Method for the preparation of a stable encapsulation system of bioactive components of royal jelly in cyclodextrins

Abstract

The invention relates to a method for preparing system for encapsulating bioactive components of royal jelly in cyclodextrins, comprising: bringing fresh royal jelly from storage temperature to a temperature; dissolving the royal jelly under stirring in a solvent comprising deionized water and one polyol, the weight ratio of polyol to water being from 10:90 to 90:10;adding at least one cyclodextrin to the solvent and stirring the resulting royal jelly / cyclodextrin / solvent mixture at a temperature between 20°C and 25°C for 10 to 120 minutes; subjecting the mixture to at least one pressure compression and decompression cycle in a sealed extraction device,; maintaining the mixture at a temperature of 5–7°C for 4 to 12 hours in a sealed container; and filtering the mixture through at least one filter having a pore size of 5 µm or less, wherein the pH of the mixture is adjusted to a value between 3.5 and 7.5.

Description

[0001] METHOD FOR THE PREPARATION OF A STABLE ENCAPSULATION SYSTEM OF BIOACTIVE COMPONENTS OF ROYAL JELLY IN CYCLODEXTRINS TECHNICAL FIELD

[0002] The present invention relates to a method for the preparation of a stable encapsulation system of bioactive components of fresh Royal Jelly (RJ) in cyclodextrins (CD). The invention relates to a method for preparing a system for encapsulating bioactive components of fresh royal jelly in cyclodextrins, The encapsulation system invention enables the simultaneous extraction of the RJ’s components and their encapsulation within CD carriers. The resulting system is intended for cosmetic use, as well as for applications related to the skin and the scalp, either as such or incorporated into a cosmetic or pharmaceutical formulation.

[0003] TECHNICAL AND SCIENTIFIC BACKGROUND

[0004] To date, neither such an encapsulation system nor its method of preparation has been disclosed.

[0005] RJ is a white, viscous, gel -like substance secreted by the hypopharyngeal glands of young worker bees. Due to its exceptional biological properties, RJ is widely used in the food and cosmetic industries. Numerous studies have demonstrated its anti-aging, anti-inflammatory, antimicrobial, neuroprotective, and immunomodulatory effects, among others. These activities are primarily attributed to the bioactive components it contains (Orsolic & Jazvinscak Jembrek, 2024).

[0006] RJ consists primarily of water (50-56%), proteins (18%), carbohydrates (15%), lipids (3-6%), minerals (1.5%), and vitamins. Approximately 185 substances have been detected in RJ based on spectroscopic analysis. The main bioactive substances are 10-hydroxy-2-decenoic acid (10-HDA) and peptides such as apisimin, royalisin, royalactin, apidaecin, defensin-1, and the jelleins. Additionally, compounds such as adenosine monophosphate, acetylcholine, polyphenols, and hormones including testosterone, progesterone, prolactin, and estradiol have been detected in RJ samples (Nagai & Inoue, 2004; Orsolic & Jazvinscak Jembrek, 2024; Ramadan & Al-Ghamdi, 2012; Sugiyama et al., 2012) .

[0007] 10-HDA is a major fatty acid found exclusively in RJ and it serves as a marker of its quality and is responsible for its anticancer, antibacterial, whitening, immunomodulatory and anti-inflammatory properties (Blum et al., 1959; Y. F. Chen et al., 2018; Peng et al., 2017; Townsend et al., 1961; Vucevic et al., 2007). Regarding the skin, evidence indicates that 10-HDA enhances collagen synthesis and inhibits the synthesis of metalloproteinases, making it a molecule of interest for anti-aging applications (Fujii et al., 1990; Koya-Miyata et al., 2004; Yang et al., 2010). Previous studies from our laboratory have provided evidence that 10-HDA encapsulated in cyclodextrins and liposomes regulates extracellular matrix (ECM) homeostasis by modulating both collagen production and deposition through multiple regulatory pathways, thereby contributing to enhanced functionality of human fibroblasts (Spanidi et al., 2022). Furthermore, this molecule acts as a tyrosinase inhibitor (Dynek et al., 2008). Changes in the levels of proteins associated with melanin production in melanoma cell lines, together with the observed increase in the mean skin whitening index in vivo in mice, further support the whitening effect of 10-HDA (Peng et al., 2017).Polyphenols, which are also present in royal jelly, constitute important bioactive molecules with antioxidant and photoprotective activity in the skin. Royal jelly exhibits significant physicochemical stability issues depending on storage time and temperature. The ideal storage temperature for royal jelly is -20 °C, whereas higher temperatures induce color changes and degradation of its components due to Maillard reactions, enzymatic reactions, and lipid-protein interactions. Other factors affecting the stability of royal jelly include:

[0008] 1. Exposure to light, as ultraviolet radiation can degrade proteins and fatty acids, reducing antioxidant properties;

[0009] 2. pH levels, since royal jelly is naturally acidic (pH ~3.5- 4.5), and changes in pH can destabilize proteins and enzymes;

[0010] 3. Oxygen, and

[0011] 4. Moisture, as oxidation can reduce levels of unsaturated fatty acids, and high humidity can promote microbial growth and spoilage (C. Chen & Chen, 1995; Rizki et al., 2023).

[0012] To minimize such degradation, royal jelly should be stored in dark containers at low temperatures (Sabatini et al., 2009).

[0013] To prevent the oxidation of royal jelly, several techniques are employed, such as lyophilization or encapsulation (Patent CN1323587A).

[0014] Other techniques in which the use of royal jelly has been investigated include coating nanoparticles and dendritic structures for antimicrobial applications (Mendoza-Resendez et al., 2014), as well as its protective effect against the toxic impact of silver nanoparticles (Dalfardi et al., 2019). Additionally, it has been shown that vacuum packaging and nitrogen flushing can reduce its oxidative degradation.

[0015] Furthermore, fresh royal jelly is more prone to degradation compared to lyophilized forms. This adds a costly step to its handling.

