A soluble microcrystal with firming and anti-wrinkle effects, its preparation method and application

Abstract

This invention discloses a soluble microcrystal with firming and anti-wrinkle effects, its preparation method, and its application, belonging to the field of cosmetic skincare technology. The soluble microcrystal comprises the following components: collagen, hyaluronic acid, modified hyaluronic acid, bee venom peptide, niacinamide, linoleic acid, phospholipids, cholesterol, etc. Hyaluronic acid is modified with EDTA and polyethylene glycol diamine to obtain modified hyaluronic acid. Modified hyaluronic acid exhibits good biocompatibility and a multi-branched structure, enabling it to form a three-dimensional cross-linked network structure between molecules or with collagen, greatly improving the mechanical strength of the microcrystal needles. The microcrystals are prepared through two steps: conventional drying and freeze-drying, ensuring rapid water solubility and drug release. The liposomes formed by linoleic acid, phospholipids, and cholesterol contain water and active ingredients, which can greatly promote the transdermal absorption of the active ingredients.

Description

Technical Field

[0001] This invention relates to the field of beauty and skincare technology, specifically to a soluble microcrystal with firming and anti-wrinkle effects, its preparation method, and its application. Background Technology

[0002] As we age and our exposure to ultraviolet radiation increases, the amount of hyaluronic acid (HA) in our skin gradually decreases, leading to a series of problems such as skin aging, decreased elasticity, and wrinkles. To combat these skin problems, cosmetics often add hyaluronic acid, utilizing its unique moisturizing and repairing abilities to improve skin texture.

[0003] Hyaluronic acid is a naturally occurring substance in the skin with a powerful water-absorbing capacity, capable of absorbing and retaining hundreds of times its own weight in water. When hyaluronic acid is added to the skin in cosmetics, it can be absorbed through the skin, increasing its moisture content. This moisturizing effect not only keeps the skin surface hydrated but also penetrates deep into the skin, providing skin cells with ample moisture and nutrients, thereby nourishing the skin and improving its texture.

[0004] Hyaluronic acid of different molecular weights exhibits varying transdermal absorption capabilities. Low molecular weight hyaluronic acid (typically referring to molecules with a molecular weight less than 300 kDa) possesses excellent transdermal absorption capabilities; its smaller molecular structure allows it to more easily penetrate the stratum corneum and sebum film, reaching the epidermis and even the dermis. Soluble patches with an array structure of microcrystalline protrusions (i.e., microneedles) are made using sodium hyaluronate or hyaluronic acid as the main excipient carrier, carrying the active ingredients. These patches promote scalp absorption.

[0005] Human skin is divided into the epidermis and the dermis. The epidermis includes the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. The stratum corneum is the outermost layer of the epidermis and is the main barrier for the transdermal transport of most substances. Microneedles can help penetrate the stratum corneum and promote transdermal absorption.

[0006] Existing technology CN118846363A discloses a microneedle for transdermal delivery of macromolecular drugs. The microneedle tip layer is prepared by repeated injection molding and drying of a pure protein solution with a concentration of 40 mg / mL. A backing layer is prepared by injecting 15% hyaluronic acid to increase the drug loading capacity of the transdermal protein microcrystals and avoid the safety risks associated with using high molecular weight polymers. However, the repeated injection molding and drying process indicates that the microneedles are prone to shrinkage and deformation during drying. Even when fully dried, the product still faces the technical challenge of strongly adsorbing moisture from the skin, which contradicts the moisturizing purpose of cosmetic skincare.

[0007] It is evident that existing technologies still have some unresolved technical problems, summarized as follows: 1. The backing of soluble microneedle patches uses water-soluble materials. Current microcrystal (or microneedle) preparation technologies rely on thoroughly drying the microcrystal patch to ensure its strength. However, the low water content of the backing after drying results in poor adhesion to the skin, making it difficult for the skin to absorb the active ingredients in the backing material. 2. To ensure the absorbability of the microcrystal tips, the tips need a certain water content, but this leads to low needle strength. To ensure tip strength and maintain needle shape, insoluble or high-molecular-weight excipients are added during manufacturing, resulting in poor absorption of the soluble material. 3. Microcrystals are prone to tip dulling due to temperature changes during room temperature storage. For example, high temperatures in summer can easily cause microcrystal softening, making room temperature storage inconvenient. Summary of the Invention

[0008] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a soluble microcrystal with firming and anti-wrinkle effects, its preparation method and application. The soluble microcrystal has the advantages of high microcrystal hardness, not easily softened at room temperature, low irritation, and easy solubility, and also has the technical effects of moisturizing and whitening. It can be used to prepare cosmetics, skin care products and medical beauty products.

