invention relates to a process for the production of a stabilizing system for the encapsulation and delivery of polyphenols derived from grape leaves of a specified variety and propolis of a specific composition, which stabilizing system provides protection and controlled release of the polyphenols, and to its cosmetic and other uses, both in its original form and after incorporation into cosmetic or pharmaceutical formulations, characterized by the fact that the components of the system are extracted and simultaneously encapsulated in liposomes, cyclodextrins and combined liposome-cyclodextrin carriers, which show a controlled release of the components. Background Art

[0002] Until today, no similar products or similar production methods have emerged. Specifically, grape leaves constitute a by-product of the enological process. Agricultural by-products are often a source of valuable bioactive ingredients. The study of the biological value of by-products is crucial for sustainable agriculture, but also for the biological properties that by-products can impart to cosmetics, pharmaceuticals and foods. In viticulture, grape leaves are a large volume by-product that is removed and treated as waste during the enological process, with their application limited at best to limited edible products and at worst to the preparation of soil conditioners.

[0003] Given its importance in enology, the vine (Vitis vinifera L.) is considered one of the most important fruit cultivations in the world, occupying a global area of ​​more than 7.5 Mha (FAO 2016) and with a production of 80 million tonnes in 2018 (FAOSTAT 2020). Currently, there are about 2000 different V. vinifera varieties used for grape production worldwide. About 50% of the world's grape production is used for wine production, an industry of great strategic importance to the economies of many countries, 30% of grape production is intended for commercialization of fresh grapes, and the rest is dried as raisins or consumed as grape juice or must.

[0004] The fruit is the most studied product of the Vitis genus due to its wide range of uses. Grape leaves, while plentiful, have been significantly less studied, and have been shown to contain a variety of phenolic compounds and antioxidants that have been used in traditional medicine to treat bleeding, inflammation, diarrhea, and liver complications caused by diabetes. Due to the large quantities of grape leaves that are not used—or used for low-value products—grape leaves constitute a byproduct with great potential for utilization.

[0005] The phenolic profile may vary depending on the species of Vitis genus, but the main bioactive phenolic compounds of the leaves are the flavonoids (+)-catechin, (-)-epicatechin, apigenin, kaempferol, quercetin, myricetin, quercetin-4′-glucoside and rutin, and the resveratrol-like compounds cis- and trans-resveratrol monomers, piceid and astringin (Katalinic et al., 2013).

[0006] Propolis is a resinous substance produced by bees and exhibits antimicrobial and antioxidant activity, which can be used in the preparation of functional foods and cosmetics and in traditional medicine. Depending on the flora of the geographical area of ​​the beehive and the period of collection, the composition of propolis varies. Propolis contains an average of 50% resin, 30% wax, 10% aromatic compounds, 5% pollen and 5% various other components. The bioactive components of propolis are polyphenols, terpenes, steroids, as well as sugars and amino acids. Its main polyphenols are flavonoids, mainly quercetin, galangin, luteolin, apigenin and phenolic acids, mainly caffeic acid, caffeic acid phenethyl ester, ferulic acid, and p-coumaric acid. The complex combination of the structure of propolis and its composition that varies with the collection area and season make the standardization of propolis and its extracts extremely difficult (Stavropoulou et al., 2021).

[0007] Polyphenols from both Vitis and propolis constitute important bioactive molecules, primarily with antioxidant, anti-inflammatory and photoprotective effects on the skin (Sangiovanni et al., 2019, Karapetsas et al., 2019, Letsiou et al., 2020). Despite their beneficial properties, both propolis and Vitis polyphenols present specific challenges that limit their use and effectiveness.

[0008] In principle, the stability of polyphenols against non-enzymatic degradation during processing and their shelf life are influenced by several parameters, such as molecular structure, pH, temperature, oxygen, light, processing conditions and the presence of interactions and / or other compounds and components. In general, polyphenols are unstable molecules and are affected by the extraction conditions, which are usually either carried out with long intervals of wetting with the extraction solvent or by using high extraction temperatures. High temperatures, long times and the use of high-energy techniques (such as microwave or ultrasound-assisted extraction) may lead to the degradation of susceptible polyphenols and a decrease in the quality of the final extract. Polyphenols are easily oxidized and leave black spots when deposited on the skin, produce an unpleasant odor - which limits their topical use - and also lose their activity due to degradation. (Munin and Edwards-Lévy).

[0009] In addition, propolis and grape leaf polyphenols are relatively lipophilic, and the solvent required all shows toxicity in oral and topical application.The most common solvent for extraction is various alcohols, such as methanol and ethanol.Among these, the shortcoming that methanol shows is that, although it is an effective solvent for polyphenols, it also has high toxicity.Ethanol is also an effective solvent for polyphenols, but its shortcoming that shows is that it is relevant with skin damage and local irritation, so ethanol is not suitable for topical application, and oral administration shows known toxicity problem.