[0016] The above methodologies do not specifically address the dermatological use of royal jelly, which presents certain challenges. Beyond the requirement that royal jelly be in a form that allows both incorporation of its components into cosmetic formulations and protection of sensitive components from degradation, it is also necessary to ensure sufficient duration of contact between royal jelly components and target skin cells to achieve activity in the skin.

[0017] It is well known from the literature that the formation of inclusion complexes of bioactive substances with cyclodextrins provides advantages such as increased water solubility, protection from oxidation, and prevention of molecular degradation. Cyclodextrin has been used for the encapsulation of royal jelly followed by lyophilization to enhance its stability (Szejtli, 1988). Additionally, Jialin et al. (Jialin et al., 2018) reported several successful attempts to incorporate royal jelly into P-cyclodextrins and optimize the process; lyophilized royal jelly was also used in the present study.Liposomes are also typical structures for encapsulating bioactive substances, aiming to modify solubility, protect against degradation, facilitate substance delivery into deeper skin layers, and alter the release profile of the encapsulated compounds.

[0018] The combination of cyclodextrins with liposomes in a single system has been developed by our team, as disclosed in patent WO2021240183A1. This invention can provide the combined advantages of both carriers for the stabilization of royal jelly, enabling controlled release of 10-HDA and polyphenols.

[0019] The present invention aims to overcome the stability limitations of fresh royal jelly, providing immediate release of its 10-HDA and polyphenols. This offers the advantage of rapid restoration in compromised skin and scalp tissues upon application, either as a standalone product or after incorporation into cosmetic and pharmaceutical formulations that can benefit from immediate activity, such as rinse-off products, masks, and fast-acting creams and serums.

[0020] Specifically, in the present invention, for the first time, simultaneous extraction and encapsulation of bioactive components from fresh, unprocessed royal jelly is achieved in a hydroxypropyl-P-cyclodextrin system, which consists exclusively of natural and safe components and provides immediate release of the incorporated polyphenols and 10-HDA.

[0021] The objective of the present invention is the preparation of a stable system for the immediate release of royal jelly components, using an innovative method in which the components are simultaneously extracted and incorporated into cyclodextrin carriers. Upon interaction with the conditions of the skin and scalp — specifically the pH and body temperature — the system releases the royal jelly components while the encapsulated compounds are maintained and protected within the system for at least ten times the shelf life of the raw material. The final formulation is suitable for dermatological and other applications, leveraging the bioactive properties of polyphenols and 10-HDA, either as such or following incorporation into cosmetic or pharmaceutical formulations.

[0022] The advantages and novel features of the method are as follows:

[0023] □ The process is carried out in a single vessel, using fresh royal jelly without any prior processing. This represents a significant improvement over existing encapsulation methods, which employ lyophilized royal jelly — a raw material already considerably more stable than fresh royal jelly. It is an exceptionally simple method that can be easily scaled up, as the required infrastructure consists essentially of extraction equipment, homogenizers, and standard cartridge filters.

[0024] □ The time required for the formation of the dispersion system is short, and the energy input is minimal, resulting in a final production cost that remains proportionally very low.

[0025] □ The optimized extraction and simultaneous incorporation into cyclodextrins provides very high extraction efficiency of polyphenols and 10-HDA. The encapsulation efficiency of the polyphenol system is >99%, and that of 10-HDA is >90%.□ The interaction of P-cyclodextrins using the applied method produces complexes that are stable under appropriate storage conditions. Upon interaction with the target sites (skin and scalp), the complexes dissociate, and 10-HDA and polyphenols are immediately released from the cyclodextrin cavities, providing instantaneous activity at the tissue with which they interact.

[0026] □ Only safe ingredients compatible with the skin, scalp, and mucous membranes are used.

[0027] □ The final extract is standardized for total polyphenols and 10-HDA.

[0028] □ The colloidal system is self-preserving for a period of 3 years at 5-7 °C and for 1 year at room temperature, and does not require the addition of preservatives despite its high water content. This makes the present invention the first, to the knowledge of the inventors, capable of maintaining fresh bioactive royal jelly molecules at room temperature for such an extended period. This stability is attributed to the very high content of polyphenols and 10-HDA, which exhibit antimicrobial properties.

[0029] □ The individual components of the system are protected from oxidation or degradation induced by light and elevated temperature, due to the formation of inclusion complexes with cyclodextrins. Additionally, for the same reason, they are also protected from interactions with other components when the system is used as an ingredient in cosmetic or pharmaceutical formulations.

[0030] □ The system can be used directly on human skin, scalp, and mucous membranes, or it can be incorporated into cosmetic or pharmaceutical formulations for topical administration. In cosmetic or pharmaceutical preparations, for example in gel formulations, it provides transparent solutions when required.

[0031] Disclosure of the Invention

[0032] In order for the present invention to be fully understood by people skilled in the relevant technical field, a detailed description of the method for preparing a dispersion system of fresh royal jelly in cyclodextrin carriers is provided below.

[0033] To prepare the system, fresh royal jelly, stored at 5-7 °C, is allowed to reach room temperature for the shortest possible period of time. The minimum required concentration of hydroxy dedecenoic acid (10-HDA) in the royal jelly is greater than 1.6% w / w, as determined by the method of Zhou et al., 2007.

[0034] Subsequently, the royal jelly is gradually dissolved under stirring in a solvent system consisting of deionized water and either vegetable-derived 1,3-propanediol, glycerol, or vegetable-derived butylene glycol, in a ratio of 1,3-propanediol (or glycerol or butylene glycol) to water ranging from 10 / 90 to 90 / 10. The concentration of royal jelly in the solvent system ranges from 0.2% to 18% w / w. Hydroxypropyl-P-cyclodextrin is pre-dissolved in deionized water at a concentration ranging from 2% to 21% w / w.