[0009] The present invention is achieved through the following technical solution: providing a soluble microcrystal, characterized in that, by weight, it comprises the following components: 20-30 parts collagen, 10-20 parts hyaluronic acid, 20-30 parts modified hyaluronic acid, 3-5 parts bee venom peptide, 1-3 parts whitening component, 5-10 parts linoleic acid, 10-20 parts phospholipid, and 3-5 parts cholesterol.

[0010] The modified hyaluronic acid has the following structure (I):

[0011] The ratio of m:n is 1:(1-10), where m and n in the structural formula only represent the number of carboxyl groups grafted onto hyaluronic acid, and are not used to limit the position of the grafted carboxyl groups in hyaluronic acid, nor do they indicate that the modified hyaluronic acid is a block compound, where z is an integer from 1 to 100.

[0012] The modified hyaluronic acid is prepared as follows:

[0013] S1: EDTA-modified polyethylene glycol diamine (NH2-PEG9-NH2): Dissolve EDTA (10 mmol) in 1 L of pH 5.5 phosphate buffer, add EDC (10 mmol) and NHS (11 mmol), stir at room temperature for 2 hours to form EDTA-NHS ester solution.

[0014] S2: Dissolve 10 mmol of polyethylene glycol diamine (NH2-PEG9-NH2) in phosphate buffer at pH 7.4, add EDTA-NHS ester solution dropwise under stirring, and continue the reaction for 24 hours to obtain EDTA-PEG-NH2 solution.

[0015] S3: Hyaluronic acid (HA) (50 mmol carboxyl group) was dissolved in 3 L phosphate buffer (pH 6.0), and EDC (10 mmol) and NHS (10 mmol) were added. The mixture was stirred at room temperature for 1 hour to form an HA-NHS ester solution. EDTA-PEG-NH2 solution was added to the HA-NHS ester solution, and the reaction was stirred for 24 hours. The reaction solution was transferred to a dialysis bag (MWCO 3 kDa) and purified by dialyzing with deionized water. After dialysis, the solution was dried and pulverized to obtain modified hyaluronic acid powder.

[0016] The molar ratio of EDTA, EDC, and NHS used above is 1:(0.9-1.1):(0.9-1.2), preferably 1:1:1.1.

[0017] The molar ratio of polyethylene glycol diamine to EDTA is 1:0.9-1.1, preferably 1:1.

[0018] The molar ratio of hyaluronic acid to polyethylene glycol diamine, calculated based on carboxyl group content, is (1-10):1.

[0019] The phospholipid is selected from at least one of dioleoylphosphatidylserine sodium (DOPS), egg yolk lecithin, soybean lecithin, dioleoyl L-α-phosphatidylethanolamine (DOPE), 1,2-dioleoyl lecithin (DOPC), dipalmitoylphosphatidylcholine (DPPC), and distearate phosphatidylcholine (DSPC).

[0020] The whitening ingredient is selected from at least one of niacinamide, vitamin C, ursolic acid, azelaic acid, and glutathione.

[0021] This invention also provides a method for preparing soluble microcrystals: A mixed aqueous solution is prepared by adding a prescribed amount of collagen, hyaluronic acid, modified hyaluronic acid, and bee venom peptide to sterile deionized water. Under low-temperature water bath heating conditions, phospholipids and cholesterol are dissolved in ethanol. Linoleic acid and whitening ingredients are added to the phospholipid ethanol solution, and the mixture is rapidly stirred. An equal volume of sterile deionized water is added, and the mixture is stirred at high speed to form a suspension. An equal volume of sterile deionized water is added to the suspension, and the mixture is homogenized under high pressure to prepare liposomes containing whitening ingredients. The liposomes are added to the prepared mixed aqueous solution and mixed evenly to obtain a microcrystal preparation solution. Under vacuum conditions, the microcrystal preparation solution is injected into a microcrystal mold, completely covering the needle tip area of ​​the mold. The mixture is allowed to stand at room temperature until it dries into an elastic gel. It is then freeze-dried for 0.5-2 hours to obtain microcrystals. The microcrystals prepared by the above method include a base layer and needles, wherein the needle length is 50-600 μm, preferably 100-500 μm, or 200-500 μm.