[0010] In addition to extraction and stability-related issues, propolis and grape leaf polyphenols present further challenges that limit their use.

[0011] A limiting factor for the topical use of polyphenols is the bioavailability of their components, the depth of penetration into the epidermis and the rate of penetration. It is generally believed that polyphenols from plant materials (e.g. grape leaves) show limited penetration into human skin. (Nichols, JA, & Katiyar, SK 2010). In addition, propolis polyphenols, due to their physicochemical properties, especially the lipophilicity they exhibit, are mainly concentrated in the stratum corneum when applied topically. (Alonso et al. 2014, Abla et al. 2013) A much smaller proportion penetrates deeper skin layers.

[0012] Finally, a further problem is the duration of contact of the polyphenols with the skin cells or mucous membranes, especially if long-term antioxidant protection and / or photoprotective effects and / or angiogenic effects are desired.

[0013] In order to solve the above problems, polyphenol protection, bioavailability enhancement and release rate improvement systems have been developed. These technologies such as spray drying, freeze drying, extrusion, emulsification, coacervation, cyclodextrin molecular encapsulation ( et al., 2020), liposome encapsulation (Fang and Bhandari, 2010; Figueroa-Robles et al., 2021), etc.

[0014] It is known from the literature that the formation of encapsulated complexes of bioactive substances in cyclodextrins has advantages such as increased solubility in water, protection against oxidation and protection against molecular degradation, and for these reasons complexes of propolis polyphenols with cyclodextrins have been created in the past. Cyclodextrins have been used to encapsulate polyphenols derived from plants of the genus Vitis, mainly resveratrol, but not polyphenols derived from grape leaves.

[0015] Regarding duration of action and bioavailability, controlled release systems involving propolis have been reported (DeLuca et al., Franca et al., Balata et al.), which use ethanol (with the above-mentioned toxicity issues) or use synthetic polymers as solvents. Our laboratory has created a controlled release system of propolis polyphenols with a natural non-toxic solvent (WO2017 / 089842A1). Regarding grapes, systems that lead to controlled polyphenol release rates have been reported and involve grape seed oil emulsions (Guel et al., 2019) and resveratrol release systems (Song et al., 2021; Paczkowska-Walendowska M et al., 2021). However, there are no reports of controlled release systems for encapsulating polyphenols from grape leaves.

[0016] The combination of liposomes and cyclodextrins in a unique system can provide the combined advantages of the two carriers, as described in our laboratory's patent, which created a method for preparing and stabilizing a controlled-release propolis colloid system (WO2017089842A1 patent). Summary of the invention

[0017] The present invention is directed to combining the beneficial properties of grape leaf and propolis polyphenols in a delivery system.

[0018] Specifically, the present invention aims to overcome the shortcomings of the prior art and proposes a method for producing a combined delivery system of grape leaves and propolis polyphenols for various purposes, characterized in that grape leaves of asyrtiko / athiri / aidani in a ratio of 25-50% / 25-50% / 25-50% are used, the grape leaves are chopped into small pieces (<0.8 mm), mixed until completely homogenized, and then dispersed at a rate of 1 kg / min in a stirring medium containing an extraction solvent system at 500-3000 rpm. The invention relates to a method for preparing a liposome system ...

[0019] More specifically, the present invention utilizes the special properties of two carriers: the ability of cyclodextrins to encapsulate polyphenols and enhance the permeability of polyphenols through the skin and the ability of liposomes to encapsulate a large number of ingredients with different polarity levels for local delivery of ingredients and control of their release rate. The encapsulation technology allows a two-stage release of ingredients: a rapid phase that releases mainly propolis polyphenols and a prolonged phase that releases mainly grape leaf polyphenols. The procedures for extracting and encapsulating polyphenols from the two natural products in the combined system are carried out in a single container.

[0020] The object of the present invention is to prepare a system for encapsulation and delivery of grape leaf and propolis polyphenols, wherein the grape leaf and propolis polyphenols are extracted and simultaneously encapsulated in a liposome-cyclodextrin combination carrier. The system, when under suitable conditions, exhibits controlled release of the ingredients, while under suitable storage conditions, it keeps the components encapsulated and protected in the combination system. The final preparation is suitable for dermatology and other uses, taking advantage of the bioactive properties of the polyphenols, either by themselves or after integration into cosmetic or pharmaceutical formulations.

