Preparation and application of active packaging carrier

Abstract

The invention discloses the preparation and application of an active encapsulation carrier, and belongs to the field of cosmetic preparations. The extraction raw materials include β-cyclodextrin, 1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimidazole, 1,6-hexanedithiol, nanogold, and maqui fruit extract. The encapsulation method provided by the invention can improve the encapsulation rate of the encapsulation carrier for the maqui fruit extract.

Description

Technical Field

[0001] The invention belongs to the field of toiletry and makeup products, and in particular relates to the preparation and application of a maqui fruit bag carrier. Background Art

[0002] Maquiberry, also known as maqui berry or Chilean wineberry, is a dark purple wild berry originating from the Patagonia Plateau. The unique geographical environment and climatic conditions of the plateau make maquiberry rich in a variety of biologically active ingredients, especially anthocyanins, whose content far exceeds that of other common fruits. Anthocyanins are a class of powerful natural antioxidants with significant antioxidant, anti-inflammatory, anti-cancer and cardiovascular health protection and other biological effects. Therefore, maquiberry extract has broad application prospects in the fields of health foods, functional beverages, cosmetics, and medical and health products.

[0003] However, maqui fruit extract faces some challenges in practical application. First, its active ingredients such as anthocyanins are sensitive to light, heat and oxygen, which can easily lead to degradation and inactivation, thus affecting the stability and efficacy of the product. Secondly, when maqui fruit extract is directly applied to products, there may be problems such as low solubility and poor bioavailability, which limits its application effect. Therefore, it is particularly important to develop a packaging technology that can effectively protect the active ingredients of maqui fruit extract and improve its stability and bioavailability.

[0004] In recent years, the application of nanotechnology in the fields of medicine and functional food has become increasingly widespread. Among them, the encapsulation system constructed by modified β-cyclodextrin and nanogold has attracted much attention due to its unique physicochemical properties and biocompatibility. β-cyclodextrin is a cyclic oligosaccharide with a cavity structure of "hydrophilic outside and hydrophobic inside", which can encapsulate a variety of small molecules to improve their stability and solubility. Nanogold is widely used in drug carriers and biosensors due to its good stability and biocompatibility. Combining the two to construct a new type of encapsulation system is expected to provide a new solution for the protection and application of maqui fruit extract.

[0005] In summary, this study aims to develop an encapsulation system constructed by modified β-cyclodextrin and nanogold to efficiently encapsulate the maqui fruit extract. By optimizing the preparation process of the encapsulation system, the stability, solubility and bioavailability of the active ingredients in the maqui fruit extract can be improved, providing technical support for its wide application in the fields of health foods, functional beverages, cosmetics, and pharmaceutical health products. Summary of the invention

[0006] The present invention provides the preparation and application of an active encapsulation carrier, using 1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimidazole to activate as many primary hydroxyls in beta-cyclodextrin as possible to enhance its encapsulation rate, and then using nano-gold to connect with the thiol groups in the activated beta-cyclodextrin to enhance its ability to resist binding with specific molecules and its stability.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] Firstly, β-cyclodextrin was activated and functionalized using 1-ethyl-3-(3-dimethylaminopropyl)carbonyldiimidazole and N-hydroxysuccinimide.

[0009] S1: Mix 1 mol of β-cyclodextrin, 1 mol of 1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimidazole, and 1 mol of N-hydroxysuccinimide in an ethanol solution environment at pH 4.5-6, react at a temperature of 20-25° C., react for 1-4 hours, evaporate the solvent, and obtain product A.

[0010] Reaction mechanism: The hydroxyl group of β-CD can be activated by EDC and N-hydroxysuccinimide (NHS) to form an active ester.

[0011] Reaction equation: β-CD-OH+NHS+EDC→β-CD-O-CO-NH-S

[0012] S2: 1,6-hexanedithiol and N-hydroxysuccinimide are mixed, and then reacted in an anhydrous pyridine environment for 16-24 hours to obtain product B; 4-dimethylaminopyridine is used as a catalyst, the reaction temperature is 25±2°C, and the molar ratio of 1,6-hexanedithiol to N-hydroxysuccinimide is 1:1-1.2.

[0013] Reaction Mechanism: 1,6-Hexanedithiol can react with N-hydroxysuccinimide to form thiol esters.

[0014] Reaction equation: HS-(CH2)6-SH+NHS→HS-(CH2)6-S-NH2

[0015] S3: Dissolve product A in a sodium carbonate aqueous solution containing 20-30% mass volume fraction of ethanol, and then add product B, heat to 40-50°C under a nitrogen atmosphere for 5-7 hours; then extract with ethyl acetate, filter after extraction to obtain a precipitate, and wash the precipitate to obtain product C.