[0022] This invention also provides a soluble microcrystal, characterized in that, by weight, it comprises the following components: 20-30 parts collagen, 10-20 parts hyaluronic acid, 20-30 parts modified hyaluronic acid, 5-10 parts linoleic acid, 10-20 parts phospholipids, and 3-5 parts cholesterol; optionally, 0.01-20 parts of the following functional components may be added, wherein the functional components are selected from at least one of the following: bee venom peptide DNA sodium, acetyl hexapeptide-8, arginine / lysine polypeptide, milk extract, grape fruit cell extract, and exosomes.

[0023] Beneficial effects

[0024] This invention provides a soluble microcrystal and its preparation method, uniquely utilizing modified hyaluronic acid as the microcrystal matrix. The modified hyaluronic acid is prepared by grafting polyethylene glycol diamine (PEG) with ethylenediaminetetraacetic acid (EDTA). Both PEG and EDTA are strongly hydrophilic and biocompatible components. Furthermore, the modified hyaluronic acid is rich in N and O branches, which can promote the formation of hydrogen bonds and other intermolecular forces between polymers, constructing a three-dimensional network structure. This significantly improves the material's mechanical strength, elastic modulus, and water retention and absorption properties. The modified hyaluronic acid can also form effective intermolecular forces with collagen and melilotoxins, forming a three-dimensional network structure, improving water retention and microcrystal strength, reducing the amount of microcrystal matrix material used, and greatly increasing drug loading capacity.

[0025] The introduction of EDTA endows HA with a strong metal ion chelating ability, which can remove Fe from the skin cell microenvironment. 2+ Cu 2+ Metal ions can inhibit the generation of free radicals and oxidation reactions. For example, copper ions competitively chelate tyrosinase, which can effectively inhibit the activity of tyrosinase, reduce the production of melanin, and achieve a whitening effect.

[0026] This invention provides a two-stage drying method. First, after the microcrystalline component solution is molded, it is dried at room temperature until it reaches a gel state. This facilitates the uniform solidification of the microcrystalline matrix, forming a three-dimensional network structure and improving mechanical strength. Second, the secondary drying employs freeze-drying. The freezing process helps maintain the shape of the microcrystals, preventing the microcrystalline matrix from shrinking and deforming due to water loss during the drying process. Simultaneously, controlling the vacuum time during freeze-drying ensures that the microcrystals retain some moisture while maintaining sufficient hardness. After freeze-drying, the microcrystals exhibit a relatively mesoporous structure, which is more conducive to water absorption and dissolution, promoting the release of active ingredients.

[0027] The microcrystals described in this invention are not completely dried; the liposomes still contain water, as does the microcrystal matrix. Linoleic acid, which is a readily flowing liquid at room temperature, interspersed between the phospholipid bilayers and is highly hydrophobic, helps increase the density of the liposome membrane, improves encapsulation ability, reduces water loss during freeze-drying, minimizes absorption of skin tissue fluid by the microcrystals, and improves applicability. The biocompatibility of the liposomes facilitates the entry of the contained active ingredients into cells, promoting transdermal absorption.

[0028] The microcrystals described in this invention have the technical effects of high microcrystal hardness, resistance to softening at room temperature, low irritation, easy solubility, moisturizing and whitening. Furthermore, the microcrystals can be prepared into microcrystal needles and microcrystal backings in one step, making them easy to produce industrially. Attached Figure Description

[0029] Figure 1 Chemical structural formula of modified hyaluronic acid.

[0030] Figure 2 Infrared spectrum of modified hyaluronic acid (amide bond peak position 1655 cm⁻¹) -1 ).

