Process for the preparation of a colloidal system for the stabilization and controlled release of royal jelly components for various uses

Abstract

Process for the preparation of a stable and controlled release colloidal system of components of royal jelly, wherein said system is characterized by extracting the components of royal jelly and simultaneously encapsulating in a liposome-cyclodextrin combined carrier, to obtain a controlled release, taking advantage of the particular characteristics of both carriers, namely: the ability of cyclodextrins to encapsulate polyphenols and their enhanced skin penetration capacity, and the ability of liposomes to encapsulate a large number of components of various degrees of polarity, for local component transport and controlled release rate.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method for the preparation of a stable colloidal royal jelly dispersion system providing controlled release of the ingredients, characterized by extracting the ingredients of royal jelly and simultaneously encapsulating them in a liposome-cyclodextrin combined carrier, showing controlled release of the ingredients. The system thus prepared is intended for cosmetic use and other uses, in itself or incorporated in cosmetics or drugs.

[0002] BACKGROUND AND PRIOR ART

[0003] Up to now, such a product or a method for its preparation has not been known. In particular, royal jelly is a white, viscous substance with a gel-like texture secreted by the royal gland of young worker bees. Due to its excellent biological properties, royal jelly is widely used in the food, supplement and cosmetic industries. Many studies have shown anti-aging, anti-inflammatory, antimicrobial, anticancer and anti-diabetic properties. These activities are mainly attributed to the bioactive ingredients it contains (Ramanathana et al., 2018).

[0004] Royal jelly is mainly composed of water (50-56%), proteins (18%), carbohydrates (15%), lipids (3%-6%), minerals (1.5%) and vitamins. Based on spectroscopic analysis, approximately 185 substances have been detected in royal jelly. The main bioactive substances are royalactin and 10-hydroxy-2-decenoic acid (10-HDA). Substances such as adenosine monophosphate, acetylcholine, polyphenols and hormones (such as testosterone, progesterone, prolactin and estradiol) are also detected in royal jelly samples and exhibit biological activity (Nagai et al., 2004; Sugiyama et al., 2012, Ramadan et al., 2012).

[0005] 10-HDA is the main fatty acid in royal jelly. Since it is present only in royal jelly, it is an indicator of its quality when proven anticancer, antibacterial and immunomodulatory, anti-inflammatory, vasodepressor properties (Townsend et al., 1961; Blum et al., 1959., Vucevic et al., 2007). As for the skin, there are indications of enhanced collagen synthesis and indications of inhibition of metalloproteinase synthesis, in fact making it a molecule of interest for anti-aging uses (Fujii et al., 1990; Koya-Miyata et al., 2004; Yang et al., 2010). Moreover, it is an effective tyrosinase inhibitor, conferring skin whitening properties (Dynek et al.; 2008).

[0006] Polyphenols are also important bioactive molecules, with skin activities of antioxidant and photoprotective nature.

[0007] Royal jelly shows significant physicochemical stability problems that depend on time and storage temperature. The ideal storage temperature for royal jelly is -20°C, while higher temperatures cause color changes and component degradation due to Maillard reactions, enzymatic reactions and lipid-protein reactions (Chen C et al., 1995).

[0008] To minimize the occurrence of this degradation, royal jelly should be stored in dark containers and refrigerators (Sabatini et al., 2009).

[0009] To avoid the degradation of royal jelly, several technological developments have been used, such as lyophilization or encapsulation (CN1323587A patent).

[0010] Other technological developments in which royal jelly applications have been studied are nanoparticle coatings and dendritic structures for antimicrobial applications (Mendoza-Resendez, 2014) and can introduce a protective activity in the toxic effects of silver nanoparticles on the hippocampus (Dalfardi et al., 2019).

[0011] The above technological developments do not explicitly involve the dermatological use of royal jelly, which presents some challenges. In addition to the fact that royal jelly should be in a state that allows its components to be dispersed in a cosmetic form, on the one hand, and to protect sensitive components from degradation, it should also ensure the duration of the contact of the royal jelly components with the target cells, so that their activity on the skin can be achieved.

