Stable supramolecular collagen hyaluronic acid body as well as preparation method and application thereof

Abstract

The invention discloses a stable supramolecular collagen hyaluronic acid body as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, constructing a supramolecular solvent system by taking betaine as a hydrogen bond receptor and taking polyol / sodium hyaluronate as a hydrogen bond donor, and preparing the stable supramolecular collagen hyaluronate from the supramolecular solvent and lipidosome colostrum prepared from lecithin, cholesterol, ceramide, collagen, polyol and water. According to the invention, collagen and sodium hyaluronate are not in direct contact in space in a form of lipidosome wrapping, the problems that flocculent precipitates are easily generated and the system is unstable when collagen and sodium hyaluronate are commonly used can be solved, and a dual delivery system formed by the supramolecular solvent and lipidosome has excellent human body permeability and efficacy.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to a stable supramolecular collagen hyaluronic acid body, a preparation method and an application thereof. Background Art

[0002] Collagen is a biopolymer and the primary component of animal connective tissue. It is also the most abundant and widely distributed functional protein in mammals, enjoying widespread applications in food, medicine, tissue engineering, cosmetics, and other fields. Recombinant human type III collagen is derived through genetic engineering techniques, using optimized and recombinant expression of the original human collagen type III gene sequence. It is highly consistent with the amino acid sequence of natural human collagen. Recombinant human type III collagen has high bioactivity, biocompatibility, and good water solubility. It can be used to repair skin damage, improve stratum corneum hydration, increase skin elasticity, improve skin firmness, and reduce wrinkles.

[0003] Hyaluronic acid (HA), a high-molecular-weight mucopolysaccharide composed of N-acetylglucosamine and D-glucuronic acid, possesses potent moisturizing properties, capable of binding 1,000 times its own weight in water molecules. It is known as the "strongest moisturizer." Hyaluronic acid possesses high biocompatibility, keeping skin moisturized, smooth, delicate, supple, and elastic. It has anti-wrinkle, anti-wrinkle, beauty, and health benefits, as well as repairing the skin's physiological functions. Sodium hyaluronate, the sodium salt form of HA, readily hydrolyzes to HA. However, sodium hyaluronate possesses superior water solubility, stability, and penetrating properties, making it a popular moisturizer and skin conditioner in cosmetics.

[0004] Collagen, hyaluronic acid, and its sodium salt can all promote cell activation and proliferation, accelerating the repair process. However, due to the different charge properties of hyaluronic acid, its sodium salt, and collagen, as well as the isoelectric point, pH, and ionic strength, the two are prone to produce flocculent precipitation when mixed in water, affecting the uniformity, structure, and performance of the mixture, thereby limiting the combined use of the two. In addition, the water-soluble large-molecule recombinant human type III collagen has difficulty penetrating the lipid structure between stratum corneum cells, is easily degraded, and has a short duration of action. It urgently needs to rely on the help of a carrier to penetrate the stratum corneum, enhance penetration, and prolong its duration of action to achieve significant results.

[0005] CN110075006 A effectively prevents the formation of flocculent polyionic precipitates when hyaluronic acid and type II collagen interact in aqueous solution by adding polyglutamate, resulting in a uniform, transparent aqueous solution. However, type II collagen is primarily found in cartilage, vitreous body, and intervertebral discs, making it difficult to achieve skin care benefits. Furthermore, the aforementioned combination approach fails to address the transdermal delivery of large-molecule collagen.

[0006] CN 114699335 A provides a stable collagen-hyaluronic acid miscible system and its preparation method. By modifying hyaluronic acid with cationic cellulose and then mixing it with a collagen solution, a uniform miscible system is obtained. This miscible system has good stability and does not produce precipitation. However, the addition of cationic cellulose makes the system viscous and unclear, which affects its application in aqueous products and prevents efficient transdermal penetration of macromolecular collagen.

[0007] Supramolecular solvents, based on weak non-covalent interactions, are widely used due to their amphiphilic nature, which significantly improves penetration efficiency. On the one hand, due to their weak chemical interactions and small physical size, supramolecular solvents can easily penetrate the brick-wall structure of the skin barrier, promoting the transport of active ingredients between cells. On the other hand, the amphiphilic nature of supramolecular solvents allows the hydrophilic and hydrophobic groups to introduce active ingredients between different skin layers according to the principle of like dissolves like, thus improving penetration efficiency.

[0008] CN115317398A provides a method for preparing an ionic liquid collagen preparation, which promotes collagen penetration by constructing a L-carnitine citrate supramolecular solvent delivery system; CN115010949A promotes the penetration of active peptides with a molecular weight of 1900-3000Da by constructing a supramolecular structure delivery system formed by γ-aminobutyric acid and α-hydroxypropionic acid (LA); currently, most studies use small molecules to construct supramolecular solvents as carriers for delivering macromolecular substances (such as collagen, etc.), and the supramolecular solvents are all single systems. There are no relevant research reports on supramolecular solvent systems constructed using hyaluronic acid macromolecules as hydrogen bond donors. Summary of the Invention

[0009] To address the shortcomings of the prior art, the present invention provides a stable supramolecular collagen hyaluronate, preparation method, and application. This invention utilizes betaine as a hydrogen bond acceptor and polyol / sodium hyaluronate as a hydrogen bond donor to construct a supramolecular solvent system. This system not only maximizes the active effects of betaine and sodium hyaluronate, but also synergistically promotes the penetration of macromolecular substances (such as collagen), thereby overcoming the problems of flocculent formation caused by the combined use of collagen and sodium hyaluronate in aqueous solutions and the poor skin permeability of macromolecular water-soluble collagen.