[0031] Figure 3 Microcrystalline electron micrographs of Example 4 and Comparative Example 5. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some preferred embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the embodiments are conventional or common methods in the art, and the materials and reagents used are commercially available unless otherwise specified.

[0034] The raw materials used in the examples and comparative examples are described below:

[0035] Collagen: Purchased from Chenguang Biotech;

[0036] Hyaluronic acid: Hunan Bloomage Biotechnology (300kDa);

[0037] Polyethylene glycol diamine (NH2-PEG4-NH2, NH2-PEG9-NH2, NH2-PEG) 19 -NH2): Shanghai Yanfen Biochemical Technology Co., Ltd.;

[0038] Bee venom peptide: purchased from North China Pharmaceutical Co., Ltd. (for injection);

[0039] EDTA (diethylaminetetraacetic acid): Xi'an Tianyi Biotechnology;

[0040] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC: purchased from Jier Biochemical (Shanghai) Co., Ltd.;

[0041] N-hydroxysuccinimide (NHS): Purchased from Hubei Xinghengkang Chemical Technology Co., Ltd.;

[0042] Phospholipids (HSPE): Purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.

[0043] Linoleic acid: purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.;

[0044] Cholesterol: Purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.

[0045] Unless otherwise specified, all components and raw materials used in the embodiments and comparative examples of this invention are commercially available, and the same type of components and raw materials are used in each parallel experiment.

[0046] The preparation method of modified hyaluronic acid is as follows:

[0047] S1: EDTA-modified polyethylene glycol diamine (NH2-PEG9-NH2): Dissolve EDTA (10 mmol) in 1 L of pH 5.5 phosphate buffer, add EDC (10 mmol) and NHS (11 mmol), stir at room temperature for 2 hours to form EDTA-NHS ester solution.

[0048] S2: Dissolve 10 mmol of polyethylene glycol diamine (NH2-PEG9-NH2) in phosphate buffer at pH 7.4, add EDTA-NHS ester solution dropwise under stirring, and continue the reaction for 24 hours to obtain EDTA-PEG-NH2 solution.

[0049] S3: Dissolve HA (50 mmol carboxyl group) in 3 L phosphate buffer (pH 6.0), add EDC (10 mmol) and NHS (10 mmol), and stir at room temperature for 1 hour to form an HA-NHS ester solution. Add EDTA-PEG-NH2 solution to the HA-NHS ester solution and stir for 24 hours. Transfer the reaction solution to a dialysis bag (MWCO 3 kDa) and dialyze to deionized water for purification; after dialysis, dry and pulverize to obtain modified hyaluronic acid powder for later use.

[0050] Example 1

[0051] Preparation method of soluble microcrystals:

[0052] Prescription: 20 parts collagen, 10 parts hyaluronic acid, 20 parts modified hyaluronic acid, 3 parts bee venom peptide, 1 part nicotinamide, 5 parts linoleic acid, 10 parts phospholipids, and 3 parts cholesterol.

[0053] Step 1: Prepare a mixed aqueous solution (modified hyaluronic acid mass concentration of 1%) by adding 20 equivalents of collagen, 10 equivalents of hyaluronic acid, 20 equivalents of modified hyaluronic acid, and 3 equivalents of bee venom peptide to sterile deionized water.

[0054] Step 2: Under low-temperature (50°C) water bath heating conditions, dissolve 10 equivalents of phospholipids (HSPE) and 3 equivalents of cholesterol in ethanol (the amount used should result in a phospholipid concentration of 1 g / ml). Add 5 equivalents of linoleic acid and 1 equivalent of nicotinamide to the phospholipid ethanol solution, stir rapidly, add an equal volume of sterile deionized water, and stir with a high-speed blender to form a suspension. Add an equal volume of sterile deionized water to the suspension, and homogenize using a high-pressure homogenizer (model: ATS AH-D150). Homogenize 3 times to prepare liposomes containing the active ingredients linoleic acid and nicotinamide.