[0012] It is known from the literature that the formation of encapsulated complexes of biologically active substances in cyclodextrins offers advantages such as increased solubility in water, protection against oxidation and protection against molecular degradation. Cyclodextrins have been used to encapsulate royal jelly, followed by lyophilization to increase its stability (Szejtli, 1988).

[0013] Liposomes are also standard encapsulation structures for biologically active substances, with the aim of changing the solubility, preventing degradation, taking the substance deeper into the skin and changing the release rate of the encapsulated substance.

[0014] The combination of the above carriers into a single system can provide the combined advantages of both carriers, as described in the patent of our laboratory, creating a method of preparation and a stable colloidal propolis system that exhibits controlled release (WO2017089842A1 patent).

[0015] The present invention aims to eliminate the stability drawbacks of royal jelly, but also aims to control the release of its components, more particularly the 10-HDA and polyphenols it contains. In particular, in the present invention, the simultaneous extraction and encapsulation of royal jelly components in a liposome-cyclodextrin combined system is implemented for the first time, which is composed entirely of natural and safe ingredients, and which exhibits controlled release of the encapsulated polyphenols and 10-HDA.

[0016] More specifically, the present application takes advantage of the peculiar characteristics of the two carriers, i.e. the ability of cyclodextrins to encapsulate polyphenols and their enhanced skin penetration ability, and the ability of liposomes to encapsulate a large number of ingredients of various degree of polarity, for the local transport and controlled release rate of the components. More specifically, liposomes are used to encapsulate 10-HDA due to the physico-chemical affinity of 10-HDA with the fatty chains of the liposome phospholipids. The process of extraction and encapsulation of the components of royal jelly (RJ) into the combined system takes place in a single container.

[0017] The aim of the present application is to prepare a stable colloidal system of royal jelly dispersion with innovative methods, in which the components of royal jelly are extracted and simultaneously encapsulated in a liposome-cyclodextrin carrier. When the system is present in suitable conditions, it exhibits a controlled release of the components, while the encapsulated components are preserved and protected in the combined system by suitable storage conditions. The final composition is suitable for dermatological and other uses, exploiting the bioactive properties of polyphenols and 10-HDA, either as such or after incorporation in cosmetic or pharmaceutical forms.

[0018] The advantages and novel features of the method are the following:

[0019] • It is a particularly simple method for the production of a complex combined royal jelly delivery system, with a very simple preparation method. The steps of formation of inclusion complexes between hydroxypropyl-β-cyclodextrin or β-cyclodextrin and the active components of royal jelly, and the step of encapsulation of royal jelly in liposomes, in free form or in the form of complexes with cyclodextrins, take place in a single container during extraction.

[0020] • The time required for colloid formation is short and the energy supply is minimal, so that the final production costs are proportionally kept at a very low level.

[0021] • By virtue of the simultaneous extraction of specific different carriers (liposomes and cyclodextrins), very high extraction yields of polyphenols and 10-HDA are provided. The encapsulation efficiency of the polyphenol system is > 95% and that of the 10-HAD system is > 85%.

[0022] • The interaction of the two carriers with polyphenols and 10-HDA provides a controlled and even prolonged release of the components of royal jelly under suitable conditions. Under storage conditions, the components are kept protected in their interior.

[0023] • The combination of the lipid composition of the liposomes and the components is such a combination that avoids the problem of fusion and aggregation of the liposomes, which results in a system of excellent stability from the physico-chemical point of view (mean hydrodynamic diameter, z-potential, polydispersity index, pH) and from the point of view of the preservation and protection of polyphenols inside the liposomes during storage.

[0024] • Only safe and skin and mucosa compatible ingredients are used.

[0025] • The final extract is titrated to total polyphenols and 10-HDA.

[0026] • The colloidal system is self-sustaining at a temperature of 5-7°C for a period of 1 year and does not require the addition of preservatives, despite its high water content. This is due to the very high polyphenol content, which exhibits antimicrobial properties.

[0027] • Due to the double encapsulation in liposomes and cyclodextrins, the ingredients of the colloidal system are protected from oxidation or degradation caused by light and temperature increases. In addition, for the same reason, in the case where the colloidal system is used as a component in a cosmetic or pharmaceutical preparation, it can also be protected from interaction with other components.