[0021] According to other optional features of the production method, it may optionally include one or more of the following features alone or in combination:

[0022] - Grape leaves were collected before spraying and dried in the absence of light to a moisture value of <8% and a total ash of <10%, wherein each variety should contain more than 2000 mg / l gallic acid in total polyphenols after 10% dissolution by weight in ethanol.

[0023] - The extraction solvent system consists of deionized water and vegetable 1,3-propylene glycol or glycerol, the ratio of 1,3-propylene glycol (or glycerol) / water is: 15 / 85 to 80 / 20, wherein hydroxypropyl-β-cyclodextrin or β-cyclodextrin has been pre-dissolved in deionized water at a concentration of 2-10% w / w,

[0024] - liposome systems are composed of 30%-95% phosphatidylcholine, 4%-10% phosphatidylserine, 0%-3% lysophosphatidylcholine, 0%-3% phosphatidylinositol, 0%-22% cholesterol, 0-10% cholate, and their solvent is 1,3 propanediol or glycerol, with ratios ranging from 20 / 80 to 80 / 20% w / w,

[0025] -Deionized water quality requirements: <=1μS / cm at 25℃,

[0026] - Add the grape leaf / cyclodextrin-solvent system to the liposome system at a rate of 10 ml / sec, so that the liposome system obtains a concentration of 1.0-7.5% w / w, and adjust the pH of the system to a range of 5.0-8.0 and stir at 3000 rpm for 2 hours,

[0027] - Then the propolis / cyclodextrin system was added to the stirred container at a rate of 10 ml / sec at a ratio of 10-17%, and the pH was readjusted to the range of 5.0-8.0 after the addition was completed, and stirred at 4000 rpm for 3 hours,

[0028] The propolis / cyclodextrin system was then added to the now agitated container at a rate of 100 ml / sec at a ratio of 5-20% - and at 1000 rpm for 10 minutes, and after the addition was complete, the system pH was finally adjusted to the range of 3.5-6.2. This procedure resulted in a portion of the propolis-cyclodextrin complex being encapsulated in the liposomes and a portion being free in the suspension, showing different release rates,

[0029] The mixture was then placed in a sealed container to stand at 5-7°C for 24 hours, then cold filtered through a cartridge filter array with pore sizes of 10μm-5μm-1μm-0.45μm, and the pH was retested and, if necessary, readjusted to a pH value in the range of 3.5-6.2

[0030] The system is then allowed to reach room temperature and the average hydrodynamic diameter of the particles is measured, which should range from 200 nm to 600 nm with a polydispersity index of less than 0.7, wherein if outside these limits a second filtration is performed at room temperature through a cartridge filter array with a pore size of 0.45 μm - 0.2 μm and then the total polyphenol content is measured, which must be > 1600 mg / LGAE.

[0031] The release rate of the encapsulated polyphenols in a buffer solution of pH 7.2 at 37°C was then determined, which was 25-50% within 30 minutes, 50-75% within 8 hours, and completely released within 24 hours, and the system was stored in a dark, light-proof container at a temperature of 5-7°C, where it remained stable for 1 year.

[0032] - For in vitro study of propolis polyphenol release, the specified amount of colloid was placed in a dialysis bag with MW=1000 molecular exclusion, which was placed in distilled water at pH=7.2 and temperature 37°C under gentle agitation, where samples were collected at specified time points and their polyphenol concentrations were measured, where the amount of water removed was replaced with distilled water at pH=7.2 and temperature 37°C to maintain chamber conditions.

[0033] The advantages and innovative elements of the method of the present invention are as follows:

[0034] A complex combination system for the administration of polyphenols from grape leaves and propolis is prepared using a particularly simple production method. The procedures for the formation of encapsulation complexes between cyclodextrin and polyphenols and the encapsulation of polyphenols in liposomes, either in free form or in the form of complexes with cyclodextrin, are carried out during the extraction in a single vessel.

[0035] • Due to the physicochemical properties of the selected cyclodextrins and phospholipids, almost all (>95%) polyphenols, both less and more lipophilic, are encapsulated in the water-soluble system.

[0036] The time required for creating the colloid is short and the energy supply required is small, so the final production costs are kept commensurately at a very low level.

[0037] The interaction of the two carriers with the polyphenols provides controlled release under appropriate conditions - the propolis polyphenols are released quickly, while the grape leaf polyphenols are released over a prolonged period. Under storage conditions, the polyphenols are protected within the carriers.

[0038] • By varying the ratio between phospholipids and cyclodextrins, the polyphenol release rate can be modified, which is very useful, depending on the application of the invention.