[0010] The technical solutions of the present invention are as follows:

[0011] The first aspect of the present invention provides a supramolecular solvent characterized by using sodium hyaluronate as a hydrogen bond donor. Furthermore, the initial components, by weight, include 0.1-8.0 parts betaine, 0.05-3 parts sodium hyaluronate, and an appropriate amount of water. The supramolecular solvent is obtained by dissolving, mixing, heating and stirring the reaction, and removing the water by rotary evaporation.

[0012] Among them, the molecular weight of sodium hyaluronate is 1KDa-1300KDa.

[0013] The supramolecular solvents are divided into single supramolecular solvents and double supramolecular solvents.

[0014] The initial components and weight percentages of the single supramolecular solvent are: 0.1-8.0 parts betaine, 0.05-3 parts sodium hyaluronate, and an appropriate amount of water. The preparation method is as follows: dissolve the sodium hyaluronate in an appropriate amount of water and add the betaine, then heat and stir in a waterbath to react. After the reaction is complete, rotary evaporation for 20-60 minutes is performed to obtain the betaine-sodium hyaluronate supramolecular solvent. Preferably, the stirring temperature is 30-50°C, the reaction time is 3-10 hours, and the rotary evaporation temperature is 50-60°C.

[0015] The initial components and weight percentages of the dual supramolecular solvent are: 0.1-8.0 parts betaine, 0.1-5.0 parts polyol, 0.05-3 parts sodium hyaluronate, and an appropriate amount of water. Preparation method: Disperse sodium hyaluronate evenly in the polyol, dissolve the sodium hyaluronate and polyol in an appropriate amount of water, add betaine, and mix. Heat and stir in a waterbath to react. After the reaction is complete, rotary evaporation for 20-60 minutes is performed to obtain the betaine-sodium hyaluronate / polyol dual supramolecular solvent. Preferably, the stirring temperature is 30-50°C, the reaction time is 3-10 hours, and the rotary evaporation temperature is 50-60°C. The polyol is a combination of one or more of propylene glycol, glycerin, dipropylene glycol, butylene glycol, and 1,3-propanediol.

[0016] The present invention also provides the use of the supramolecular solvent in preparing stable supramolecular collagen hyaluronic acid bodies.

[0017] The second aspect of the present application provides a stable supramolecular collagen hyaluronic acid body, which comprises, by weight, 0.1-2.0 parts of lecithin, 0.01-0.4 parts of cholesterol, 0.01 parts of ceramide, 0.05-0.5 parts of collagen, 10-30 parts of an aqueous solution of the supramolecular solvent prepared by the above method (sodium hyaluronate content 0.05-3%), 5-10 parts of polyol, and the remainder water (to make up 100 parts).

[0018] Preferably, the lecithin is one of egg yolk lecithin, soybean lecithin or hydrogenated lecithin.

[0019] Preferably, the polyol is one or more combinations of glycerol, butylene glycol and 1,3-propylene glycol.

[0020] Preferably, the ceramide is ceramide NP.

[0021] Preferably, the collagen is recombinant type III collagen.

[0022] Its preparation method adopts a two-step preparation method:

[0023] S1: Dissolve lecithin, cholesterol, and ceramide in a polyol to form a lipid phase, dissolve collagen in an appropriate amount of water to obtain a collagen aqueous phase, and then add the lipid phase to the collagen aqueous phase under stirring to form liposome colostrum; prepare a supramolecular solvent with water to obtain an aqueous supramolecular solvent solution containing 0.05-3% sodium hyaluronate;

[0024] S2: The liposome colostrum obtained in step S1 is placed in a water bath at 40-50° C., and an isothermal supramolecular solvent aqueous solution as shown in step S1 is added under stirring, and the mixture is then homogenized to obtain a stable supramolecular collagen hyaluronic acid body.

[0025] Preferably, the mixing temperature of the lipid phase in step S1 is 75-85°C; the mixing temperature of the collagen aqueous phase is 45-55°C.

[0026] The beneficial effects of the present invention are:

[0027] 1. A supramolecular solvent system is constructed with betaine as a hydrogen bond acceptor and polyol / sodium hyaluronate as a hydrogen bond donor. On the one hand, it can exert the active effects of betaine and sodium hyaluronate, and on the other hand, the supramolecular system synergistically promotes the penetration of macromolecular substances (collagen, etc.).

[0028] 2. By encapsulating collagen in liposomes, the collagen and sodium hyaluronate are not in direct contact in space, which can solve the problem of flocculent precipitation and system instability caused by the joint use of collagen and sodium hyaluronate.