[0028] • The colloidal system can be used directly on human skin and mucosa, or can be incorporated into a cosmetic or pharmaceutical preparation for topical or systemic administration. In fact, in a cosmetic or pharmaceutical preparation, it provides a transparent liquid, if necessary, for example in the production of gels.

[0029] • The colloidal system can be used as a dietary supplement, a pharmaceutical preparation, a nutraceutical and as a functional food, with appropriate formation. SUMMARY

[0030] In order for the person skilled in the art to fully understand our invention, we describe in detail hereafter the method for the preparation of the dispersion system of fresh royal jelly in a liposome / cyclodextrin combined carrier.

[0031] To prepare the system, fresh royal jelly maintained at 5-7°C is placed to reach room temperature in the shortest possible time. The concentration of hydroxydecenoic acid (10-HDA) in the royal jelly is the minimum requirement of > 1.8% w / w as determined by the method of Koshio et al.

[0032] The royal jelly is then gradually dissolved under stirring in a solvent system consisting of deionized water with vegetable 1,3-propanediol or glycerol or vegetable butylene glycol in a ratio of 1,3-propanediol (or glycerol or butylene glycol) / water: 5 / 95 to 90 / 10. The concentration of royal jelly in the solvent system ranges between 0.5% and 6.0% w / w. Hydroxypropyl-β-cyclodextrin or β-cyclodextrin or γ-cyclodextrin is pre-dissolved in deionized water at a concentration of 1-22% w / w.

[0033] The system royal jelly / cyclodextrin / solvent system is subjected to stirring (1000-3000 rpm) for 30-90 minutes at a temperature of 20°C to 26°C.

[0034] The quality requirement for the deionized water is <= 1 μS / cm at 25°C, which meets the European Pharmacopoeia specification for parenteral pharmaceutical product manufacturing. This quality requirement is necessary for the complete absence of charge in the final formulation, which can cause the lipid membrane of the liposome to agglomerate and eventually reduce the product stability.

[0035] The preparation of the deionized water used in our invention is as follows:

[0036] The water from the water supply system is introduced into the raw water tank (volume 2 m 3 ) through an automatic turbidity filter to remove turbidity and solid particles and activated carbon to remove chlorine and organic charges, then an antiscalant is added to remove its hardness. Before introducing it into the main unit of the reverse osmosis unit, it passes through a 1 micron cartridge filter.

[0037] The completely treated water for reverse osmosis is introduced into the reverse osmosis unit with a capacity of 350 lt / h, recovery 70%. The water produced from this unit is stored in a 5 m 3 volume stainless steel tank. The water is supplied from this tank to the deionization device with a suitable water pump unit and driven online to the extraction tank by ultraviolet radiation. To avoid stagnant water in the network, the water is returned to the tank under constant circulation.

[0038] The preparation of the liposome system is carried out in a separate container. The liposome system consists of 50%-95% phosphatidylcholine, 2%-10% phosphatidylethanolamine, 0%-3% lysophosphatidylcholine, 0%-3% phosphatidylinositol, 0%-3% phosphatidic acid, 0%-22% cholesterol and 0-17% bile salts in 1,3-propanediol or glycerol solvent. The above components are in the ratio range from 20 / 80 to 80 / 20% w / w with 1,3-propanediol or glycerol. Then, the system Royal Jelly / Cyclodextrin / Solvent system is added to the liposome system at a ratio of 1.0-7.5% w / w at a rate of 10 ml / sec. After completing the addition, the system pH is adjusted to a range between 5.0-8.2 and 0.1-0.2% w / w concentration of disodium ethylenediaminetetraacetate or tetrasodium glutamate diacetate is added. The system is stirred at 4000 rpm for 4 hours.

[0039] The mixture is then left in a gas-tight sealed container at 5-7°C for 24 hours. The colloidal system is then subjected to cold filtration through an array cartridge filter with a pore size of 5 μm-1 μm-0.45 μm and the pH is retested and, if necessary, adjusted to the 5.0-8.2 range.