[0035] The royal jelly / cyclodextrin / solvent system is subjected to stirring at 1000-3000 rpm for 10-120 minutes at a temperature ranging from 20 °C to 25 °C.A method for preparing a system for encapsulating bioactive components of royal jelly in cyclodextrins, comprising the steps of: a) bringing fresh royal jelly from a storage temperature of 5-7°C to a temperature between 17°C and 23°C;b) dissolving the royal jelly under stirring in a solvent comprising deionized water and at least one polyol selected from vegetable 1,3 -propanediol, glycerol or vegetable butylene glycol, the weight ratio of polyol to water being from 10:90 to 90: 10;c) adding at least one cyclodextrin to the solvent and stirring the resulting royal jelly / cyclodextrin / solvent mixture at a temperature between 20°C and 25°C for 10 to 120 minutes; d) subjecting the mixture to at least one pressure compression and decompression cycle in a sealed extraction device, at a pressure between 0 and 10 bar and at a temperature between 10°C and 25°C;e) maintaining the mixture at a temperature of 5-7°C for 4 to 12 hours in a sealed container; and f) filtering the mixture at a temperature below 10°C through at least one filter having a pore size of 5 pm or less, wherein the pH of the mixture is adjusted to a value between 3.5 and 7.5, thereby obtaining a system in which bioactive components of royal jelly are encapsulated in cyclodextrins.

[0036] According to an embodiment, the invention aims a method for the preparation of a stable system of inclusion of bioactive components of royal jelly in cyclodextrins, which is characterized by the fact that, in the first stage to protect the system, fresh royal jelly, kept at 5-7° C, is allowed to reach room temperature, for the shortest possible time; in a second stage, the royal jelly is gradually dissolved under stirring, in a solvent system consisting of deionized water, properly treated, and either vegetable 1,3 -propanediol or glycerol or vegetable butylene glycol at a ratio of 1,3 -propanediol (or glycerol or butylene glycol) / water: 10 / 90 to 90 / 10, in the third stage, the royal jelly / cyclodextrin / solvent system is stirred (1000-3000 rpm) for 10-120 minutes and at a temperature of 20°C to 25°C, in a fourth step, the system is placed in a bag filter with pores with a diameter of 450 nm to 1000 nm, which is then placed in an extraction device where the pressure and temperature are controlled, so that the pressure ranges from 0 bar to 10 bar, while the temperature from 10°C to 25°C and one to twelve compression and decompression cycles are performed with the decompression phase lasting from 1 minute to 5 minutes, in the fifth stage, the mixture is left in a tightly sealed container at 5-7 °C for 4-12 hours, in the sixth stage, the system is cold filtrated through an array of cartridge filters, with pores of 5 pm-1 pm-0.45 pm, and the pH is controlled and adjusted in the range of 3.5-7.5, in the seventh stage, the total content of polyphenols and the total content of 10-hydroxy decenoic acid in the system are measured, in the eighth stage, the release rate of polyphenols and 10-hydroxy decenoic acid is determined in a buffer solution at pH 7.2 at 37°C and the system is stored in a dark container at a temperature of 5-7°C or room temperature, depending on the application that will follow.

[0037] Preferably, the invention aims a method of preparation of a stable system of encapsulation of bioactive components of royal jelly in cyclodextrins and preferably in solvent, which is characterized by the fact that : in the first stage to protect the system, fresh royal jelly, kept at 5-7° C, is allowed to reach room temperature, preferably 17-23 °C; in a second stage, the royal jelly is gradually dissolved under stirring, in a solvent system consisting of deionized water, and either vegetable 1,3-propanediol or glycerol or vegetable butylene glycol at a ratio of 1,3-propanediol (or glycerol or butylene glycol) / water: 10 / 90 to 90 / 10, in the third stage, the royal jelly / cyclodextrin / solvent system is stirred at 1000-3000 rpm for 10-120 minutes and at a temperature of 20°C to 25°C,in a fourth step, the royal jelly / cyclodextrin / solvent system is placed in a bag filter with pores with a diameter of 450 nm to 1000nm, which is then placed in an extraction device where the pressure and temperature are controlled, so that the pressure ranges from 0 bar to 10 bar, while the temperature from 10°C to 25°C and one to twelve compression and decompression cycles are performed with a decompression phase lasting from 1 minute to 5 minutes, in the fifth stage, the royal jelly / cyclodextrin / solvent system is left in a hermetically sealed container at 5-7 °C for 4-12 hours, in the sixth stage, the royal jelly / cyclodextrin / solvent system is cold filtrated through an array of cartridge filters, with pores of 5 pm,l pm and 0.45 pm, and the pH is controlled and adjusted in the range of 3.5-7.5, in the seventh stage, the total content of polyphenols and the total content of 10-hydroxy decenoic acid in the royal jelly / cyclodextrin / solvent system are measured, preferably by HPLC (Zhou et al. 2007), in the eighth stage, the release rate of polyphenols and 10-hydroxy decenoic acid is determined in a buffer solution at pH 7.2 at 37°C and the royal jelly / cyclodextrin / solvent system is stored in a dark container at a temperature of 5-7°C or room temperature.

[0038] According to other optional features of the method, the method may optionally include one or more of the following features, alone or in combination:

[0039] in order to produce the deionized water, a water from a water supply network first enters in a raw water tank, through a pumping unit, passes through an automatic turbidity filter to remove turbidity and solid particles, and through activated carbon to remove chlorine and organic charge and then an anti-scaling agent is dosed to bind its hardness, while water then passes through a 1 micron cartridge filter and then, in reverse osmosis, enters a reverse osmosis unit producing 3501 / h with a recovery of 70%, while the water produced in the unit is tanked in a stainless steel tank, water is led to a extraction tank through UV unit,

[0040] the minimum concentration requirement of 10-hydroxy decenoic acid in royal jelly, used to create the stable system is greater than 1.6% w / w, according to the method of Zhou et al, 2007 Journal of AO AC International. 90. 244-9. 10.1093;

[0041] the concentration of royal jelly in the solvent ranging between 0.2% and 18% w / w, and cyclodextrin, preferably hydroxypropyl-P-cyclodextrin, is pre-dissolved in deionized water, at a concentration of 2% - 21% w / w,

[0042] a quality requirement of deionized water, in order to achieve the formation of stable cyclodextrin-royal jelly complexes is: less than or equal to 1 pS / cm at 25°C.