[0055] Step 3: Add the liposomes from Step 2 to the mixed aqueous solution prepared in Step 1, mix thoroughly to obtain the microcrystal preparation solution. Under vacuum conditions, inject the microcrystal preparation solution into the microneedle mold, ensuring the injection volume completely covers the tip area of ​​the mold. Let it stand at room temperature for 2.5 hours until it dries into an elastic gel. Freeze-dry, slowly cool to -20°C, and vacuum-dry for 0.5 hours to obtain microcrystals.

[0056] Examples 2-4

[0057] Examples 2-4 were prepared using the same method as Example 1, the only difference being the adjustment of the amount of each component.

[0058] Comparative Examples 1-5

[0059] Comparative Examples 1 and 2 were prepared using the same methods as Examples 1 and 4, respectively, except that the modified hyaluronic acid was replaced with an equal amount of hyaluronic acid.

[0060] Comparative Example 3 was prepared using the same method as Example 2, except that linoleic acid was reduced in the composition.

[0061] Comparative Example 4 was prepared using the same method as Example 3, except that the number of bee venom peptides was reduced.

[0062] Comparative Example 5 has the same components as Example 4, except that: after injecting the microcrystal preparation solution into the microneedle mold, it was left to stand at room temperature for 2.5 hours until it dried into an elastic gel, and then dried at room temperature for another 4 hours.

[0063] Table 1. Components and parts by mass of Examples 1-4 and Comparative Examples 1-5

[0064]

[0065]

[0066] Performance tests were conducted on Examples 1-4 and Comparative Examples 1-5 to verify the results.

[0067] (1) Skin irritation test:

[0068] Skin irritation tests were conducted on the whitening skincare products used in the examples and comparative examples. The test subjects were nude mice (BALB / c-nu test). Ten mice (five male and five female) were randomly assigned to each sample. Sample microcrystals (0.5cm × 0.5cm) were adhered to the backs of mice (0.5cm × 0.5cm) for 0.5 hours, twice daily (9:00 AM and 6:00 PM) for four consecutive weeks. Mouse behavior and skin condition were observed daily. Scoring was performed according to the standards in the table below.

[0069] Mouse skin condition Rating / points Normal skin 5 No irritation, normal skin tone 4 Mild irritation (redness, etc.) 3 Rash and swelling, 2 Ulcers, blisters, poor mobility, death 0

[0070] Table 2 Results of skin irritation test 4 weeks later

[0071]

[0072] After a 4-week trial, Table 2 above shows that the microcrystals prepared in Examples 1-4 and Comparative Examples 1-6 have good biocompatibility and do not cause skin irritation in mice.

[0073] Safety evaluation of human trial use: Human skin trial use was used for testing. The specific test methods refer to the "Cosmetic Safety Technical Specifications" (2015 edition), as detailed in the table below.

[0074] Classification Skin reaction 0 No response 1 faint erythema 2 Erythema, infiltration, and papules may be present. 3 Erythema, infiltration, edema, papules, vesicles 4 Erythema, edema, bullae

[0075] A total of 180 subjects, aged 18-56 years with a mean age of 32 years, were randomly divided into 9 groups. Microcrystalline samples 1 from Examples 1-4 and Comparative Examples 1-5 were applied to the outer forearm for 0.5 hours, once daily, and followed up for 4 weeks. The experimental results (see Table 3) showed that samples from Examples 1-4 and Comparative Examples 1-5 all showed negative reactions (grade 0) and did not induce erythema on the skin.

[0076] Table 3. Experimental Results

[0077] Number of people in the project Level 0 Level 1 Level 2 Level 3 Level 4 Example 1 20 0 0 0 0 Example 2 20 0 0 0 0 Example 3 20 0 0 0 0 Example 4 20 0 0 0 0 Comparative Example 1 20 0 0 0 0 Comparative Example 2 20 0 0 0 0 Comparative Example 3 20 0 0 0 0 Comparative Example 4 20 0 0 0 0 Comparative Example 5 20 0 0 0 0

[0078] (2) Determine the water content of the microcrystals

[0079] Examples 1-4 and Comparative Examples 1-5 were fully dried in a drying oven at 50°C for 48 hours, and the weights W1 before drying and W2 after drying were recorded. The water content of the microcrystals was (W1-W2) / W1. The experimental results are shown in Table 4.