[0040] The system is then allowed to reach room temperature and the average hydrodynamic particle diameter is measured, which should range between 220 nm and 650 nm, with a polydispersity index ranging from 0.25 to 0.68. If the measured values are outside the limits, a second filtration is then performed at room temperature through an array cartridge filter with a pore size of 0.45 μιη - 0.2 μιη.

[0041] Once the values of the average hydrodynamic diameter and the polydispersity index are within the specifications, the total content of polyphenols and the total 10-HDA content in the system are evaluated.

[0042] The release rate of the polyphenols and 10-HDA is therefore determined at 37°C in a buffer at pH 7.2, and the colloidal system is stored in dark containers at a temperature of 5-7°C, remaining stable for 1 year.

[0043] Based on the above method, in order for the system of the present application to be suitable for its intended use, the total content of polyphenols should be greater than 200 mg / L GAE, while the total content of 10-HDA should be greater than 0.04% w / w.

[0044] The release (cumulative release) of the polyphenols at pH 7.2 and a temperature of 37°C in 8 hours is 25%-75%, while the system practically releases all the encapsulated polyphenols in 24 hours. The respective values for 10-HDA are 40%-80% in 8 hours, while the system practically releases the total amount of encapsulated 10-HDA in 12 hours.

[0045] In order for the present application to be fully understood, we continue with the following examples:

[0046] Example 1

[0047] RJ is dispersed at a concentration of 2% in a solvent system consisting of water and the vegetable 1,3-propanediol at a ratio of 1,3-propanediol / water: 50 / 50, after reaching ambient temperature. In the aqueous phase, the hydroxypropyl-β-cyclodextrin is pre-dissolved at a concentration of 9%.

[0048] When the system is added with the liposome suspension at a concentration of 3.0% per weight, under vigorous stirring (3000 rpm) and at a temperature of 20°C. The procedure is as described in the disclosure of the present application.

[0049] The values exhibited by the final colloidal suspension after filtration are shown in Table 1.

[0050] Table 1

[0051]

[0052]

[0053] Example 2

[0054] RJ was dispersed at a concentration of 5% in a solvent system consisting of water and vegetable 1,3-propanediol at a ratio 1,3-propanediol / water: 75 / 25, after reaching the ambient temperature. In the aqueous phase, hydroxypropyl-β-cyclodextrin was pre-dissolved at a concentration of 12%.

[0055] When the system was added with the liposome suspension at a concentration of 3.5% per weight, under vigorous stirring (3000 rpm) and at a temperature of 20°C. This step was carried out as described in the disclosure of the present application.

[0056] The values exhibited by the final colloidal suspension after filtration are shown in Table 1.

[0057] Table 1

[0058] Parameter Value Mean particle hydrodynamic diameter 120-250 nm Polydispersity index <0.68 10-HDA 0.08% w / w Total polyphenols (gallic acid equivalent) 430 mg / L Cumulative release of 10-HDA over 8 hours 60-75 Cumulative release of polyphenols over 8 hours 40-65

[0059] The determination of the average hydrodynamic diameter and the polydispersity index was carried out by dynamic light scattering.

[0060] References cited in the description

[0061] Ramanathana A.N.K.G., Nair A.J., Sugunan V.C, A review on Royal Jelly proteins and peptides, Journal of Functional Foods, 44, 2018, 255-264 5

[0062] Nagai T., Inoue R. Preparation and the functional properties of water extract and alkaline extract of royal jelly. Food Chemistry. 2004; 84(2): 181-186

[0063] Sugiyama T., Takahashi K., Mori H. Royal jelly acid, 10-hydroxy-trans-2-decenoic 10 acid, as a modulator of the innate immune responses. Endocrine, Metabolic & Immune Disorders-Drug Targets. 2012; 12(4): 368-376

[0064] Ramadan M.F., Al-Ghamdi A. Bioactive compounds and health-promoting properties of royal jelly: a review. Journal of Functional Foods, 4, 1, 2012, 39-52 15