[0039] The combination of ingredients and lipid composition of the liposomes is such in order to avoid fusion and liposome aggregation problems, resulting in excellent stability of the system both from a physicochemical point of view (mean hydrodynamic diameter, zeta potential, polydispersity index, pH) and from the retention of polyphenols inside the liposomes during storage.

[0040] · Use only ingredients that are safe and friendly to the skin and mucous membranes.

[0041] The final extract was titrated to total polyphenols.

[0042] The system preserves itself for 1 year at a temperature of 5-7°C and without the need for added preservatives, despite the fact that it contains a large amount of water. This is due to the high polyphenol content of the system, which exhibit, among other things, antimicrobial properties.

[0043] Due to the quality of the water used, there is no need to add chelating agents.

[0044] Due to the double encapsulation in liposomes and cyclodextrins, the sensitive polyphenols in this system are protected from oxidation or degradation due to light and temperature increases. In case the system is used as an ingredient in cosmetic or pharmaceutical formulations, the polyphenols are also protected from interaction with other ingredients for the same reason.

[0045] The system can be used directly on human skin and / or mucous membranes, or integrated into cosmetic or pharmaceutical formulations for topical or systemic administration. In cosmetic or pharmaceutical formulations, it even provides clear liquids, if necessary, for example in jelly preparations.

[0046] • The system can be used with appropriate formulations as food supplements, pharmaceutical formulations, nutraceuticals and as functional food ingredients.

[0047] The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0048] Figure 1 : Bar graph of collagen-I quantification obtained from cell counting and specific signal detection in the control group (*p<0.05§p<0.1 - t-test for binary comparison of stress groups; "Control" or "CTRL": cells not exposed to UVA and not treated with the propolis and grape leaf polyphenols combination delivery system, "Stress": cells exposed to stress but not treated with the polyphenols combination delivery system, "Stress+Active": cells exposed to stress and treated with the polyphenols combination delivery system).

[0049] Figure 2 : Bar graph of collagen-III quantification obtained from cell counting and specific signal detection in the control group (*p<0.05, t-test for binary comparison of stress groups; "Control" or "CTRL": cells not exposed to UVA and not treated with the propolis and grape leaf polyphenols combination delivery system, "Stress": cells exposed to stress but not treated with the polyphenols combination delivery system, "Stress+Active": cells exposed to stress and treated with the polyphenols combination delivery system).

[0050] Figure 3 : is a bar graph of elastin quantification obtained from cell counting and specific signal detection in the control group (§p<0.1 - t-test for binary comparison of stressed groups; "Control" or "CTRL": cells not exposed to UVA and not treated with the propolis and grape leaf polyphenols combination delivery system, "Stress": cells exposed to stress but not treated with the polyphenols combination delivery system, "Stress+Active": cells exposed to stress and treated with the polyphenols combination delivery system). DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to understand our invention, we will now describe in detail the production method of the grape leaf and propolis polyphenol delivery system in a liposome / cyclodextrin combination carrier.

[0052] The grape leaves were harvested before spraying and dried in the absence of light to show moisture values ​​<8% and total ash <10%. The respective varieties preferably contained >2000 mg / l of total polyphenols in gallic acid equivalents after dissolving 10% by weight in ethanol and subsequently measuring in a spectrophotometer using the Folin-Ciocalteu method.

[0053] Propolis can be used without prior treatment, provided that it preferably contains >1800 mg / l total polyphenols in gallic acid equivalents after dissolving the propolis at 10% by weight in ethanol and subsequently measuring by the Folin-Ciocalteu method in a spectrophotometer. The ratio of grape leaf to propolis varies from 75% / 25% to 50% / 50%.

[0054] The deionized water quality requirement is: <=1 μS / cm at 25°C, which exceeds the European Pharmacopoeia specifications for the preparation of parenteral drugs. This quality is necessary in order to completely eliminate charges in the final formulation, which could cause the lipid membrane of the liposomes to aggregate and ultimately lead to a decrease in product stability.

[0055] The deionized water used in the present invention is prepared as follows:

[0056] Water from the water supply network enters the raw water tank (volume 2m 3 ), through a suitable pump assembly, through an automatic turbidity filter to remove turbidity and solid particles, through activated carbon to remove chlorine and organic loads, and then dosing antiscalant to limit its hardness. Before entering the central assembly of the reverse osmosis unit, the water passes through a 1 micron cartridge filter.

[0057] The fine treated water for reverse osmosis enters a reverse osmosis unit with a capacity of 350 lt / h and a recovery rate of 70%. The water produced by the reverse osmosis unit is stored in a 5m 3 From this tank, the water is fed to the deionizer via a suitable pumping assembly and driven online via the UV to the extraction tank. To avoid water accumulation in the network, the water is continuously circulated and returned to the tank.

