Soothing oat base-hyaluronic acid co-assemblies, methods of making and use thereof

Abstract

The application belongs to the field of biological medicine and cosmetics, and particularly relates to a soothing oat alkaloid-hyaluronic acid co-assembly and a preparation method and application thereof. The method comprises the following steps: adding dihydro-oat alkaloid into a hyaluronic acid compound solution, mixing to obtain a primary mixture, adding a cyclodextrin compound, homogenizing, centrifuging, filtering, and drying to obtain soothing oat alkaloid-hyaluronic acid co-assembly (AHC). The co-assembly inclusion method is used to improve the water solubility of Ave by more than 1200 times, and the synergistic effect of Ave and HA makes the anti-inflammatory, anti-allergic and anti-photoaging effects of AHC significantly better than those of free Ave and HA with the same molar. In addition, compared with the same products on the market, the AHC of the application has lower cytotoxicity, better safety and stability when applied to skin care products, and can fully meet the product demand of mitochondrial energy skin care; compared with the precursor mixture, the redness and itching relieving effect is significantly improved.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine, beauty and personal care, and specifically relates to a soothing oat base-hyaluronic acid co-assembly, its preparation method and application. Background Technology

[0002] Dihydroavenanthramide D (Ave) is an active ingredient extracted from oats. Ave possesses significant anti-inflammatory, anti-allergic, and antipruritic effects. It can inhibit the degradation of NF-κB-α, a nuclear factor directly related to inflammation, and prevent the phosphorylation of the p65 protein subunit on NF-κB, thereby blocking the process of cellular inflammation. It is a potent non-steroidal anti-inflammatory and antipruritic natural ingredient with a potency comparable to hydrocortisone, but without the side effects of steroid hormones. In addition, Ave has strong antioxidant properties, effectively resisting free radical damage to the skin and preventing skin aging. Therefore, Ave is widely used in personal care and cosmetic products, including various cosmetics such as baby skin antipruritic creams, eczema creams, sensitive skin repair masks, insect bite care products, shampoos, and shower gels. However, Ave has poor water solubility; its solubility in water at room temperature is less than 30 μg / mL. This limits its application in water-based products and affects its absorption efficiency on the skin, reducing product efficacy. Furthermore, Ave is pH sensitive and easily degraded.

[0003] To address the issue of Avene's poor water solubility, researchers have developed metal salts, such as potassium, zinc, magnesium, calcium, and organic salts, to improve their solubility in cosmetic formulations. However, salt forms of Avene still exhibit pH sensitivity; at high concentrations or under specific pH conditions, precipitation or separation may occur, negatively impacting formulation stability. Another approach is nanoencapsulation technology. Nanoencapsulation can encapsulate Avene within nanocarriers, making them more stable in water-based systems, less prone to precipitation, and significantly increasing absorption efficiency and bioavailability. However, traditional nanoencapsulation technologies, such as liposomes, polymer nanoparticles, and nanoemulsions, suffer from complex formulations, cumbersome processes, high production costs, low drug loading, and poor stability. Furthermore, the extensive use of surfactants may pose potential biotoxicity issues.

[0004] Co-assembly delivery technology is an emerging drug delivery method that utilizes the physicochemical properties of active ingredients to spontaneously form nanoscale structures under specific conditions, thereby achieving efficient drug delivery. Co-assembly is a molecular aggregate formed through weak non-covalent interactions such as hydrogen bonds, van der Waals forces, π-π stacking, electrostatic interactions, and coordination bonds. It does not alter the original molecular structure and therefore has minimal impact on biological activity. Compared to traditional nano-formulations, co-assembly technology is simpler and more environmentally friendly, requiring no complex preparation processes or specialized equipment. Cyclodextrin (CD) is a cyclic oligosaccharide formed by multiple glucose units linked by α-1,4 glycosidic bonds, possessing unique inclusion properties. Through inclusion, CD can improve the stability and solubility of active ingredients, reduce irritation, and exert a controlled-release effect. However, CD inclusion is subject to strict requirements on the size of the guest molecule. If the guest molecule is too large, it cannot enter the cavities of the cyclodextrin to form a stable inclusion complex. Furthermore, CD has a limited drug loading capacity. In the prior art, in order to encapsulate the drug as much as possible in the CD cavity, the amount of CD used is often much greater than the amount of drug used, resulting in a viscous system, restricted drug diffusion, insufficient encapsulation, and low yield. Summary of the Invention

[0005] To overcome the aforementioned shortcomings of existing technologies, this invention provides a soothing oat alkali-hyaluronic acid co-assembly, its preparation method, and its applications. The preparation method provided by this invention utilizes active ingredient co-assembly technology to improve the inclusion efficiency and stability of CD on Ave, and has certain practical application value.