[0065] Townsend G.F, Brown W.H, Felauer E.E, Hazlett B. Studies on the in vitro antitumor activity of fatty acids. IV. The esters of acids closely related to 10-hydroxy-2-decenoic acids from royal jelly against transplantable mouse leukemia. Can J Biochem Physiol. 1961; 39: 1765-1770 20

[0066] Blum M.S, Novak A.F, Taber S. 10-Hydroxy-2Δ-decenoic acid, an antibiotic found in royal jelly. Sci. 1959; 130: 452-453

[0067] Vucevic D, Melliou E, Vasilijic S, Gasic S, Ivanovski P, Chinou I, Colic M. Fatty acids 25 isolated from royal jelly modulate dendritic cell-mediated immune response in vitro. Int Immunopharmacol. 2007; 7: 1211-1220

[0068] Fujii A. Kobayashi S. Kuboyama N, et al. Augmentation of wound healing by royal jelly (RJ) in streptozotocin-diabetic rats. Jpn J Pharmacol. 1990; 53: 331-337 30

[0069] Koya-Miyata S. Okamoto I. Ushio S. Iwaki K. Ikeda M. Kurimoto M. Identification of a collagen production-promoting factor from an extract of royal jelly and its possible mechanism. Biosci Biotechnol Biochem. 2004; 68: 767-773 11

[0070] Yang X.Y., Yang D.S., Wei-Zhang, et al. 10-Hydroxy-2-decenoic acid from Royal jelly: A potential medicine for RA. J Ethnopharmacol. 2010; 128: 314-321

[0071] Dynek J.N, Chan S.M, Liu J, Zha J, Fairbrother W.J, Vucic D. Microphthalmia-5 associated transcription factor is a critical transcriptional regulator of melanoma inhibitor of apoptosis in melanomas. Cancer Res. 2008; 68: 3124-3132

[0072] Chen C., Chen S.Y., Changes in protein components and storage stability of Royal Jelly under various conditions, Food Chemistry, 54, 2, 1995, 195-200

[0073] Sabatini, A.G., Marcazzan, G.L., Caboni, M.F., Bogdanov, S., Almeida-Muradian, L.B. (2009): Quality and standardisation of Royal Jelly. J. Api Prod. Api Med. Sci. 1(1), 1-6

[0074] Mendoza-Resendez R., A.G., Castro E.D.B et al., Synthesis of Antibacterial Silver-based Nanodisks and Dendritic Structures Mediated by Royal 15 Jelly, RSC Advances 4(4), 2014, 1650

[0075] Dalfardi M., Taghavi M.M., Kohbanani M., S. et al., Protective and modulatory effects of royal jelly used against the induced changes in silver nanoparticles on the hippocampus of male rats, Nanomedicine Journal, 6, 2, 2019, 136-141 Szejtli, J., Cyclodextrin technology, 1988, pp.322

[0076] Koshio S., Almeida-Muradian L., HPLC aplication for 10-HDA determination in pure royal jelly and honey with royal jelly, Química Nova 26(5), 2003, 670-673