[0058] To prepare the system, grape leaves are first cut into small portions (<0.8 mm) in the ratio of asyrtiko / athiri / aidani 25-50% / 25-50% / 25-50% and mixed until completely homogenized. Then, they are dispersed into the solvent system at a rate of 1 kg / min, stirring at 500–3000 rpm. The concentration of grape leaves can be 1.8% to 8% by weight, depending on its polyphenol content. The extraction solvent system consists of deionized water and vegetable 1,3-propylene glycol or glycerol, with the ratio of 1,3-propylene glycol (or glycerol) / water being: 15 / 85 to 80 / 20. Hydroxypropyl-β-cyclodextrin or β-cyclodextrin has been pre-dissolved in deionized water at a concentration of 2-10% w / w.

[0059] After cooling at -20°C for 24 hours, the propolis is crushed into small particles (<1 mm). In a second container, the crushed propolis is dispersed into the solvent system at a rate of 1 kg / min and stirred at 500–3000 rpm. The propolis concentration can be 1% to 12% by weight, depending on its polyphenol content and on the desired release rate of the system. The extraction solvent system consists of deionized water and vegetable 1,3-propylene glycol or glycerol, with the ratio of 1,3-propylene glycol (or glycerol) / water being: 15 / 85 to 80 / 20. Hydroxypropyl-β-cyclodextrin or β-cyclodextrin has been pre-dissolved in deionized water at a concentration of 2-10% w / w.

[0060] The preparation of the liposome system is carried out in the third container. The liposome system consists of the following:

[0061] -30%-95% phosphatidylcholine

[0062] -4%-10% phosphatidylserine

[0063] -0%-3% lysophosphatidylcholine

[0064] -0%-3% Phosphatidylinositol

[0065] -0%-22% cholesterol

[0066] -0-10% bile salt

[0067] Their solvent is 1,3 propylene glycol or glycerol. The ratio of the above components to 1,3 propylene glycol or glycerol ranges from 20 / 80 to 80 / 20% w / w.

[0068] Then the grape leaf / cyclodextrin-solvent system was added to the liposome system at a rate of 10 ml / sec, so that the liposome system had a concentration of 1.0-7.5% w / w. After the addition, the pH of the system was adjusted to a range of 5.0-8.0 and stirred at 3000 rpm for 2 hours.

[0069] Then the propolis / cyclodextrin system was added to the stirred container at a rate of 10 ml / sec at a ratio of 10-17%. After the addition was completed, the pH of the system was adjusted to a range of 5.0-8.0 and stirred at 4000 rpm for 3 hours.

[0070] Finally, the propolis / cyclodextrin system was added to the now agitated container at a rate of 100 ml / sec at a ratio of 5-20%, and stirred for another 10 minutes at 1000 rpm. After the addition was completed, the system pH was finally adjusted to the range of 3.5–6.2. By the protocol of these steps, it was ensured that part of the propolis-cyclodextrin complex would be encapsulated in the liposomes and part would be free in the suspension. These parts showed different release rate characteristics.

[0071] The mixture was then placed in a sealed container to stand for 24 hours at 5-7° C. The system was then cold filtered through a cartridge filter array with pore sizes of 10 μm–5 μm–1 μm-0.45 μm, and the pH was retested and, if necessary, readjusted to a pH in the range of 3.5–6.2.

[0072] The system is allowed to reach room temperature and the average hydrodynamic diameter of the particles is measured, which should range from 200 nm to 600 nm with a polydispersity index of less than 0.7. If these values ​​are outside the above limits, a second filtration is performed at room temperature through a cartridge filter array with a pore size of 0.45 μm–0.2 μm.

[0073] If the mean hydrodynamic diameter and the polydispersity index values ​​are within the specification range, the total polyphenol content is measured. Although the method according to the invention allows to obtain a combination of liposomes and cyclodextrins that is particularly effective in, for example, protecting, increasing the bioavailability of phenolic compounds and regulating the release rate of phenolic compounds, it should be taken into account that the higher the concentration of phenolic compounds, the more effective the combined delivery system is in providing a biological effect, as reported in the Examples section. In a specific embodiment, the total polyphenol content of the combined delivery system of the invention is better than 1600 mg / L gallic acid equivalent (GAE).

[0074] Determining the gallic acid equivalent (GAE) content is a well-known method for estimating the total phenolic compound content of a composition. Gallic acid equivalents can be determined, for example, using the Folin-Ciocalteu method described by Chun and Kim (2004).

[0075] The release rate of the polyphenols was then determined in a buffer solution at pH 7.2 at 37°C, and the system was stored in a dark container at a temperature of 5-7°C, where it remained stable for 1 year.