[0080] Table 4. Water content of microcrystals (%)

[0081]

[0082] Examples 1-4 utilize liposomes to encapsulate effective combinations and water, significantly increasing the water content of microcrystals. The water content is positively correlated with the ratio of liposome components used. Compared to traditional microcrystals (microneedles), there is no need to use a completely drying method to ensure the strength of the needle, which can effectively reduce the absorption of water from skin cells during use. In Comparison 3, the water content is reduced compared to Comparative Example 2, which is related to the lack of linoleic acid, a component that can improve the encapsulation performance of the liposome bilayer.

[0083] (3) Testing microcrystal hardness

[0084] The strength of the microcrystals was tested using a Shimadzu universal testing machine. The microcrystals were fixed on a horizontal testing platform, and a P / 6 type flat-headed stainless steel cylindrical probe was used to apply vertical pressure along the microneedle axis at a constant speed of 0.1 mm / s and an excitation force of 0.01 N. The entire mechanical response from probe contact with the tip until reaching the predetermined termination height (i.e., the total length of the 400 μm microcrystal) was recorded in detail to evaluate the mechanical properties, deformation characteristics, and product stability of the microcrystals. The microneedle strength of the test samples was measured at 0 days at 30°C and 40°C, and after 4 months of sealed storage at 30°C.

[0085] Table 5. Strength (N) of microcrystals at different temperatures.

[0086]

[0087]

[0088] Comparing Comparative Examples 1 and 2 with Examples 1-4, it can be seen that the modified hyaluronic acid used in Examples 1-4 significantly improves the strength of the microneedles, and they still maintain good strength at 40°C. This indicates that the microcrystals described in this invention still possess extremely high strength at high temperatures and can be effectively delivered transdermally. However, at 40°C, the strength of the microcrystals in Comparative Examples 1 and 2 decreases significantly, reducing their transdermal penetration performance. With the human body temperature approaching 37°C, high summer temperatures or human contact with the microcrystals may severely affect the use of hyaluronic acid as a matrix. The microcrystal strength results at 30°C after 4 months show that the strength of the microcrystals in Examples 1-4 remains consistent with that at day 0, indicating that the microcrystals described in this invention have good stability, are easy to store at room temperature, and are convenient for transportation. The strength of the microcrystals in Comparative Example 3 significantly decreases after 4 months, showing that linoleic acid can effectively stabilize the encapsulation performance of liposomes, preventing water leakage and thus reducing the strength of the microcrystal needles. This may be due to the easy flow and strong hydrophobicity of linoleic acid, which enhances the encapsulation performance of liposomes. Compared with Example 4, Comparative Example 5 showed an increased strength of microcrystals. However, the needles of the microcrystals in Comparative Example 5 shrank after drying, resulting in a shorter and stouter needle body, thus slightly increasing the strength.

[0089] (4) Microcrystalline solubility test

[0090] A constant temperature water bath at 37℃ was used to simulate the process of dissolving microcrystals in the human body. The microcrystal patch was immersed in the bath, and the time (min) required for the microcrystal needle to completely dissolve was recorded.

[0091] Table 6 Dissolution time of microcrystals

[0092]

[0093] As shown in Table 6, comparing Comparative Example 1 with Example 1, and Comparative Example 2 with Example 4, the dissolution times of the microcrystals are similar. This indicates that after conventional drying and freeze-drying, the modified hyaluronic acid and hyaluronic acid as the microcrystal matrix maintain the original shape and interpenetrating molecular network structure of the microcrystals, increasing the intermolecular gaps and improving the dissolution rate. However, the microcrystals in Comparative Example 5, which only underwent conventional drying, shrank, resulting in tighter intermolecular bonds and making it difficult for water to penetrate into the microcrystals, thus significantly prolonging the dissolution time.

[0094] (5) Human skin anti-wrinkle test

[0095] Ninety volunteers aged 30-50 years were selected as test subjects. Microcrystals from Examples 1-4 and Comparative Examples 1-5 were applied to the outer corner of the left eye of the test subjects. The microcrystals were removed after 10 minutes of application. The application was performed twice a day, once in the morning and once in the evening, for a total of 4 weeks. The wrinkle depth (D1) before and after application and the wrinkle depth (D2) after 4 weeks of application were recorded. The wrinkle improvement rate was then calculated. The formula for calculating the wrinkle improvement rate is: Improvement rate (%) = (D1-D2) / D1×100%.