[0029] 3. The dual delivery system formed by supramolecular solvent and liposome has excellent human permeability and efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 1 are infrared spectra of Examples 1-5 and Comparative Example 1 of the present invention; wherein, Figure a is an infrared spectrum of sodium hyaluronate, betaine, and glycerol alone; and Figure b is an infrared spectrum of the supramolecular solvent shown in Examples 1-5 and Comparative Example 1;

[0031] Figure 21 is a graph showing the particle size distribution of Example 7 (Fig. a) and Example 10 (Fig. b) of the present invention;

[0032] Figure 3 Figure 7 is a Raman permeation graph of Example 7 (a) of the present invention, Comparative Example 2 (b), Comparative Example 6 (c), and Comparative Example 8 (d) when applied to human skin for 8 hours;

[0033] Figure 4 This is a bar graph of the type I collagen content in the in vitro skin tissue under the action of Example 7, Example 10 and Comparative Examples 2, 6 to 8 of the present invention;

[0034] in, Figure 4 In the table, compared with the blank group, the significance is indicated by #, 0.01 < P < 0.05 is indicated by #, 0.001 < P < 0.01 is indicated by ##, 0.0001 < P < 0.001 is indicated by ###, and P < 0.0001 is indicated by ####; compared with the negative control group, the significance is indicated by *, 0.01 < P < 0.05 is indicated by *, 0.001 < P < 0.01 is indicated by **, 0.0001 < P < 0.001 is indicated by ***, and P < 0.0001 is indicated by ****. DETAILED DESCRIPTION

[0035] In order to better understand the present invention, the following is further explained in conjunction with the examples. However, the protection scope of the present invention is not limited to the following examples, and the examples should not be regarded as limiting the protection scope of the present invention.

[0036] The sodium hyaluronate used in the Examples and Comparative Examples was purchased from Shandong Baifu Freda Pharmaceutical Co., Ltd. The collagen was type III collagen with a molecular weight of approximately 70 kDa, and the preparation method was described in Patent ZL202410290161.2. All other reagents and materials, unless otherwise specified, were commercially available.

[0037] Examples 1-4 provide a betaine-sodium hyaluronate / polyol dual supramolecular solvent and its preparation method, and Example 5 provides a betaine-sodium hyaluronate single supramolecular solvent and its preparation method. Examples 1-5 have active efficacy and can be used as carriers to deliver macromolecular substances, promoting their transdermal absorption. For comparison, Comparative Example 1 is also provided, which differs from Example 2 in that the sodium hyaluronate content exceeds a specific range.

[0038] Example 1: Preparation of Betaine-Sodium Hyaluronate / Polyol Dual Supramolecular Solvent

[0039] Preparation method: 0.05g sodium hyaluronate (1KDa~10KDa) is evenly dispersed in 0.1g glycerol, 99.75g water is taken to dissolve the sodium hyaluronate and glycerol phase, 0.1g betaine is added and mixed, and then stirred in a 30°C water bath for 10 hours. After maintaining a rotary evaporation treatment at 50°C for 20 minutes, the water is removed to obtain a betaine-hyaluronic acid / polyol dual supramolecular solvent.

[0040] Example 2: Preparation of Betaine-Sodium Hyaluronate / Polyol Dual Supramolecular Solvent

[0041] Preparation method: 3g of sodium hyaluronate (200KDa-400KDa) is evenly dispersed in 5g of glycerol, 84g of water is taken to dissolve the sodium hyaluronate and glycerol phase, 8g of betaine is added and mixed, and then stirred in a 50°C water bath for 6 hours. After maintaining a rotary evaporation treatment at 55°C for 60 minutes, the water is removed to obtain a betaine-sodium hyaluronate / polyol dual supramolecular solvent.

[0042] Example 3: Preparation of Betaine-Sodium Hyaluronate / Polyol Dual Supramolecular Solvent

[0043] Preparation method: 2g of sodium hyaluronate (1000KDa~1300KDa) is evenly dispersed in 5g of 1,3-propylene glycol, 85g of water is taken to dissolve the sodium hyaluronate and 1,3-propylene glycol phase, 8g of betaine is added and mixed, and then stirred in a 50℃ water bath for 3 hours. After maintaining a rotary evaporation temperature of 60℃ for 40 minutes, the water is removed to obtain a betaine-sodium hyaluronate / polyol dual supramolecular solvent.

[0044] Example 4: Preparation of Betaine-Sodium Hyaluronate / Polyol Dual Supramolecular Solvent

[0045] Preparation method: 0.5g sodium hyaluronate (200KDa~400KDa) is evenly dispersed in 4g glycerol, 90.5g water is taken to dissolve the sodium hyaluronate and glycerol phase, 5g betaine is added and mixed, and then stirred in a 45°C water bath for 6 hours. After maintaining a rotary evaporation treatment at 50°C for 60 minutes, the water is removed to obtain a betaine-sodium hyaluronate / polyol dual supramolecular solvent.

[0046] Example 5: Preparation of Betaine-Sodium Hyaluronate Single Supramolecular Solvent

[0047] Preparation method: Dissolve 0.5g of sodium hyaluronate (200KDa-400KDa) in 94.5g of water, add 5g of betaine and mix, then stir and react in a 45°C water bath for 6 hours. Maintain a rotary evaporation at 55°C for 60 minutes and remove the water to obtain a betaine-sodium hyaluronate monomolecular solvent.