Claims

1. A method for preparing a colloid-stabilized system of royal jelly components, characterized in that: Preparation of the royal jelly component delivery system: - in a first stage, fresh royal jelly is stored at 5-7°C and contains 10-hydroxydecenoic acid at a concentration of > 1.8% w / w, as determined by the method of Koshio et al., described in the reference Koshio S., Almeida-Muradian L., HPLC aplication for 10-HDA determination in pure royal jelly and honey with royal jelly, Química Nova 26(5), 2003, 670-673, and is brought to room temperature; - in the second stage, it is gradually dissolved in a solvent system under stirring, wherein the solvent system consists of deionized water and vegetable 1,3-propylene glycol in a ratio of 1,3-propylene glycol / water: 5 / 95 to 90 / 10, wherein hydroxypropyl-β-cyclodextrin is pre-dissolved in deionized water at a concentration of 1%-22% w / w, and the concentration of royal jelly in the solvent system ranges from 0.5% to 6.0% w / w; - In the third stage, the royal jelly / cyclodextrin / solvent system is stirred at 1000-3000 rpm for 30-90 minutes at a temperature of 20°C to 26°C; at the same time, the royal jelly / cyclodextrin / solvent system is added to the liposome system at a concentration of 1.0-7.5% w / w at a rate of 10 ml / sec. After the addition is completed, the pH of the system is set in the range of 5.0-8.2, and 0.1-0.2% w / w concentration of disodium edetate or tetrasodium glutamate diacetate is added, and the system is stirred at 4000 rpm for 4 hours; wherein the liposome system is in a 1,3-propylene glycol solvent at a concentration range of 20 / 80 to 80 / 20%. w / w preparation, the liposome system consists of 50%-95% phosphatidylcholine, 2%-10% phosphatidylethanolamine, 0%-3% lysophosphatidylcholine, 0%-3% phosphatidylinositol, 0%-3% phosphatidic acid, 0%-22% cholesterol and 0-17% bile salts, - In the fourth stage, the mixture is stored in an airtight sealed container at 5-7°C for 24 hours, and then the colloidal system is cold filtered through an array of cartridge filters with pore sizes of 5 μm-1 μm-0.45 μm, and the pH is retested and readjusted to the range of 5.0-8.2; - In the fifth stage, the system is brought to room temperature and the mean hydrodynamic particle diameter is measured, which should be between 220 nm and 650 nm, with a polydispersity index ranging from 0.25 to 0.

68. If the measured value is outside the tolerances, a secondary filtration is then performed at room temperature through an array of cartridge filters with a pore size of 0.45 μm-0.2 μm; - In the final stage, after finding the values ​​of the mean hydrodynamic diameter and the polydispersity index within the specifications, the total content of polyphenols and the total content of 10-hydroxydecenoic acid were measured, while continuously, the release rate of polyphenols and 10-hydroxydecenoic acid was measured at 37°C in a buffer of pH 7.2 and the colloidal system was stored in a dark container at a temperature of 5-7°C, where it is stable for up to 1 year.

2. The method for preparing the colloid stabilization system of royal jelly components according to claim 1, characterized in that The quality requirement of the deionized water is: <= 1 μS / cm at 25°C.

3. The method for preparing a colloid-stabilized system of royal jelly components according to claim 1 or 2, characterized in that For the preparation of deionized water, water from the water supply network is introduced into a tank using a pump unit, passes through automatic turbidity and activated carbon filters, then antiscalant is added to remove its hardness, then enters the main unit of a reverse osmosis unit with a capacity of 350 lt / h, with a recovery rate of 70% after the first pass, passes through a 1 micron cartridge filter and is then stored in a stainless steel tank, from which it is supplied to the deionization device using a system.

4. The method for preparing a colloid-stabilized system of royal jelly components according to claim 1 or 2, characterized in that The total content of polyphenols in the system should be greater than 200 mg / L GAE.

5. The method for preparing a colloid-stabilized system of royal jelly components according to claim 1 or 2, characterized in that The total content of 10-hydroxydecenoic acid in the system should be greater than 0.04% w / w.

6. The method for preparing a colloid-stabilized system of royal jelly components according to claim 1 or 2, characterized in that The cumulative release of polyphenols at pH 7.2 and 37°C within 8 hours was 25%-75%, and the encapsulated polyphenols were completely released from the system within 24 hours.

7. The method for preparing a colloid-stabilized system of royal jelly components according to claim 1 or 2, characterized in that The release of 10-hydroxydecenoic acid at pH 7.2 and 37°C was 40%-80% within 8 hours, and the encapsulated 10-hydroxydecenoic acid was completely released from the system at 12 hours.

8. A stable colloidal dispersion of royal jelly, prepared according to the method according to any one of claims 1 to 7, characterized in that The total content of polyphenols is greater than 200 mg / L GAE, and the total content of 10-hydroxydecenoic acid is greater than 0.04% w / w. At the same time, the cumulative release of polyphenols at pH 7.2 and 37°C within 8 hours ranges from 25% to 75%, and the complete release occurs within 24 hours; while the release of 10-hydroxydecenoic acid within 8 hours ranges from 40% to 80%, and the complete release occurs within 12 hours.

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