[0016] Reaction mechanism: The activated 1,6-hexanedithiol reacts with the activated ester on β-CD to form a thioether bond.

[0017] Reaction equation: β-CD-O-CO-NH-S+HS-(CH2)6-S-NH2→β-CD-O-(CH2)6-S-(CH2)6-SH+NH3

[0018] Furthermore, gold nanoparticles were prepared;

[0019] S4: Dissolve 1 part by mass of the maqui fruit extract in 1 L of sterile deionized water, add 0.1-0.3 parts by mass of chloroauric acid solution, stir for 24 hours until the nano-gold solution turns wine red, then centrifuge and freeze-dry to obtain nano-gold powder.

[0020] Secondly, prepare the activated β-cyclodextrin modified with nano-gold

[0021] S5: Mix 1 part by mass of nano-gold powder with 4-6 parts by mass of product C, stir for 24 hours, then add ethyl acetate and stir for 10 minutes, centrifuge, wash the solid obtained by centrifugation with ethyl acetate-ethanol (ethyl acetate accounts for 40%-50% by volume) solution 3-5 times, and vacuum dry to obtain product D.

[0022] Reaction mechanism: The thiol part of 1,6-hexanedithiol can form Au-S bonds with the gold atoms on the surface of nanogold.

[0023] Reaction equation:

[0024] Au+β-CD-O-(CH2)6-S-(CH2)6-SH→Au-S-(CH2)6-S-(CH2)6-O-β-CD

[0025] Finally, product D was used to encapsulate the maqui fruit extract.

[0026] S6: 1 part by mass of the maqui fruit extract and 0.1-0.5 parts by mass of the product D are mixed in a 20-40% (v / v) ethanol solution, heated to 60°C under a nitrogen atmosphere, stirred for 2-5 hours, and then the ethanol is evaporated, washed, and centrifuged to obtain the final product E.

[0027] In the present invention, 1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimidazole, 1,6-hexanedithiol, N-hydroxysuccinimide and nano-gold are used as raw materials to activate and functionalize β-cyclodextrin, and then the modified β-cyclodextrin is loaded on the maqui fruit extract.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] First, 1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimidazole is used to activate β-cyclodextrin. There are many primary hydroxyl groups in the β-cyclodextrin molecule. With the activation of primary hydroxyl groups, more natural active substances can be combined in the β-cyclodextrin cavity. Then, nanogold is prepared using maqui fruit extract. Nanogold prepared using natural products can show more active functions than nanogold prepared by traditional processes. In addition, nanogold prepared using plant extracts may show effects that traditional nanogold does not have, such as antibacterial, anti-inflammatory, strong penetrability, etc. Nanogold prepared from different plant extracts may have different effects. After the nanogold is connected to the activated β-cyclodextrin, the strong antioxidant property of the nanogold and the encapsulation capacity of β-cyclodextrin can make the maqui fruit extract loaded in the modified β-cyclodextrin retain its active efficacy as much as possible. The stability of the nanogold is improved after the nanogold is combined with the modified β-cyclodextrin. The strong electronegativity of the nanogold surface enhances the surface activity of β-cyclodextrin, allowing it to carry more drug molecules. Then, the activated 1,6-hexanedithiol forms a thioether bond with the activated ester on β-CD. The thioether bond has a stronger polarity than the carbon-carbon bond, so the carbon chains at both ends of the thioether bond are easily entangled with each other, thereby forming a network cross-linking, and then forming a stable three-dimensional network structure. This network structure can further increase the loading amount of the maqui fruit extract in it. Finally, given the strong penetration ability of the nanogold itself, the active substances can also reach the deep layer of the skin more effectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the active package carrier structure. DETAILED DESCRIPTION

[0031] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples, wherein the terms used in the examples are for describing specific embodiments and do not constitute limitations on the scope of protection of the present invention.

[0032] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0033] Some raw materials and their sources are as follows:

[0034] Maqui fruit extract: homemade;

[0035] The solvents used in the experiments were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0036] Example 1

[0037] Ester extraction of Maqi fruit.

[0038] S1: Wash the maqui fruit, remove impurities, freeze-dry, grind into powder, and store in a sealed and dark place at 5-8℃ before use.

[0039] S2: The maqui fruit powder was subjected to ester extraction using ethyl acetate, and then filtered to obtain a filtrate A1 and a residue B1.

[0040] S3: freeze-drying the ester extraction product A1 to obtain freeze-dried powder A2.