[0048] Comparative Example 1: Preparation of Betaine-Sodium Hyaluronate / Polyol Dual Supramolecular Solvent with Excess Sodium Hyaluronate

[0049] Preparation method: 4g of sodium hyaluronate (200KDa-400KDa) is evenly dispersed in 5g of glycerol, 83g of water is taken to dissolve the sodium hyaluronate and glycerol phase, 8g of betaine is added and mixed, and then stirred in a 50°C water bath for 6 hours. Maintain 55°C for 60 minutes after rotary evaporation to obtain a betaine-sodium hyaluronate supramolecular solvent.

[0050] The supramolecular solvent prepared above was combined with liposomes to prepare supramolecular collagen hyaluronan bodies for the co-delivery of collagen, namely Examples 6 to 10. Comparative Examples 2 to 8 were also provided, wherein Comparative Example 2 was a collagen liposome, and Comparative Examples 3 to 5 differed from the Examples in that the preparation process or one of the substances in the system was changed beyond a specific range to prepare dual supramolecular collagen hyaluronan bodies, Comparative Example 6 was a physical mixture system of a collagen solution and a dual supramolecular solvent, Comparative Example 7 was a mixed aqueous solution of collagen and sodium hyaluronate, and Comparative Example 8 was a collagen solution.

[0051] Example 6: Preparation of Stable Dual Supramolecular Collagen Hyaluronan

[0052] Preparation method: Two-step preparation method;

[0053] S1: At 85°C, 0.1g lecithin, 0.02g cholesterol, and 0.01g ceramide were dissolved in 8g 1,3-propylene glycol to form a lipid phase. 0.05g collagen was dissolved in 81.82g water to obtain a collagen aqueous phase, which was maintained at 50°C. The lipid phase was then added to the collagen aqueous phase and sheared at 5000 rpm for 3 minutes to form liposomal colostrum. The supramolecular solvent described in Example 4 was prepared into a supramolecular solvent aqueous solution containing 0.5% sodium hyaluronate.

[0054] S2: The liposomal colostrum was placed in a 40°C water bath, and 10 g of an aqueous solution of the supramolecular solvent described in step S1 was added under stirring. The mixture was then homogenized at a pressure of 600 bar for 6 times to obtain a dual supramolecular collagen hyaluronic acid body.

[0055] Example 7: Preparation of Stable Dual Supramolecular Collagen Hyaluronan

[0056] Preparation method: Two-step preparation method;

[0057] S1: At 75°C, 0.5g of lecithin, 0.1g of cholesterol, and 0.01g of ceramide were dissolved in 8g of 1,3-propylene glycol to form a lipid phase. 71.19g of water was used to dissolve 0.2g of collagen to obtain a collagen aqueous phase, and the temperature was maintained at 45°C. The lipid phase was then added to the collagen aqueous phase and sheared at 7000 rpm for 4 minutes to form liposomal colostrum. The supramolecular solvent described in Example 4 was prepared into a supramolecular solvent aqueous solution containing 0.5% sodium hyaluronate.

[0058] S2: Place the liposome colostrum in a 45°C water bath, add 20 g of an isothermal aqueous solution of the supramolecular solvent shown in step S1 under stirring, and then homogenize the mixture at a pressure of 1000 bar for 3 times to obtain a dual supramolecular collagen hyaluronic acid body.

[0059] Example 8: Preparation of Stable Dual Supramolecular Collagen Hyaluronan

[0060] Preparation method: Two-step preparation method;

[0061] S1: At 80°C, 1.6g of lecithin, 0.32g of cholesterol, and 0.01g of ceramide were dissolved in 8g of 1,3-propylene glycol to form a lipid phase. 0.4g of collagen was dissolved in 59.67g of water to obtain a collagen aqueous phase, which was maintained at 55°C. The lipid phase was then added to the collagen aqueous phase and sheared at 8000 rpm for 5 minutes to form liposomal colostrum. The supramolecular solvent described in Example 4 was prepared into an aqueous supramolecular solvent solution containing 0.5% sodium hyaluronate.

[0062] S2: Place the liposome colostrum in a 50°C water bath, add 30 g of an aqueous solution of the supramolecular solvent shown in step S1 at an equal temperature under stirring, and then homogenize the mixture at a pressure of 800 bar for 5 times to obtain a dual supramolecular collagen hyaluronic acid body.

[0063] Example 9: Preparation of Stable Dual Supramolecular Collagen Hyaluronan

[0064] Preparation method: Two-step preparation method;

[0065] S1: At 75°C, 2g of lecithin, 0.4g of cholesterol, and 0.01g of ceramide were dissolved in 8g of 1,3-propylene glycol to form a lipid phase. 0.5g of collagen was dissolved in 69.09g of water to obtain a collagen aqueous phase, which was maintained at 45°C. The lipid phase was then added to the collagen aqueous phase and sheared at 6500 rpm for 4 minutes to form liposomal colostrum. The supramolecular solvent described in Example 4 was prepared into an aqueous supramolecular solvent solution containing 0.5% sodium hyaluronate.