[0043] a continuous recirculation of the water,

[0044] a pH of the royal jelly / cyclodextrin / solvent system being regulated in the range between 3.5-7.5,

[0045] the total polyphenol content is greater than 220 mg / L of gallic acid equivalents and the total content of 10-hydroxy decenoic acid is greater than 0,03% w / w,

[0046] a cumulative release of polyphenols at pH 7,2 and at a temperature of 37 °C is between 75% - 95% in 1 minute, while a total release of encapsulated polyphenols takes place in 10 minutes,a cumulative release of 10-hydroxy decenoic acid is between 70-80% in 1 minute, while a total release of encapsulated 10-hydroxy decenoic acid takes place in 10 minutes,

[0047] the cyclodextrin is hydroxypropyl-P-cyclodextrin,

[0048] the royal jelly is present in the solvent at a concentration between 0.2% and 18% w / w, and the cyclodextrin is pre-dissolved in deionized water at a concentration between 2% and 21% w / w.

[0049] he deionized water has an electrical conductivity of < 1 pS / cm at 25°C.

[0050] he deionized water is continuously recirculated during its preparation prior to step b).

[0051] he royal jelly used has a content of 10-hydroxydecenoic acid greater than 1.6% w / w.

[0052] the encapsulated system obtained exhibits a total polyphenol content greater than 220 mg / L expressed as gallic acid equivalents and a 10-hydroxydecenoic acid content greater than 0.03% w / w.

[0053] the encapsulated system obtained releases 75-95% of the encapsulated polyphenols within 1 minute when placed in a buffer at pH 7.2 and 37°C.

[0054] the encapsulated system obtained releases 70-80% of the encapsulated 10-hydroxydecenoic acid within 1 minute when placed in a buffer at pH 7.2 and 37°C.

[0055] Figures and definitions

[0056] “Stable” means that the encapsulation system maintains, under normal storage conditions, its physical integrity and its ability to retain the encapsulated bioactive components, without substantial phase separation, precipitation, or degradation, nor significant chemical degradation of its components, to an extent that would impair its performance or intended use.

[0057] “bioactive components of royal jelly” comprise 10-HAD and polyphenols and more preferably consist in 10-HAD and polyphenols.

[0058] “room temperature” may be defined as a temperature between 17 and 23 °C, limits included.

[0059] “an anti-scaling agent” may correspond to zinc salts. Zinc salts may be dosed until the acceptable hardness is achieved.

[0060] In a preferred embodiment of the invention and the method, cyclodextrin corresponds to betacyclodextrin and more preferably to Hydroxypropyl-P-cyclodextrin. In addition cyclodextrin may correspond into the description at cyclodextrin, or cyclodextrin carriers.

[0061] “the royal jelly / cyclodextrin / solvent system” may correspond to a “mixture” in the description or “system” or “stable system” or colloidal system or dispersion system.Figure 1: In situ imaging of fluorescein diffusion in the hair cross-section. The fluorescence emission signal for fluorescein was captured using specific excitation and emission wavelengths (498 / 517 nm). Fluorescein is visualized using a color scale (low levels in dark shades, high levels in bright colors).

[0062] Figure 2. Hair Porosity. The quantitative assessment of fluorescein diffusion in hair is reported for each experimental group and is expressed as % values of fluorophore penetration into the hair shaft. Results are presented as mean ± S.D. ***, p < 0.001; **, p < 0.01 - ANOVA with Dunnett’s post-hoc test for multiple comparisons vs. the stress group, or t-test with Welch’s correction for binary comparison (95% confidence interval).

[0063] Figure 3: Imaging of isolated hair-shaft samples collected from volunteers (Volunteer I - fig.3-A) (Volunteer IV-fig.3-B) using scanning electron microscopy, before (left) and after (right) the use of the routine consisting of a shampoo enriched with 0.5% w / w RJ-IR and a hair conditioner enriched with 0.5% w / w RJ-IR. Magnification 1000* and 2000*.

[0064] Figure 4. Assessment of gene expression of SOD1 (a) and Nrf2 (b) following exposure of HFDPCs to RJ-IR 0.05% v / v. Non-parametric t-test, comparison with control. *p-value < 0.05.

[0065] Figure 5. Assessment of cell viability (a) and gene expression of the FLG gene (b) after exposure of NHEK to RJ-IR 0.05% v / v. Non-parametric t-test, comparison with control. **p-value < 0.01.

[0066] For a clearer understanding of the present invention by persons skilled in the relevant technical field, a detailed description of a method for the preparation of a stable system of inclusion (i.e. encapsulation) of bioactive components of fresh royal jelly and cyclodextrin carriers is provided below.

[0067] To prepare the system, fresh royal jelly, stored at 5-7 °C, is allowed to reach room temperature for the shortest possible period of time. The minimum required concentration of 10-HDA in the royal jelly is greater than 1.6% w / w, as determined by the method of Zhou et al., 2007.

[0068] Subsequently, the royal jelly is gradually dissolved under stirring in a solvent system consisting of deionized water and either vegetable-derived 1,3-propanediol, glycerol, or vegetable-derived butylene glycol, in a ratio of 1,3-propanediol (or glycerol or butylene glycol) to water ranging from 10 / 90 to 90 / 10. The concentration of royal jelly in the solvent system ranges from 0.2% to 18% w / w. Cyclodextrin, preferably P-cyclodextrin and more preferably Hydroxypropyl-P-cyclodextrin is predissolved in deionized water at a concentration ranging from 2% to 21% w / w.

[0069] Royal jelly dissolved in a solvent and cyclodextrin pre-dissolved in deionized water are added together (i.e. mixed together).

[0070] The royal jelly / cyclodextrin / solvent system or the system is subjected to stirring at 1000-3000 rpm for 10-120 minutes at a temperature ranging from 20 °C to 25 °C.

[0071] Subsequently, the system is placed in a bag filter having pores with a diameter ranging from 450 nm to 1000 nm. The bag filter is then positioned in an extraction device in which pressure and temperature can be controlled. The process is carried out under a pressure ranging from 0 bar to 10 bar and at atemperature between 10 °C and 25 °C. Compression and decompression cycles are performed, the number of which may range from 1 to 12, with the decompression phase lasting from 1 minute to 5 minutes.