[0041] The ethyl acetate in the ester extraction is water-saturated ethyl acetate, the extraction temperature is 40-65° C., and the extraction reflux times is 3-6 times.

[0042] Next, the B1 residue was enzymatically hydrolyzed.

[0043] T1: washing, drying and crushing the residue of maqui ester extraction to obtain residue powder B2;

[0044] T2: Use cellulase, pectinase, protease and glucanase to enzymolyze B2, and filter to obtain the enzymolysis product A3, wherein the order of addition is first adding cellulase and pectinase for enzymolysis for 3±0.5; then adding glucanase for enzymolysis for 2±0.3h; then adding protease for enzymolysis for 4±0.2h, and then obtaining the enzymolysis product A3.

[0045] The total added amount of the enzyme accounts for 8-10% of the raw material mass; the mass fraction ratio of the cellulase, pectinase, hydrolyzing protease and glucanase is 1:1-1.5:3:1.

[0046] Furthermore: fermentation of enzymatic hydrolysate A3

[0047] V1: The enzymatic hydrolysate A3 is fermented using yeast to obtain a fermentation product A4.

[0048] The yeast strain used is Pichia pastoris, the V1 fermentation starting temperature is 28±2°C, the fermentation starting pH is 5.5±0.5; the fermentation starting oxygen content is 18±2%; the strain mass percentage accounts for 8-10% of the raw material mass; and the fermentation time is 16±4h.

[0049] Finally: Fermentation product A4 undergoes secondary fermentation

[0050] V2: After filtering and sterilizing the fermentation product A4, 2-4% of the mass percentage of the filtered fermentation product A4 was added with the maqui extract A2, and then fermented with Aspergillus, and filtered to obtain the fermentation product A5 and mycelium B3.

[0051] The Aspergillus species used is Aspergillus oryzae, the fermentation starting temperature is 29±1°C, the fermentation starting pH is 5.5±0.5; the fermentation starting relative humidity is 92±2.5%; the mass percentage of the strain accounts for 6-10% of the raw material mass, the fermentation time is 48±12h, and the fermentation stirring speed is 45r / min.

[0052] V3: The mycelium B3 is enzymatically hydrolyzed using protease and cellulase, and then freeze-dried to obtain product A6.

[0053] V4: Mix A5 and A6 to obtain the final product A7.

[0054] Example 2

[0055] β-cyclodextrin activation and functionalization:

[0056] S1: Mix 1 mol of β-cyclodextrin, 1 mol of 1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimidazole, and 1 mol of N-hydroxysuccinimide in a 50-60 wt% ethanol solution at a pH of 4.5-6, at a reaction temperature of 20-25° C., for 1-4 h, and evaporate the solvent to obtain product A.

[0057] 1,6-Hexanedithiol Activation:

[0058] S2: 1,6-hexanedithiol and N-hydroxysuccinimide are mixed, and then reacted in an anhydrous pyridine environment for 16-24 hours to obtain product B; 4-dimethylaminopyridine is used as a catalyst, the reaction temperature is 25±2°C, and the molar ratio of 1,6-hexanedithiol to N-hydroxysuccinimide is 1:1.

[0059] Product A combines with product B:

[0060] S3: Dissolve 1 mass part of product A in a sodium carbonate aqueous solution containing 20-30% mass volume fraction of ethanol, and then add 1 mass part of product B, heat to 40-50°C under a nitrogen atmosphere, and heat for 5-7h; then extract with ethyl acetate, filter after extraction to obtain a precipitate, and wash the precipitate to obtain product C.

[0061] Preparation of gold nanoparticles:

[0062] S4: Dissolve 1 part by mass of the maqui fruit extract in 1 L of sterile deionized water, add 0.3 parts by mass of chloroauric acid solution, stir for 24 hours until the nano-gold solution turns wine red, then centrifuge and freeze-dry to obtain nano-gold powder.

[0063] β-cyclodextrin modified, activated and functionalized with nano-gold:

[0064] S5: Mix 1 part by mass of nano-gold powder with 5 parts by mass of product C, stir for 24 hours, then add ethyl acetate and stir for 10 minutes, centrifuge, wash the solid obtained by centrifugation with ethyl acetate-ethanol (ethyl acetate accounts for 40%-50% by volume) solution for 3-5 times, and vacuum dry to obtain product D.

[0065] Package:

[0066] S6: Mix 1 part by mass of the maqui fruit extract with 0.5 part by mass of the product D, stir for 24 hours, then add ethyl acetate and stir for 10 minutes, centrifuge, wash the solid obtained by centrifugation with ethyl acetate-ethanol (ethyl acetate accounts for 40%-50% by volume) solution for 3-5 times, and vacuum dry to obtain product E.