[0066] S2: The liposome colostrum was placed in a 50°C water bath, and 20 g of an aqueous solution of the supramolecular solvent described in step S1 was added under stirring. The mixture was then homogenized at a pressure of 1000 bar for three times to obtain a dual supramolecular collagen hyaluronan body.

[0067] Example 10: Preparation of Stable Single Supramolecular Collagen Hyaluronan

[0068] Preparation method: Two-step preparation method;

[0069] S1: At 75°C, 0.5g of lecithin, 0.1g of cholesterol, and 0.01g of ceramide were dissolved in 8g of 1,3-propylene glycol to form a lipid phase. 71.19g of water was used to dissolve 0.2g of collagen to obtain a collagen aqueous phase, and the temperature was maintained at 45°C. The lipid phase was then added to the collagen aqueous phase and sheared at 6500 rpm for 4 minutes to form liposomal colostrum. The supramolecular solvent described in Example 5 was prepared into a supramolecular solvent aqueous solution containing 0.5% sodium hyaluronate.

[0070] S2: Place the liposome colostrum in a 45°C water bath, add 20 g of an isothermal aqueous solution of the supramolecular solvent shown in step S1 under stirring, and then homogenize the mixture at a pressure of 1000 bar for 3 times to obtain a single supramolecular collagen hyaluronic acid body.

[0071] Comparative Example 2: Preparation of collagen liposomes only.

[0072] Preparation method: Specifically, at 75°C, 0.5g of lecithin, 0.1g of cholesterol, and 0.01g of ceramide are dissolved in 8g of 1,3-propylene glycol to form a lipid phase, 91.19g of water is taken to dissolve 0.2g of collagen to obtain a collagen aqueous phase and the temperature is maintained at 45°C. The lipid phase is then added to the collagen aqueous phase at 6500r / min and sheared for 4min to form liposome colostrum; the liposome colostrum is homogenized at a pressure of 1000bar for 3 times to obtain collagen liposomes.

[0073] Comparative Example 3: Dual supramolecular collagen hyaluronic acid bodies prepared by one-step method

[0074] Preparation method: A one-step preparation method is adopted, specifically: at 75°C, 0.5g lecithin, 0.1g cholesterol, and 0.01g ceramide are dissolved in 8g 1,3-propylene glycol to form a lipid phase, 71.19g water is dissolved in 0.2g collagen and 20g of the aqueous solution prepared by the dual supramolecular solvent described in Example 4 (sodium hyaluronate content is 0.5%) to obtain an aqueous phase and maintain the temperature at 45°C, and then the lipid phase is added to the collagen aqueous phase at 6500r / min and sheared for 4min to form liposome colostrum; the liposome colostrum is homogenized at a pressure of 1000bar for 3 times to form a dual supramolecular collagen hyaluronic acid body.

[0075] Comparative Example 4: Preparation of dual supramolecular collagen hyaluronic acid bodies (excess collagen)

[0076] Preparation method: Two-step preparation method;

[0077] S1: At 75°C, 2g of lecithin, 0.4g of cholesterol, and 0.01g of ceramide were dissolved in 8g of 1,3-propylene glycol to form a lipid phase. 0.6g of collagen was dissolved in 68.99g of water to obtain a collagen aqueous phase, which was maintained at 45°C. The lipid phase was then added to the collagen aqueous phase and sheared at 6500 rpm for 4 minutes to form liposomal colostrum. The supramolecular solvent described in Example 4 was prepared into an aqueous supramolecular solvent solution containing 0.5% sodium hyaluronate.

[0078] S2: The liposome colostrum was placed in a 50°C water bath, and 20 g of an aqueous solution of the supramolecular solvent described in step S1 was added under stirring. The mixture was then homogenized at a pressure of 1000 bar for three times to obtain a dual supramolecular collagen hyaluronan body.

[0079] Comparative Example 5: Preparation of dual supramolecular collagen hyaluronic acid bodies (reducing the amount of lecithin and cholesterol)

[0080] Preparation method: Two-step preparation method;

[0081] S1: At 75°C, 0.05g lecithin, 0.01g cholesterol, and 0.01g ceramide were dissolved in 8g 1,3-propylene glycol to form a lipid phase. 71.73g water was used to dissolve 0.2g collagen to obtain a collagen aqueous phase, and the temperature was maintained at 45°C. The lipid phase was then added to the collagen aqueous phase and sheared at 6500 rpm for 4 minutes to form liposomal colostrum. The supramolecular solvent described in Example 4 was prepared into a supramolecular solvent aqueous solution containing 0.5% sodium hyaluronate.

[0082] S2: The liposome colostrum was placed in a 50°C water bath, and 20 g of an aqueous solution of the supramolecular solvent described in step S1 was added under stirring. The mixture was then homogenized at a pressure of 1000 bar for three times to obtain a dual supramolecular collagen hyaluronan body.

[0083] Comparative Example 6: Preparation of a physical mixing system of collagen solution and dual supramolecular solvent

[0084] Preparation method: 79.8 g of water was used to dissolve 0.2 g of collagen and 20 g of the aqueous solution prepared by the dual supramolecular solvent described in Example 4 (the sodium hyaluronate content was 0.5%) to obtain a mixed solution.