[0072] The quality requirement for the deionized water is < 1 pS / cm at 25 °C, in compliance with the specifications of the European Pharmacopoeia for the preparation of parenteral pharmaceutical products. This quality is necessary in order to enable the formation of stable cyclodextrin-royal jelly complexes.

[0073] The preparation of the deionized water used in the present invention is carried out as follows:

[0074] The water from the water supply network enters a raw water storage tank (volume 2 m3) via a suitable pumping system and passes through an automatic turbidity filter for the removal of turbidity and suspended solids, followed by an activated carbon filter for the removal of chlorine and organic load. Subsequently, an anti-scalling agent ( preferably zinc salt) is dosed to bind water hardness. Prior to entering the central reverse osmosis unit, the water passes through a 1 -micron cartridge filter. Subsequently, having the appropriate treatment for use, the water enters the reverse osmosis unit producing 350 L / h with a recovery rate of 70%. The produced water is stored in a stainless-steel tank with a volume of 5 m3. From this tank, the water is conveyed via an appropriate pumping system to the deionizer and is then supplied online to the extraction tank through a UV unit. To prevent stagnant water in the distribution network, continuous recirculation of the water is carried out, with return to the storage tank, (with return to the tank in order to avoid stagnant water in the water treatment network).

[0075] After the addition of the components, the pH of the system is adjusted to a range between 3.5 and 7.5.

[0076] The mixture (i.e. the system) is then kept in a hermetically sealed container at 5-7 °C for 4-12 hours. Subsequently, the system is subjected to cold filtration through an array of cartridge filters with pore sizes of 5 pm, 1 pm, and 0.45 pm, and the pH is rechecked and, if necessary, readjusted to the range of3.5-7.5.

[0077] Subsequently, the total polyphenol content and the total 10-HDA content of the system are measured.

[0078] The release rate of polyphenols and 10-HDA is determined in a buffer solution at pH 7.2 at 37 °C, and the colloidal system is stored in a dark container at a temperature of 5-7 °C or at room temperature, depending on the intended application.

[0079] Based on the method described above, in order for the system of the invention to be suitable for its intended use, the total polyphenol content shall be greater than 220 mg / L gallic acid equivalents (GAE), while the total 10-HDA content shall be greater than 0.03% w / w.

[0080] The cumulative release of polyphenols at pH 7.2 and at a temperature of 37 °C is 75%-95% within 1 minute, while the system releases substantially all encapsulated polyphenols within 10 minutes. Thecorresponding values for 10-HDA are 70%-80% within 1 minute, while the system releases substantially the entire amount of encapsulated 10-HDA within 10 minutes.

[0081] In order to provide a full understanding of the present invention, the following examples are provided:

[0082] Example 1

[0083] After reaching ambient temperature, royal jelly is dispersed at a concentration of 1% in a solvent system consisting of water and vegetable-derived 1,3-propanediol at a 1,3-propanediol / water ratio of 50 / 50. In the aqueous phase, hydroxypropyl-P-cyclodextrin is pre-dissolved at a concentration of 8%.

[0084] The royal jelly / cyclodextrin / solvent system is subjected to stirring at 1000-3000 rpm for 10 minutes at a temperature of 25 °C.

[0085] Subsequently, the system is placed in a bag filter having a pore diameter of 450 nm. The bag filter is then positioned in an extraction device in which pressure and temperature can be controlled. The process is carried out at a pressure of 1 bar and a temperature of 25 °C, with three compression and decompression cycles, separated by a resting period of 1 minute.

[0086] The final dispersion system after filtration exhibits the values shown in Table 1.

[0087] Table 1

[0088]

[0089] Example 2

[0090] After reaching ambient temperature, royal jelly is gradually dissolved at a concentration of 1% in a solvent system consisting of water and vegetable-derived 1,3-propanediol at a 1,3-propanediol / water ratio of 25 / 75. In the aqueous phase, hydroxypropyl-P-cyclodextrin is pre-dissolved at a concentration of 5%.

[0091] The royal jelly / cyclodextrin / solvent system is subjected to stirring at 1000-3000 rpm for 10 minutes at a temperature of 25 °C.

[0092] Subsequently, the system is placed in a bag filter having a pore diameter of 450 nm. The bag filter is then positioned in an extraction device in which pressure and temperature can be controlled. The process is carried out at a pressure of 3 bar and a temperature of 25 °C, with seven compression and decompression cycles, separated by a resting period of 2 minutes.The final dispersion system after filtration exhibits the values shown in Table 2.

[0093] Table 2

[0094]

[0095] Demonstration of Effectiveness

[0096] The present inclusion system for the bioactive components of fresh royal jelly in cyclodextrins is intended for topical application in cosmetic products, following its incorporation into a formulation (as a solution, suspension, emulsion, or gel), and exhibits reparative and antioxidant properties. These properties are mediated by the ability of the system to achieve the following effects:

[0097] (a) When incorporated at a concentration of 0.5% w / w into hair shampoo and hair care mask formulations, used in combination as a treatment:

[0098] (i) to reduce hair fiber porosity (ex vivo experiment) under normal (baseline) conditions or under conditions of urban pollution;

[0099] (ii) to cover and fill defects in the hair shaft, thereby restoring the structural integrity of the hair (in vivo experiment, observation by scanning electron microscopy (SEM)).

[0100] (b) When incorporated at a concentration of 0.5% w / w into a facial cream, to restore skin barrier function in individuals with dry or very dry facial skin or skin requiring regeneration.

[0101] (c) When incorporated at a concentration of 0.05% w / w into a cell culture medium, to increase, in cellulo, the transcription of the antioxidant genes SOD1 and NRF2.

[0102] These results are unprecedented, as the interaction of the different and specific encapsulated polyphenols and 10-hydroxy dedecenoic acid (10-HDA) with hydroxypropyl-P-cyclodextrin in the encapsulation system of the present invention could not have been predicted.