[0067] Comparative Example 1: The difference from Example 2 is that product A is replaced by β-cyclodextrin, and the other steps follow the same procedures as Example 1.

[0068] Comparative Example 2: The difference from Example 2 is that Vc is used to reduce the nano-gold, and the maqui fruit extract is not used to reduce the nano-gold. The other steps follow the same steps as Example 1.

[0069] Comparative Example 3: Different from Example 2, nano-gold modification is not used, and the direct product C is encapsulated. The other steps are the same as those in Example 1.

[0070] Comparative Example 4: Different from Example 2, product B is not used, and only product A is loaded.

[0071] Cytotoxicity assay

[0072] Take the human immortalized epidermal cell line (HaCaT) with stable growth state and adjust the cell concentration to 1×10 5 / mL, 100μL per well. Inoculate into 96-well cell culture plates (except the outermost wells and blank control wells). Add 200μL of PBS to the outermost wells of the cell culture plate to reduce evaporation of the culture medium. Place in a carbon dioxide incubator and culture at 37 ℃±1℃, CO2 concentration 5.0%±1% until the cell fusion reaches more than 90%. Add samples, the sample concentration is 10mg / L, set up blank control and sample groups, 100 μL per well. Start adding samples (sample group) and PBS (blank control) at the same time interval between each two columns, and ensure that the intervention time of each test substance on the cells is 5 min. After 5 min, remove the test substances in turn, add PBS to each well to wash the test substances, and wash twice. When using a vacuum suction pump or a discharge gun to remove the liquid in the well, be careful to avoid sucking away the surviving cells and affecting the experimental results. 100 μL of complete medium and 10 μL of CCK-8 solution were added to each well and cultured in an incubator for about 2 hours. The absorbance (OD) value was measured at a wavelength of 450 nm and the cell viability was calculated; the results are shown in Table 1.

[0073] Formula (1) ×100%.

[0074] Table 1 Cytotoxicity test

[0075] Experimental Group HaCaT survival rate Example 2 98.94±0.37% blank （100）%

[0076] According to the experimental results, the cell survival rate remained above 90%. The combination of the present experiment had little effect on cell viability.

[0077] Packing rate test:

[0078] Accurately weigh 5 parts by mass of the maqui fruit extract and 2 parts by mass of the product C and mix them in an aqueous solution. Use ultraviolet spectrophotometry to determine the content of the maqui fruit extract in the aqueous solution at this time, record it as V1 (with rutin as the standard), heat it to 60°C, stir for 2-5 hours, and then centrifuge it. Take the supernatant and use ultraviolet spectrophotometry to determine the content of the maqui fruit extract V2 (with rutin as the standard), then take the precipitate for washing, freeze-drying, and obtain product E; divide product E into three parts E1, E2, and E3 , E1 was placed in an aqueous solution, exposed to natural light for 30 minutes, and its supernatant was centrifuged to obtain the content of the maqui fruit extract X1, E2 was placed in an aqueous solution, heated to 36-37°C in the dark for 30 minutes, and its supernatant was centrifuged to obtain the content of the maqui fruit extract X2, E3 was placed in an aqueous solution, exposed to natural light, heated to 36-37°C for 30 minutes, and its supernatant was measured to obtain the content of the maqui fruit extract X3, referring to the calculation formula:

[0079] Encapsulation efficiency V = ;

[0080] Release rate X = *100%.

[0081]

[0082] The experimental results show that Example 2 has the best encapsulation effect. Combined with Comparative Example 1, it is shown that the use of activated β-cyclodextrin and nanogold reduced by the extract of maqui fruit can effectively improve the encapsulation rate. Example 2 combined with Comparative Examples 2-3 shows that the nanogold reduced by the extract of maqui fruit is irreplaceable, and the nanogold has a certain effect on the encapsulation efficiency. This may be because the formation of gold-sulfur bonds not only changes the surface charge and hydrophilicity of the original β-cyclodextrin, thereby enhancing the binding ability of β-cyclodextrin with other molecules; and the nanogold particles reduced by plant extracts have added other properties that nanogold itself does not have, such as whitening, anti-inflammatory, antibacterial, etc., on the basis of its strong penetration. The results of Example 2 and Comparative Example 4 show that 1,6-hexanedithiol has a great influence on the encapsulation rate. After activation, 1,6-hexanedithiol is used to modify product A. The modified product contains both thiol groups that can link nanogold to form gold-sulfur bonds to enhance the encapsulation effect and thioether bonds. The thioether bonds can form a network structure due to their polar properties, linking individual molecules to form a network structure to further load more maqui fruit extract.