[0085] Comparative Example 7: Preparation of a mixed aqueous solution of collagen and sodium hyaluronate

[0086] Preparation method: Take 99.7g water and dissolve 0.2g collagen and 0.1g sodium hyaluronate to obtain a mixed solution.

[0087] Comparative Example 8: Collagen solution only

[0088] Preparation method: Take 99.8g of water and dissolve 0.2g of collagen to obtain a collagen solution.

[0089] The above examples and comparative examples were subjected to performance tests as follows: The ex vivo skin tissue used in the following tests was purchased from Guangdong Boxi Biotechnology Co., Ltd.; other reagents and materials were commercially available unless otherwise specified.

[0090] 1. Infrared Testing of the Supramolecular Solvents of Examples 1 to 5 and Comparative Example 1

[0091] 1. Experimental Method: Examples 1 to 5 and Comparative Example 1 were prepared using a Shimadzu IRSpirit-T Fourier transform infrared spectrometer in attenuated total reflection mode to determine the main functional groups of the materials. The liquid sample was injected into the liquid cell using a matching syringe. During the test, the scanning range was 400-4000 cm -1 .

[0092] 2. Experimental results: Supramolecular solvents with different proportions are prepared through hydrogen bond interactions. The amino groups in betaine form hydrogen bond interactions with the hydroxyl and carboxyl groups in glycerol or hyaluronic acid, where the amino groups are hydrogen bond acceptors and the carboxyl or hydroxyl groups are hydrogen bond donors. Infrared spectrum Figure 1 (a) shows 3331cm -1 The characteristic absorption peak at 1035cm corresponds to the hydroxyl stretching vibration peak of glycerol, betaine, and HA (shown by the left red line). -1The characteristic absorption peak at corresponds to the stretching vibration peak of the CN bond in betaine (shown by the right red line). Due to the interaction of hydrogen bonds, the CN bond shifts, and the infrared spectrum Figure 1 The dotted line in (b) indicates the offset peak of the CN bond. The infrared spectra of Examples 1-5 show that the peak at 1035 cm -1 The characteristic absorption peak at 1035 cm was shifted, indicating the formation of supramolecular solvent; the comparative example 1 did not have the 1035 cm -1 Therefore, no supramolecular solvent was formed in Comparative Example 1.

[0093] II. Stability Tests of Examples 6 to 10 and Comparative Examples 2 to 8

[0094] 1. Experimental method: The samples of Examples 6 to 10 and Comparative Examples 2 to 8 were placed at 45°C, room temperature, 4°C, -20°C, hot and cold cycles (-18°C, 4°C, 45°C, 24 hours at each temperature) and illumination conditions (4500±500 Lux) for observation.

[0095] Experimental results: As shown in Table 1, Examples 6 to 10 and Comparative Example 2 all formed stable colorless semipermeable liquids, Comparative Example 8 was a colorless transparent liquid, and under the six investigation conditions, it could maintain a stable state within 4 weeks; Comparative Example 3 was a colorless semipermeable liquid after preparation, but precipitation occurred within 24 hours at room temperature, and the material body became turbid, indicating that under this preparation process, direct contact between type III collagen and sodium hyaluronate for encapsulation was not conducive to the stability of the system; Comparative Examples 4 and 5 decreased in transparency after preparation, and precipitation occurred within 24 hours, and the material body became turbid, indicating that when the addition amount of type III collagen or phospholipid exceeded a certain range, the stability of the encapsulation system deteriorated, which was not conducive to its subsequent application; Comparative Example 6 had an initial state of The initial state was a colorless transparent liquid, and floccules appeared in the material body at the 4th week, indicating that the simple physical mixing of the supramolecular solvent and collagen could not exist as a stable system; Comparative Example 7 was a colorless transparent liquid at the beginning, and white floccules appeared in the material body at the 4th week, indicating that at this ratio, the compounding of collagen and sodium hyaluronate would form white floccules, which had poor stability and affected its direct application in aqueous products; the type III collagen used in the present invention was prepared in a concentration range of 0.05-0.5% by mass with 0.01-0.2% sodium hyaluronate according to a certain process to form a double supramolecular hyaluronic acid body, which had good stability, could solve the stability problem of the compounding of the two, and at the same time had a delivery effect.

[0096] Table 1: Stability of Examples 6 to 10 and Comparative Examples 2 to 8

[0097]

[0098]

[0099] III. Particle Size and Potential Measurement of Examples 7 and 10

[0100] 1. Experimental Method: The particle size and distribution, polydispersity index (PDI), and zeta potential of Examples 7 and 10 were measured using an Anton Paar Litesizer 500 laser particle size analyzer. The measurement angle was 90° and the experimental temperature was 25°C. The samples were filtered through a 450nm filter membrane and placed in a sample cell. The sample was equilibrated for 1 minute under the configured instrument parameters. The measurement was repeated three times, and the data was recorded. The sample was diluted 10-fold with ultrapure water before measurement to prevent interference from multiple scattering.