[0103] Documentation - Case 1

[0104] To document the use of the combined delivery system of bioactive ingredients from fresh royal jelly in cyclodextrins (hereinafter referred to as RJ-IR) in cosmetic formulations, an ex vivo study was conducted to evaluate its effectiveness against pollution-induced stress on the hair shaft (environmental pollution and UVA). The formulations used for the study were a hair shampoo base containing 0.5% v / v RJ-IR and a hair conditioner base containing 0.5% w / w RJ-IR.

[0105] METHODOLOGYIn this study, the restorative action of the shampoo combined with the conditioner enriched with 0.5% RJ-IR — used as a routine — was comparatively evaluated against a standard shampoo and conditioner routine of simple composition (Placebo), to assess their ability to reduce hair porosity induced by urban pollution (PM10 + UV-A). The evaluation was performed using the diffusion of a fluorescent probe associated with hair permeability and its damaged structural integrity.

[0106] The hair samples used in the study were exposed to urban dust (PMlO-like; Ref. ERM-CZ100; certified European Reference Material; 0.375 pg / cm2) and subsequently irradiated with UV-A (LED source, emission peak at = 365 nm; 84 J / cm2; 6 hours of irradiation) using a standardized UV emission system (OxiProteomics®).

[0107] For the routine application, the hair samples were washed with the shampoos (either the simple composition - Placebo, or the 0.5% RJ-IR enriched version) and then rinsed with ultrapure water. The conditioner (either simple composition -Placebo or enriched with 0.5% RJ-IR) was applied to wet hair samples for a contact time of 5 minutes, followed by natural air drying.

[0108] The group exposed to stress (Stress group) received no treatment other than water-washing steps and exposure to stress conditions. The Control group received no stress exposure or treatments except for rinsing with water. After the experimental procedures, the hair samples were collected for analysis, and the following results were obtained :

[0109] Table 3

[0110]

[0111] The hair samples from each experimental condition were immersed in a fluorescein solution (fluorescein; Ex. [465-490 nm] / Em. [520-530 nm]). Subsequently, the excess fluorophore wasremoved by extensive rinsing with ultrapure water, and the hair samples were collected and cryopreserved in Cryomatrix (OCT) until analysis.

[0112] Next, cross-sections of the hair samples (perpendicular to the hair shaft axis, sagittal view) with a thickness of 5 pm were obtained using a cryostat (Leica), followed by their analysis. More specifically, fluorescent images (16-bit, TIFF format) were captured across the full intensity range of the signal using a fluorescence microscope (ThermoFisher, Evos M5000) and analyzed with ImageJ software (Schneider, 2012). Image acquisition for all conditions was performed using identical imaging parameters (40X objective lens).

[0113] Quantification of fluorophore diffusion (%) was assessed as the penetration distance of the dye (evaluated at a defined fluorescence threshold) at both edges of the hair cross-section along its diameter, following the penetration axis (from cuticle to cortex):

[0114] Dye penetration (%) = (Dye penetration distance (pm) / Hair diameter (pm)) * 100

[0115] In situ detection of fluorescein diffusion was performed using fluorescence microscopy on cross-sections perpendicular to the hair axis, in oval view (Figure 1).

[0116] RESULTS

[0117] As expected, an increase in dye penetration and porosity (associated with the weakening of the structural integrity of the hair shaft) was observed upon exposure to stress (PM10 + UV-A). The application of the RJ-IR routine showed a remarkable reduction in dye penetration and beneficial effects on the structural integrity and porosity of the hair. For a better understanding of the results, see Figure 1.

[0118] Statistical analyses were performed using the “GraphPad” software (La Jolla, California, USA). One-way ANOVA with Dunnett’ s post-hoc test was applied for multiple comparisons against the stress group, or a t-test with Welch’s correction was used for pairwise comparisons (95% confidence interval).

[0119] An effectiveness value (%) was calculated for the groups using the Control group as the reference for maximum effectiveness (100%) and the Stress group as the reference for minimum effectiveness (0%): Effectiveness % (group X) = [(Dye penetration (Stress) - Dye penetration (group X)) / (Dye penetration (Stress) - Dye penetration (Control))] x 100

[0120] Table 4

[0121]

[0122]

[0123] <

[0124] < <

[0125] <

[0126]

[0127] Hair Porosity. See figure 2

[0128] The quantitative assessment of fluorescein diffusion in hair is reported for each experimental group and is expressed as % values of fluorophore penetration into the hair shaft. Results are presented as mean ± S.D. ***, p < 0.001; **, p < 0.01 - ANOVA with Dunnett’s post-hoc test for multiple comparisons vs. the stress group, or t-test with Welch’s correction for binary comparison (95% confidence interval).

[0129] The RJ-IR routine demonstrated a beneficial restorative effect (100% effectiveness), reducing hair porosity and dye penetration, and showing a significant differential performance compared to the simple-composition placebo routine. Overall, the RJ-IR routine (Shampoo + Hair Conditioner) showed significant restorative efficacy against pollution-induced alterations in hair porosity, indicating its potential benefits for the structural integrity of the hair.

[0130] Documentation - Case 2

[0131] To assess the potential beneficial effects of RJ-IR on hair structure, an additional clinical study was conducted in which five (5) volunteers used the RJ-IR routine under real-life conditions. The routine consisted of using a shampoo enriched with 0.5% w / w RJ-IR and a hair conditioner enriched with 0.5% w / w RJ-IR. Hair samples were collected from the volunteers before and after applying the RJ-IR routine, followed by examination of the samples using scanning electron microscopy (SEM).

[0132] METHODOLOGY

[0133] The tests were performed in accordance with Regulation (EC) No. 1223 / 2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products.

[0134] The study was conducted following the recommendations of the Cosmetics Europe - The Personal Care Association Guidelines:

[0135] • Guidelines for Product Testing to Assess Human Skin Compatibility, 1997Guidelines for the Evaluation of the Efficacy of Cosmetic Products, 2008

[0136] • The microscope used for this study was the Phenom SCANING.