[0083] The experimental results show that Example 2 has the best release effect. It can be seen from the data of Example 2 that both illumination and heating can increase the release rate of, and illumination and heating are performed simultaneously, but the release rate of is maximized. The possible principle of this phenomenon is that the ultraviolet and infrared rays in natural illumination irradiate the nanogold to trigger the photothermal effect of the nanogold itself, thereby increasing the ambient temperature, thereby increasing the kinetic energy of the drug molecules, causing them to escape from the cavity; in addition, due to the increase in ambient temperature, thermal responsive structural changes are triggered, thereby destroying the package carrier and promoting drug release; and heating or weakening the gold-sulfur bond, so that the stability of the overall package carrier is destroyed, resulting in drug release. Therefore, the two pathways of illumination and heating are shared to make them synergistic and promote each other, thereby further increasing the release of the drug.

[0084] Compared with Example 2, because Comparative Example 1 does not use activated β-cyclodextrin, it cannot combine with activated 1,6-hexanedithiol, and thus cannot form a gold-sulfur bond with nanogold, and cannot achieve a network structure due to its own sulfide bond. Therefore, Comparative Example 1 has a lower release rate than Example 2.

[0085] In Comparative Example 2, without considering its encapsulation rate, its release rate is almost the same as that of Example 2.

[0086] In Comparative Example 3, since it is not modified with nanogold, its photothermal effect cannot be achieved, so its release rate is greatly reduced under natural light. Its release rate is acceptable under heating, but there is no synergistic effect when light and heating coexist, so its release rate is lower than that of Example 2.

[0087] Comparative Example 4 has a lower release rate than Example 2 for similar reasons to Comparative Example 1.

[0088] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present application.

Claims

1. An active package carrier, characterized in that: A packaging system was constructed using β-cyclodextrin, 1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimidazole, 1,6-hexanedithiol, N-hydroxysuccinimide, and nano-gold to encapsulate the maqui fruit extract; The steps of preparing the encapsulation system are as follows: S1: β-cyclodextrin, 1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimidazole and N-hydroxysuccinimide are mixed in a 50-60wt% ethanol solution at a pH of 4.5-6, the reaction temperature is 20-25° C., the reaction time is 1-4 hours, and the solvent is evaporated to obtain product A; S2: 1,6-hexanedithiol and N-hydroxysuccinimide are mixed, and then reacted in an anhydrous pyridine environment for 16-24 hours to obtain product B; 4-dimethylaminopyridine is used as a catalyst, the reaction temperature is 25±2°C, and the molar ratio of 1,6-hexanedithiol to N-hydroxysuccinimide is 1:1-1.2; S3: dissolving product A in a sodium carbonate aqueous solution containing 20-30% by mass volume fraction of ethanol, and then adding product B, heating to 40-50° C. under a nitrogen atmosphere for 5-7 hours; then extracting with ethyl acetate, filtering to obtain a precipitate after extraction, and washing the precipitate to obtain product C; S4: Dissolve 1 part by weight of the maqui fruit extract in 1 L of water, add 0.1-0.3 parts by weight of chloroauric acid solution, stir for 24 hours, then centrifuge and freeze-dry to obtain nano-gold powder; S5: Mix 1 part by mass of nano-gold powder with 4-6 parts by mass of product C, stir for 24 hours, then add ethyl acetate and stir for 10 minutes, wash the solid obtained by centrifugation with ethyl acetate-ethanol solution, and vacuum dry to obtain product D; S6: The maqui fruit extract and product D are mixed in an ethanol solution, heated to 60° C. under a nitrogen atmosphere, stirred for 2-5 hours, and then the ethanol is evaporated, washed, and centrifuged to obtain the final product E.

2. The carrier according to claim 1, characterized in that: The molar ratio of β-cyclodextrin to 1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimidazole and N-hydroxysuccinimide in S1 is 1:1-1.2:1-1.

1.

3. The carrier according to claim 1, characterized in that: The mass fraction ratio of product A to product B in S3 is 1:0.5-1.

4. The carrier according to claim 1, characterized in that: The mass fraction ratio of the maqui fruit extract to chloroauric acid in S4 is 1:0.1-0.

5.

5. The carrier according to claim 1, characterized in that: The volume ratio of ethyl acetate in ethyl acetate-ethanol in the S5 is 40-50%.

6. The carrier according to claim 1, characterized in that: The mass ratio of the maqui fruit extract to the product D in S6 is 1:0.1-0.5; the ethanol solution is a 20-40% by volume ethanol aqueous solution.

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