[0076] At pH 7.2 and a temperature of 37°C, the release of polyphenols (cumulative release) was 25-50% within 30 minutes, 50-75% within 8 hours, and within 24 hours the system released almost all of the encapsulated polyphenols.

[0077] Determination of the mean hydrodynamic diameter and polydispersity index was performed by dynamic light scattering.

[0078] In vitro studies on the release of propolis polyphenols were conducted with the aid of dialysis bags:

[0079] The designated amount of colloid was placed in a dialysis bag with a molecular exclusion of MW = 1000. The dialysis bag was placed in distilled water at pH = 7.2 and a temperature of 37°C under gentle agitation. Samples were collected at designated time points and their polyphenol concentrations were measured, wherein the amount of water removed was replaced with distilled water at pH = 7.2 and a temperature of 37°C to maintain the box conditions.

[0080] In order to make the present invention fully understood, we carry out the following examples:

[0081] Example 1

[0082] Grape leaves of assyrtiko / athiri / aidani varieties 34 / 33 / 33% w / w were used. Chopped leaves were dispersed in a solvent system consisting of deionized water / vegetable 1,3 propanediol 75 / 25 at a rate of 1 kg / min at a ratio of 8%. The system was added to the liposome suspension under vigorous stirring (3000 rpm) and at a temperature of 20°C so that the latter obtained a proportion by weight of 6.0%. Hydroxypropyl-β-cyclodextrin had been previously dissolved in deionized water at a concentration of 4.5% w / w.

[0083] The propolis in a separate container was dispersed in the solvent system in the same ratio as above until the concentration reached 10% w / w. The propolis / cyclodextrin-solvent system was added to the first container at a rate of 10 ml / sec at a ratio of 6%. After the addition was completed, the system pH was adjusted to 5.0 and then stirred at 4000 rpm for 3 hours.

[0084] Then, the propolis / cyclodextrin system was added at a rate of 100 ml / sec at a ratio of 15% to the now agitated container - and agitated for a further 10 minutes at 1000 rpm. After the addition was complete, the system pH was finally adjusted to 4.0.

[0085] The protocol set out in the present disclosure is as follows.

[0086] The final suspension after filtration exhibited the values ​​shown in Table 1.

[0087] Table 1

[0088] parameter value Mean hydrodynamic particle diameter 250-320nm Polydispersity Index <0.60 Total polyphenols (gallic acid equivalent) 2150mg / L Cumulative release of polyphenols within 30 minutes 30-35 Cumulative release of polyphenols in 8 hours 70-80

[0089] Example 2

[0090] Grape leaves of assyrtiko / athiri / aidani varieties 50 / 35 / 15% w / w were used. Chopped leaves were dispersed in a solvent system consisting of deionized water / vegetable 1,3 propanediol 75 / 25 at a rate of 1 kg / min at a ratio of 8%. The system was added to the liposome suspension under vigorous stirring (3000 rpm) and at a temperature of 20°C so that the latter obtained a proportion by weight of 6.0%. Hydroxypropyl-β-cyclodextrin had been previously dissolved in deionized water at a concentration of 4.5% w / w.

[0091] The propolis in a separate container was dispersed in the solvent system in the same ratio as above until the concentration reached 10% w / w. The propolis / cyclodextrin-solvent system was added to the first container at a rate of 10 ml / sec at a ratio of 10%. After the addition was completed, the system pH was adjusted to 5.0 and then stirred at 4000 rpm for 3 hours.

[0092] Then, the propolis / cyclodextrin system was added at a rate of 100 ml / sec at a ratio of 20% to the now agitated container - and agitated for a further 10 minutes at 1000 rpm. After the addition was complete, the system pH was finally adjusted to 4.0.

[0093] The protocol set out in the present disclosure is as follows.

[0094] The final suspension after filtration exhibited the values ​​shown in Table 2.

[0095] Determination of the mean hydrodynamic diameter and polydispersity index was performed by dynamic light scattering.

[0096] Table 2

[0097] parameter value Mean hydrodynamic particle diameter 20-300nm Polydispersity Index <0.70 Total polyphenols (gallic acid equivalent) 1600mg / L Cumulative release of polyphenols within 30 minutes 45-50 Cumulative release of polyphenols in 8 hours 40 / 45

[0098] Experimental evidence for the use of a combined delivery system of propolis and grape leaf polyphenols

[0099] To document the use of a propolis and grape leaf polyphenols combined delivery system (hereafter referred to as Active) in a cosmetic form, we conducted an in vitro study to test its effectiveness against pollution (environmental pollution and UVA).