[0137] • Two types of samples were subjected to SEM testing:

[0138] • A reference sample collected before the application of the products

[0139] • A sample collected after the application of the products

[0140] OLUNTEER I (TESTER I)

[0141] Reference sample collected before, and sample collected after use of the products by Volunteer No. 1 VOLUNTEER II (TESTER II)

[0142] Reference sample collected before, and sample collected after use of the products by Volunteer No. 2 VOLUNTEER III (TESTER III)

[0143] Reference sample collected before, and sample collected after use of the products by Volunteer No. 3 VOLUNTEER IV (TESTER IV)

[0144] Reference sample collected before, and sample collected after use of the products by Volunteer No. 4 VOLUNTEER V (TESTER V)

[0145] Reference sample collected before, and sample collected after use of the products by Volunteer No. 5 RESULTS

[0146] Visualization of hair samples. See figure 3

[0147] Based on the results of the analysis, it is concluded that the hair treated with the RJ-IR routine exhibits the following changes:

[0148] • Improvement in the uniformity of the hair structure

[0149] • Improvement in the hair surface, with a smoother appearance of the cuticle (scales)

[0150] • Closing of the cuticle in the samples of Volunteers II, IV, and V

[0151] • Filling of gaps within the hair structure

[0152] Documentation - Case 3

[0153] To evaluate the potential beneficial effects of RJ-IR on human skin, we conducted an in vivo study on 10 volunteers to assess its restorative effectiveness. Volunteers applied a face cream of simple composition (placebo) and a cream enriched with 0.5% v / v RJ-IR. Organoleptic tests followed, aiming to objectively confirm the declared efficacy of the product and determine skin-condition parameters such as transepidermal water loss (TEWL).METHODOLOGY

[0154] The tests were performed in accordance with Regulation (EC) No. 1223 / 2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products.

[0155] The study was carried out based on the recommendations of the Cosmetics Europe - The Personal Care Association Guidelines:

[0156] • Guidelines for Product Testing to Assess Human Skin Compatibility, 1997

[0157] • Guidelines for the Evaluation of the Efficacy of Cosmetic Products, 2008

[0158] Cosmetic product testing on volunteers using the products under standard use conditions (home use) was conducted under the supervision of specialized personnel performing the study and a dermatologist.

[0159] The volunteers participating in the study were not given special requirements, under the assumption that the product’s effects and functional properties should be evaluated under real usage conditions. The results of the use- tests could be influenced by factors such as:

[0160] • skin type and skin condition

[0161] • genetically determined individual characteristics

[0162] • individual preferences

[0163] • lifestyle

[0164] • environmental conditions

[0165] Volunteers received one package of each of the two products (with and without RJ-IR) and were instructed to apply the products regularly for 2 weeks. According to usage instructions, the placebo face cream was applied to the left side of the face, and the face cream enriched with 0.5% v / v RJ-IR to the right side.

[0166] Volunteers selected for organoleptic tests were required to appear for measurements before starting use of the test product and on the dates specified in the study plan. They were informed of the need to avoid using other products with similar or related functions during the study. They were also instructed to discontinue product use immediately in case of any adverse symptoms or sensations in the application areas and to report them to the researcher.

[0167] Documentation of the claim requires more than 50% positive responses from subjects to the question regarding that claim.

[0168] In vivo instrument-based tests were carried out under standardized environmental conditions (temperature 20-25 °C, relative humidity 40-60%), using specialized measurement equipment from Courage & Khazaka:

[0169] • Tewameter TM HEX - Measurement of TEWLThe studies were conducted:

[0170] • before starting the application of the tested formulations

[0171] • 30 minutes after the first application of the products, in the laboratory

[0172] • after regular use for 14 days

[0173] • on a group of 10 women with dry / very dry skin

[0174] • on the facial skin area selected for each participant

[0175] • on facial skin properly prepared for testing, i.e., clean, makeup-free, and untreated by the test formulation

[0176] Confirmation of the claim based on instrument testing requires a measurable improvement in more than 50% of subjects.

[0177] RESULTS

[0178] The instrument-based tests performed demonstrated the following:

[0179] Table 5

[0180]

[0181]

[0182] Simple-composition face cream (Placebo)

[0183] -Does not contribute to reducing water evaporation from the stratum corneum / does not regenerate the epidermis / does not repair the skin’s hydrolipidic barrier / does not restore the skin’s hydrolipidic barrier.

[0184] The TEWL parameter showed no change:

[0185] -after a single use.

[0186] -after 2 weeks of use.

[0187] Cream enriched with 0.5% RJ-IR

[0188] Leaves a protective layer on the skin that helps reduce water evaporation from the stratum corneum / regenerates the epidermis / repairs the skin’s hydrolipidic barrier / restores the skin’s hydrolipidic barrier.

[0189] The TEWL parameter decreased:

[0190] -after a single use, by an average of 7% in the group. Improvement was observed in 8 out of 10 subjects. -after 2 weeks of use, by an average of 12% in the group. Improvement was observed in 10 out of 10 subjects.

[0191] In comparison, the cream enriched with 0.5% RJ-IR shows 100% greater efficacy than the simple-composition face cream (Placebo).

[0192] Documentation - Case 4

[0193] Transcriptional regulation of genes associated with antioxidant and epidermal barrier-repair activity in human dermal papilla cells of hair follicles and keratinocytes (NHEK).

[0194] METHODOLOGY

[0195] Following the evaluation of the restorative activity of the tested system on hair and skin, and after a literature review, the expression profile of the genes for superoxide dismutase-1 (SOD1) and theantioxidant transcription factor Nrf2 was examined after administration of the active ingredient to human hair follicle dermal papilla cells (HFDPCs).

[0196] Furthermore, human keratinocytes were incubated with the active ingredient, followed by evaluation of filaggrin (FLG), a structural protein essential for maintaining the epidermal barrier.

[0197] A 24-hour incubation was performed on the cell cultures, after which the above genes were quantified using real-time polymerase chain reaction (RT-PCR).