[0101] 2. Experimental results: The double and single collagen hyaluronan bodies prepared by the two-step method both exhibited a light blue opalescence and a significant Tyndall effect, indicating unique characteristics at the nanoscale; Figure 2 As shown, the average particle sizes of the double collagen hyaluronic acid body of Example 7 and the single collagen hyaluronic acid body of Example 10 are (77.57±0.68) nm and (88.70±0.93) nm, respectively, the Zeta potentials are (-26.3±1.25) mV and (-10.2±0.63) mV, respectively, and the polydispersity indices are 0.265 and 0.267, respectively; the surface charges of Examples 7 and 10 are medium negative ions, indicating that they have good stability, and PDI < 0.3, indicating that the two systems have reasonable size uniformity.

[0102] IV. Raman Penetration of Example 7 and Comparative Examples 2, 6, and 8 on Human Skin

[0103] 1. Experimental Method: Raman spectroscopy was used to track the characteristic peaks of Example 7 and Comparative Examples 2, 6, and 8 applied to human skin. Peaks that differed from the skin itself were selected for calibration. Raman images were used to analyze the permeability of human skin after application of Example 7 and Comparative Examples 2, 6, and 8, and the relative permeabilities of Example 7 and Comparative Examples 2, 6, and 8 were calculated.

[0104] 2. Experimental results: The relative permeability of Example 7 and Comparative Examples 2, 6, and 8 after 8 hours of exposure to human skin is shown in Table 2, and the visual display is as follows: Figure 3As shown. The experimental results show that after using Comparative Example 8 (collagen solution) on human skin for 8 hours, its relative permeability is 5.34%, which can only penetrate the stratum corneum and is distributed in part of the active epidermis. The relative permeability after using Comparative Example 6 (collagen solution and supramolecular solvent physical mixture) is improved but not significantly, indicating that there are limitations in using supramolecular solvent alone to deliver collagen macromolecules, and the penetration-promoting effect is not good; after using Comparative Example 2 (collagen liposomes), the relative permeability is increased to 8.74%, which is significantly improved compared to collagen solution, indicating that the liposome form has the potential to deliver collagen macromolecules; compared to Comparative Example 8, after using Example 7 in which supramolecular solvent and liposome are combined, the relative permeability of the sample on human skin is increased by 89.51%, indicating that the supramolecular collagen hyaluronic acid body provided by the present invention can effectively promote the penetration of macromolecular collagen while solving the problem of using collagen and hyaluronic acid together.

[0105] Table 2 Relative permeability of Example 7, Example 10 and Comparative Examples 2, 6, and 8 after 8 hours of action on human skin

[0106]

[0107] V. Effects of Examples 7 and 10 and Comparative Examples 2, 6 to 8 on Type I Collagen Content in Excised Skin Tissue

[0108] 1. Experimental method: UVA+UVB combined irradiation was used to establish the skin photoaging model. The blank control group and negative control group (30J / cm 2 UVA+50mJ / cm 2 UVB) and sample group (30J / cm 2 UVA+50mJ / cm 2 UVB + Example 7 / 10 / Comparative Example 2 / 6 / 7 / 8), 3 replicates per group. First, the excised skin tissue (24±2 mm 2 After culturing for 2 days, all groups except the blank control group were irradiated and dosed. The daily irradiation dose was UVA 30J / cm 2 and UVB 50mJ / cm 2During irradiation, the culture medium was discarded and replaced with PBS buffer. UVA irradiation was performed first, followed by UVB irradiation, for 4 consecutive days. Fresh culture medium was replaced after each irradiation, and surface drug administration was performed. The test liquids of different groups were added to the surface of the ex vivo skin tissue, and the added volume of the test liquid was 10 μL. After 4 consecutive days of irradiation, the ex vivo skin tissue was cultured for another 3 days, and only drug treatment was performed without irradiation. After the incubation period, the test liquid remaining on the surface of the ex vivo skin tissue was cleaned and the residual liquid was removed. The tissue was fixed with 4% paraformaldehyde, then embedded and sectioned. After dewaxing, COL-I was stained with conventional immunofluorescence staining and photographed under a fluorescence microscope within 24 hours. Three images of different fields of view were collected, and the target signals in the images were analyzed for relative IOD using Ipwin32 image analysis software, and the average value was calculated. The relative IOD average value was calculated using the blank control group as the benchmark to characterize the relative expression level of the protein. The test concentrations of the samples in Examples 7 / 10 and Comparative Examples 2 / 6 / 7 / 8 were the original solution, and the test solution of the blank control group and the negative control group was EpiGrowth culture solution.

[0109] 2. Experimental results: Type I collagen is the main component of collagen fibers in the dermis, accounting for more than 80%, and plays a role in supporting and protecting the skin. Maintaining the activity and content of type I collagen is important for maintaining the youthful state of the skin. As shown in Table 3 and Figure 4 As shown, after ultraviolet stimulation, the type I collagen content in the negative control group was significantly lower than that in the blank control group (P < 0.01), proving that the skin photoaging model was successfully established. After ultraviolet stimulation, compared with the negative control group, the type I collagen content was significantly increased after adding the systems of Examples 7, 10 and Comparative Examples 2, 6-8 to the in vitro skin tissue photoaging model (P < 0.05). Among them, the improvement rates of Examples 7 and 10 on type I collagen were 198.21% and 184.11%, respectively, and the improvement rates of Comparative Examples 2, 6-8 on type I collagen were 124.93%, 125.87%, 105.10%, and 55.55%, respectively. From the above results, it can be seen that when collagen is used alone in Comparative Example 8, it has a certain effect of increasing the content of type I collagen, but compared with Comparative Example 7, the effect is better when collagen is used in combination with sodium hyaluronate; the improvement rate of type I collagen content in Examples 7 and 10 is much higher than that in Comparative Examples 2, 6 to 8, indicating that the double or single supramolecular collagen hyaluronic acid body in the present invention can significantly increase the content of type I collagen, provide mechanical support for the skin, and improve skin elasticity and toughness.