[0100] The induction and regulation of collagen-I, collagen-IIIa, and elastin were evaluated in human dermal fibroblasts by in situ immunofluorescence microscopy analysis.

[0101] Prior to treatment with fibroblasts, the system was diluted in culture medium, and freshly prepared solutions were diluted to the desired final concentration (0.1%).

[0102] Human dermal fibroblasts were plated in 96-well plates at an initial density of 10,000 cells / well and maintained under optimal growth conditions for 2 days. On the first day, particles (ERMCZ100, similar to PM10-PAH; 1 μg / cm 2 ) were added to the cells belonging to the "stress" group and the "stress + activity" group. After 1 hour of exposure, UVA (LED light source, 365nm, 3J / cm 2 ) irradiated cells. Immediately after irradiation, the medium was renewed and active (0.1%) was added to the cells and kept in contact for 24 h. The control group received only medium renewal. After 24 h of incubation, the cells were fixed on the plates and analyzed by in situ immunofluorescence detection of collagen-I, collagen-IIIa and elastin.

[0103] Cells were incubated with primary antibodies specific for the biomarkers of interest (Table 3) in a solution of BSA in PBS. Excess primary antibodies were removed by a series of washing steps, and then cells were incubated with secondary antibodies conjugated with fluorophores (Table 3), and DAPI (4',6-diamidino-2-phenylindole) was used to label the nuclei. Finally, antibodies and excess DAPI were removed by a series of PBS washing steps.

[0104] Table 3

[0105]

[0106] Biomarkers and Antibodies

[0107] A series of images of each biomarker were collected using an epifluorescence microscope (ThermoFisher, Evos M5000) using strictly identical acquisition time and resolution (40x objective) for each biomarker. Images of all levels of fluorescence signal intensity were collected and recorded (0 to 65535; 16-bit .TIFF format) and then analyzed using ImageJ software (Rasband, NIH).

[0108] For each biomarker, each image was quantified by integrating the specific fluorescent signal exceeding an intensity threshold and then normalizing it relative to the assessment area and cell number (calculated from DAPI nuclei detection images).

[0109] Taking into account the "stressed" group at minimum productivity (0%) and the untreated group (control) at maximum productivity (100%), the effectiveness values ​​(%) of the experimental groups were obtained.

[0110] like Figure 1-3 Illustrated, the presence of active ingredients:

[0111] · resulted in an increase in collagen-I levels (44% effectiveness) compared to stress conditions, (§: p<0.1; *: p<0.05),

[0112] Counteracts stress-induced changes in collagen-IIIa levels (100% effectiveness) (*: p<0.05),

[0113] · Resulted in an increase in elastin levels (79% effectiveness) compared to stress conditions (§p<0.1).

[0114] The results obtained show that the tested activities present a beneficial effect on collagen-I, collagen-IIIa and elastin, in contrast to the changes induced in skin cells exposed to stress (pollution).

[0115] References

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Claims

1. A method for producing a combined delivery system of grape leaves and propolis polyphenols, characterized in that: The method comprises: - a step of chopping grape leaves in the ratio of 25-50% / 25-50% / 25-50% w / w of asyrtiko / athiri / aidani varieties, and wherein the grape leaves are chopped into pieces smaller than 0.8 mm, - a step of mixing the chopped grape leaves until completely homogenized, then - a step of dispersing at a rate of 1 kg / min in a first container stirred at 500-3000 rpm, the first container containing an extraction solvent system comprising deionized water, - A step of cooling the propolis at -20°C for 24 hours, - a step of crushing propolis into particles smaller than 1 mm, - a step of dissolving 10% by weight of propolis in ethanol without prior treatment, provided that it contains more than 1800 mg / l of total polyphenols in gallic acid equivalent after dissolution, and then dispersing it at a rate of 1 kg / min in a second container stirred at 500-3000 rpm, the second container containing an extraction solvent system comprising deionized water, and then - A placing step, which is carried out in a third container where the liposome system has been produced, and comprises placing the grape leaf system and the propolis system together in a ratio of 75-25% to 50-50%.

2. The method for producing the combined delivery system of grape leaves and propolis polyphenols according to claim 1, characterized in that: The method comprises a step of collecting the grape leaves, followed by a step of spraying and drying under lightless conditions to a moisture value of less than 8% and a total ash of less than 10%, wherein each variety contains more than 2000 mg / l of total polyphenols in gallic acid equivalents after a step of dissolving 10% by weight of propolis in ethanol.