[0096] Table 7. Anti-wrinkle improvement rate of microcrystals

[0097]

[0098]

[0099] As shown in Table 7 above, Comparative Examples 1 and 2 showed similar wrinkle improvement rates to Examples 1 and 4, respectively. Compared to Comparative Example 2, Comparative Example 3 showed a slightly lower skin improvement rate, possibly due to the low water content in the microcrystals, which increased the absorption of moisture from the skin and reduced the transdermal absorption of active ingredients such as bee venom peptides by cells. Compared to Example 3, Comparative Example 4 showed a significantly lower wrinkle improvement rate, indicating that the combination of bee venom peptides with collagen and hyaluronic acid can significantly improve the anti-wrinkle effect. Compared to Example 4, Comparative Example 5 showed a reduced anti-wrinkle effect, which is related to the drying method of the microcrystals. The microcrystals in Comparative Example 5 shrank due to drying, resulting in a significant reduction in the length of the microcrystal needles, leading to a significant decrease in transdermal depth and reduced transdermal delivery performance.

[0100] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A soluble microcrystal, characterized in that, By weight, it includes the following components: 20-30 parts collagen, 10-20 parts hyaluronic acid, 20-30 parts modified hyaluronic acid, 3-5 parts bee venom peptide, 1-3 parts whitening component, 5-10 parts linoleic acid, 10-20 parts phospholipids, and 3-5 parts cholesterol; the whitening component is niacinamide. The modified hyaluronic acid is prepared as follows: S1: Dissolve 10 mmol EDTA in 1 L of pH 5.5 phosphate buffer, add 10 mmol EDC and 11 mmol NHS, stir at room temperature for 2 hours to form EDTA-NHS ester solution; S2: Dissolve 10 mmol of polyethylene glycol diamine in phosphate buffer at pH 7.4, add EDTA-NHS ester solution dropwise under stirring, and continue the reaction for 24 hours to obtain EDTA-PEG-NH2 solution; S3: Dissolve 50 mmol of hyaluronic acid (HA) based on carboxyl group content in 3 L of pH 6.0 phosphate buffer, add 10 mmol of EDC and 10 mmol of NHS, and stir at room temperature for 1 hour to form an HA-NHS ester solution; add EDTA-PEG-NH2 solution to the HA-NHS ester solution and stir for 24 hours; transfer the reaction solution to a dialysis bag and purify by dialyzing with deionized water; after dialysis, dry and pulverize to obtain modified hyaluronic acid powder; The microcrystals were prepared by the following method: Step 1: Prepare a mixed aqueous solution by adding collagen, hyaluronic acid, modified hyaluronic acid, and bee venom peptide to sterile deionized water, so that the mass concentration of modified hyaluronic acid is 1%; Step 2: Under low-temperature (50°C) water bath heating conditions, phospholipids and cholesterol are dissolved in ethanol to achieve a phospholipid concentration of 1 g / ml. Linoleic acid and nicotinamide are added, stirred, and an equal volume of sterile deionized water is added. The mixture is stirred to form a suspension. An equal volume of sterile deionized water is added to the suspension, and the mixture is homogenized using a high-pressure homogenizer. The homogenization cycle is repeated 3 times to prepare liposomes containing the active ingredients linoleic acid and nicotinamide. Step 3: Add the liposomes from Step 2 to the mixed aqueous solution prepared in Step 1, mix evenly to obtain the microcrystal preparation solution; under vacuum conditions, inject the microcrystal preparation solution into the microneedle mold, the injection amount completely covering the tip area of ​​the mold, let it stand at room temperature for 2.5h, wait for it to dry into an elastic gel, freeze dry, slowly cool to -20℃, vacuum dry for 0.5h, and you will get the product.

2. The application of the soluble microcrystals as described in claim 1 in the preparation of skin care products or medical aesthetic products.

Images