[0198] For total RNA isolation (500 ng) and cDNA synthesis, the Nucleospin RNA kit (Macherey-Nagel, Duren, Germany) and PrimeScript-RT reagent kit (Takara Bio, Otsu, Japan) were used, respectively. The RT-PCR method and the gene analysis procedure were conducted following standard Laboratory protocols. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was selected as the reference gene for normalization of expression levels. Comparisons were made between two conditions:

[0199] • cells not incubated with the active ingredient (control), and

[0200] • cells incubated with the RJ-IR system.

[0201] RESULTS

[0202] According to the results shown in the figure 4, an enhancement of gene expression for stress-response factors was observed in HFDPCs.

[0203] Additionally, incubation of human epidermal keratinocytes (NKHEK) with RJ-IR at 0.05% v / v did not affect cell viability and led to upregulation of mRNA levels of filaggrin (FLG) (Figure 5).

Claims

CLAIMS1. A method of preparation of a stable system of encapsulation of bioactive components of royal jelly in cyclodextrins, which is characterized by the fact that :-in the first stage, fresh royal jelly, kept at 5-7° C, is allowed to reach room temperature, preferably between 17-23 °C;-In a second stage, royal jelly is gradually dissolved under stirring, in a solvent consisting of deionized water, and either vegetable 1,3-propanediol or glycerol or vegetable butylene glycol at a ratio of 1,3-propanediol (or glycerol or butylene glycol) / water: 10 / 90 to 90 / 10, cyclodextrin is pre-dissolved in deionized water;-In the third stage, the royal jelly / cyclodextrin / solvent system is stirred at 1000-3000 rpm for 10-120 minutes and at a temperature of 20°C to 25°C,-In a fourth step, the royal jelly / cyclodextrin / solvent system is placed in a bag filter with pores with a diameter of 450 nm to 1000 nm, which is then placed in an extraction device where the pressure and temperature are controlled, so that the pressure ranges from 0 bar to 10 bar, while the temperature from 10°C to 25°C and one to twelve compression and decompression cycles are performed with a decompression phase lasting from 1 minute to 5 minutes,-In the fifth stage, the royal jelly / cyclodextrin / solvent system is left in a hermetically sealed container at 5-7 °C for 4-12 hours,-In the sixth stage, the royal jelly / cyclodextrin / solvent system is cold filtrated through a cold filtration with an array of cartridge filters, with pores of 5 pm,l pm and 0.45 pm, and the pH is controlled and adjusted in the range of 3.5 -7.5,-In the seventh stage, the total content of polyphenols and the total content of 10-hydroxy decenoic acid in the royal jelly / cyclodextrin / solvent system are measured, preferably by HPLC (Zhou et al. 2007), -In the eighth stage, the release rate of polyphenols and 10-hydroxy decenoic acid is determined in a buffer solution at pH 7.2 at 37°C and the royal jelly / cyclodextrin / solvent system is stored in a dark container at a temperature of 5-7°C or room temperature.

2. A method of preparation of a stable system of encapsulation of bioactive components of royal jelly in cyclodextrins, according to the claim 1, which is characterized by the fact that, in order to produce the deionized water, a water from a water supply network first enters in a raw water tank, through a pumping unit, passes through an automatic turbidity filter to remove turbidity and solid particles, and through activated carbon to remove chlorine and organic charge and then an anti-scaling agent is dosed to bind its hardness, while water then passes through a 1 micron cartridge filter and then, in reverse osmosis, enters a reverse osmosis unit producing 350 1 / h with a recovery of 70%, while the water produced in the unit is tanked in a stainless steel tank, water is led to a extraction tank through UV unit.

3. A method of preparation of a stable system of encapsulation of bioactive components of royal jelly in cyclodextrins, according to claim 1, which is characterized by the fact that, the minimum concentration requirement of 10-hydroxy decenoic acid in royal jelly, used to create the stable system is greater than 1.6% w / w, according to the method of Zhou et al, 2007 Journal of AO AC International.

90. 244-9. 10.1093.

4. A method of preparation of a stable system of encapsulation of bioactive components of royal jelly in cyclodextrins, according to claim 1, which is characterized by the concentration of royal jelly in the solvent ranging between 0.2% and 18% w / w, and cyclodextrin, preferably hydroxypropyl-P-cyclodextrin, is pre-dissolved in deionized water, at a concentration of 2% - 21% w / w.

5. A method of preparation of a stable system of encapsulation of bioactive components of royal jelly in cyclodextrins, according to claims 1 and 2, which is characterized by the fact that a quality requirement of deionized water, in order to achieve the formation of stable cyclodextrin-royal jelly complexes is: less than or equal to 1 pS / cm at 25°C.

6. A method of preparation of a stable system of the encapsulation of bioactive components of royal jelly in cyclodextrins, in accordance with claims 1 and 2, which is characterized by a continuous recirculation of the water.

7. A method of preparation of a stable system of encapsulation of bioactive components of royal jelly in cyclodextrins, in accordance with claim 1, which is characterized by a pH of the royal jelly / cyclodextrin / solvent system being regulated in the range between 3.5-7.5.. A method of preparation of a stable system of encapsulation of bioactive components of royal jelly in cyclodextrins, in accordance with claim 1, which is characterized by the fact that, the total polyphenol content is greater than 220 mg / L of gallic acid equivalents and the total content of 10-hydroxydecenoic acid is greater than 0,03% w / w.

9. A method of preparation of a stable system of encapsulation of bioactive components of royal jelly in cyclodextrins, in accordance with claim 1, which is characterized by the fact that, a cumulative release of polyphenols at pH 7,2 and at a temperature of 37 °C is between 75% - 95% in 1 minute, while a total release of encapsulated polyphenols takes place in 10 minutes.

10. A method of preparation of a stable system of encapsulation of bioactive components of royal jelly in cyclodextrins, in accordance with claim 1, which is characterized by that a cumulative release of 10-hydroxy decenoic acid is between 70-80% in 1 minute, while a total release of encapsulated 10-hydroxy decenoic acid takes place in 10 minutes.

Images