3. The method for producing the combined delivery system of grape leaves and propolis polyphenols according to claim 1, characterized in that: The extraction solvent system consists of deionized water and plant 1,3-propylene glycol or glycerol, and the ratio of 1,3-propylene glycol (or glycerol) / water is: 15 / 85 to 80 / 20, wherein hydroxypropyl-β-cyclodextrin or β-cyclodextrin has been pre-dissolved in the deionized water of the extraction solvent at a concentration of 2-10% w / w.

4. The method for producing the combined delivery system of grape leaves and propolis polyphenols according to claim 1, characterized in that: The liposome system is composed of 30%-95% phosphatidylcholine, 4%-10% phosphatidylserine, 0%-3% lysophosphatidylcholine, 0%-3% phosphatidylinositol, 0%-22% cholesterol, 0-10% cholate, and their solvent is 1,3 propanediol or glycerol, with a ratio ranging from 20 / 80 to 80 / 20% w / w.

5. The method for producing the combined delivery system of grape leaves and propolis polyphenols according to claims 1 to 4, characterized in that: The deionized water quality requirement is: less than or equal to 1 μS / cm at 25°C.

6. The method for producing the combined delivery system of grape leaves and propolis polyphenols according to claims 1 to 4, characterized in that: The production method comprises: - a step of adding the grape leaves in the solvent system with cyclodextrin to the liposome system at a rate of 10 mL / sec, so that the liposome system obtains a concentration of 1.0-7.5% w / w, - a step of adjusting the pH of the system to a range of 5.0-8.0, and - A stirring step at 3000 rpm for 2 hours. - adding the propolis in the system solvent with cyclodextrin at a ratio of 10-17% at a rate of 10 ml / sec to the stirred second container, and after the addition is completed, - a step of adjusting the pH to a range of 5.0-8.0, and - A stirring step at 4000 rpm for 3 hours. - a step of adding a propolis / cyclodextrin solvent system at a ratio of 5-20% at a rate of 100 ml / sec into a stirred third container, - A further stirring step of 10 minutes at 1000 rpm, and after the addition is complete, - a step of adjusting the pH of the system to a range of 3.5-6.2, which results in a portion of the propolis-cyclodextrin complex being encapsulated in the liposomes and a portion being free in the suspension, showing different release rates, - a step of placing the mixture in a sealed container at 5-7°C for 24 hours, then - a step of cold filtering the system through an array of cartridge filters with pore sizes of 10 μm, then 5 μm, then 1 μm, then 0.45 μm, and - a step of retesting the pH and, if necessary, re-adjusting the pH to within the range of 3.5-6.2, then allowing the system to reach room temperature, and - a step of measuring the average hydrodynamic diameter of the particles, said average hydrodynamic diameter should range from 200 nm to 600 nm with a polydispersity index of less than 0.7, wherein outside these limits, - a second filtration step at room temperature through a cartridge filter array with a pore size of 0.45 μm and then 0.2 μm, then - A step of measuring the total polyphenol content, which must be greater than 1600 mg / L gallic acid equivalent - a step of determining the release rate of the encapsulated polyphenol in a buffer solution of pH 7.2 at 37°C, the release rate being 25-50% within 30 minutes, 50-75% within 8 hours, and complete release occurring within 24 hours, and - A step of storing the system in a dark container at a temperature of 5-7°C.

7. The method for producing the combined delivery system of grape leaves and propolis polyphenols according to claims 1 to 6, characterized in that: The method comprises the steps of studying the release of propolis polyphenols in vitro, the steps comprising - Place the specified amount of colloid in a dialysis bag with a molecular exclusion of MW = 1000 and place it in distilled water at pH = 7.2 and a temperature of 37°C under gentle agitation. - Collect samples at designated time points and - The polyphenol concentration thereof was measured, wherein the amount of water removed was replaced with distilled water at pH = 7.2 and temperature 37°C to maintain the container conditions.

8. A cosmetic, nutraceutical or pharmaceutical composition comprising a combined delivery system of grape leaves and propolis polyphenols obtained by the method of any one of the preceding claims 1-7.

9. Use of the cosmetic composition according to claim 8 for counteracting stress-induced changes in skin cells.

10. Use of the cosmetic composition according to claim 8 for protecting skin cells against pollution.

11. Cosmetic use of the combined delivery system of grape leaves and propolis polyphenols obtained by the method of any one of the preceding claims 1 to 7, comprising applying the combined delivery system to human skin and / or mucous membranes.

12. A combined delivery system of grape leaves and propolis polyphenols obtained by the method of any one of the preceding claims 1 to 7 for use in protecting skin cells against UVA and / or against particles.

13. A combined delivery system of grape leaves and propolis polyphenols obtained by the method of any one of the preceding claims 1 to 7 for use in protecting skin cells against pollution.

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