[0110] Table 3 Effects of Example 7, Example 10 and Comparative Examples 2, 6 to 8 on the content of type I collagen in ex vivo skin tissue

[0111] Group Relative IOD average SD Improvement rate P-value Blank control group 1.00 0.08 / / Negative control group 0.50 0.07 / 0.0012## Example 7 1.50 0.02 198.21％ <0.0001**** Example 10 1.43 0.05 184.11％ <0.0001**** Comparative Example 2 1.13 0.01 124.93％ 0.0001*** Comparative Example 6 1.14 0.09 125.87％ 0.0007*** Comparative Example 7 1.03 0.03 105.10％ 0.0003*** Comparative Example 8 0.78 0.02 55.55％ 0.0026**

Claims

1. A supramolecular solvent, characterized in that: Sodium hyaluronate is used as a hydrogen bond donor.

2. The supramolecular solvent according to claim 1, wherein The initial components include, by weight, 0.1 to 8.0 parts of betaine, 0.05 to 3 parts of sodium hyaluronate and an appropriate amount of water. The supramolecular solvent is obtained by dissolving, mixing, heating and stirring the reaction and removing water by rotary evaporation.

3. The supramolecular solvent according to claim 1 or 2, wherein: The molecular weight of the sodium hyaluronate is 1KDa-1300KDa.

4. The supramolecular solvent according to claim 3, wherein The supramolecular solvent is divided into a single supramolecular solvent or a double supramolecular solvent; the initial components and weight parts of the single supramolecular solvent are: 0.1-8.0 parts of betaine, 0.05-3 parts of sodium hyaluronate, and an appropriate amount of water; the initial components and weight parts of the double supramolecular solvent are: 0.1-8.0 parts of betaine, 0.1-5.0 parts of polyol, 0.05-3 parts of sodium hyaluronate, and an appropriate amount of water, and the polyol is a combination of one or more of propylene glycol, glycerin, dipropylene glycol, butylene glycol, and 1,3-propylene glycol.

5. The supramolecular solvent according to claim 4, wherein: The preparation method of the single supramolecular solvent comprises the following steps: dissolving sodium hyaluronate in water and adding betaine to mix, then heating and stirring in a water bath for reaction, and performing rotary evaporation after the reaction to obtain the betaine-sodium hyaluronate supramolecular solvent.

6. The supramolecular solvent according to claim 4, wherein The preparation method of the dual supramolecular solvent comprises the following steps: uniformly dispersing sodium hyaluronate in a polyol, dissolving the sodium hyaluronate and the polyol in water, adding betaine and mixing, then heating and stirring in a water bath for reaction, and subjecting the mixture to rotary evaporation after the reaction is complete to obtain the betaine-sodium hyaluronate / polyol dual supramolecular solvent.

7. Use of the supramolecular solvent according to any one of claims 1 to 6 in preparing stable supramolecular collagen hyaluronan bodies.

8. A stable supramolecular collagen hyaluronic acid body, characterized by: In parts by weight, 0.1-2.0 parts of lecithin, 0.01-0.4 parts of cholesterol, 0.01 parts of ceramide, 0.05-0.5 parts of collagen, 10-30 parts of an aqueous solution of the supramolecular solvent according to any one of claims 2-6, 5-10 parts of a polyol, and water to make up to 100 parts; The aqueous solution of the supramolecular solvent is prepared by using water to prepare the supramolecular solvent into an aqueous solution having a sodium hyaluronate content of 0.05 to 3%.

9. The supramolecular collagen hyaluronic acid body according to claim 8, characterized in that: The lecithin is one of egg yolk lecithin, soybean lecithin or hydrogenated lecithin; The polyol is one or more combinations of glycerol, butanediol and 1,3-propylene glycol; The ceramide is ceramide NP; The collagen is recombinant type III collagen.

10. The method for preparing supramolecular collagen hyaluronic acid according to claim 8 or 9, characterized in that: A two-step preparation method is used: S1: Dissolving lecithin, cholesterol, and ceramide in a polyol to form a lipid phase, dissolving collagen in water to obtain a collagen aqueous phase, and then adding the lipid phase to the collagen aqueous phase under stirring to form liposome colostrum; preparing a supramolecular solvent in water to prepare an aqueous solution of the supramolecular solvent containing 0.05-3% sodium hyaluronate, and setting aside; S2: The liposome colostrum obtained in step S1 is placed in a water bath at 40-50° C., and an aqueous solution of the supramolecular solvent shown in step S1 is added at an isothermal temperature under stirring. The mixture is then homogenized to obtain a stable supramolecular collagen hyaluronan body.

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