[0006] According to a first aspect of the present invention, the present invention provides a soothing oat alkaloid-hyaluronic acid co-assembly (abbreviated as AHC); the co-assembly comprises the following components: dihydrooat alkaloid (Ave), hyaluronic acid compound (HA), and cyclodextrin compound (CD); the mass ratio of dihydrooat alkaloid, hyaluronic acid compound and cyclodextrin compound is 1:(1-5):(5-20).

[0007] In some embodiments, the mass ratio of dihydroagar alkaloids, hyaluronic acid compounds, and cyclodextrin compounds is 1:(1-2.5):(5-10).

[0008] In some embodiments, the mass ratio of dihydroaerial alkaloids, hyaluronic acid compounds, and cyclodextrin compounds is 1:1:5.

[0009] In some embodiments, the mass ratio of dihydroaerial alkaloids, hyaluronic acid compounds, and cyclodextrin compounds is 1:1:10.

[0010] In some embodiments, the mass ratio of dihydroaerial alkaloids, hyaluronic acid compounds, and cyclodextrin compounds is 1:2.5:5.

[0011] Specifically, this invention addresses the solubility and stability issues of ave (Ave) through the organic combination of linear polysaccharides (HA) and cyclic polysaccharides (CD). The co-assembly of HA and ave achieves solubilization of ave, while CD inclusion enhances the system's stability. Based on the good compatibility between HA and CD, both high ave solubility and system stability are simultaneously achieved.

[0012] In some embodiments, the cyclodextrin compounds include one or more of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, γ-cyclodextrin, sulfobutyl ether-β-cyclodextrin, and hydroxypropyl-α-cyclodextrin.

[0013] In some embodiments, the cyclodextrin compound is hydroxypropyl-β-cyclodextrin.

[0014] In some embodiments, the cyclodextrin compound is methyl-β-cyclodextrin.

[0015] In some embodiments, the hyaluronic acid compound includes one or more of hyaluronic acid and acetylated hyaluronic acid;

[0016] In some embodiments, the hyaluronic acid compound is hyaluronic acid;

[0017] In some embodiments, the hyaluronic acid compound is acetylated hyaluronic acid;

[0018] In some implementations, the molecular weight of the hyaluronic acid compound is less than 1500 kDa.

[0019] In some implementations, the molecular weight of the hyaluronic acid compound is 5-50 kDa.

[0020] In some implementations, the molecular weight of the hyaluronic acid compound is 5 kDa.

[0021] In some embodiments, the molecular weight of the hyaluronic acid compound is 50 kDa.

[0022] The hyaluronic acid compound (HA) used in this invention is a linear polysaccharide with multiple effects, including moisturizing, anti-oxidation, promoting normal epidermal cell differentiation, and enhancing skin barrier function. In addition to the above-mentioned biological effects, hyaluronic acid compounds can also form nanoparticles or gels through co-assembly technology, thereby stabilizing, solubilizing, and sustaining the release of other active ingredients. Therefore, this invention selects hyaluronic acid compounds with a molecular weight of less than 1500 kDa.

[0023] According to a second aspect of the present invention, the present invention provides a method for preparing a soothing oat base-hyaluronic acid co-assembly, the method comprising the following steps:

[0024] Dihydroagar alkaloids were added to a solution of hyaluronic acid compounds and mixed (stirred until completely dissolved) to obtain a primary mixture. Cyclodextrin compounds were added to the primary mixture and mixed to obtain a secondary mixture. The mixture was homogenized, centrifuged to obtain the supernatant, filtered to obtain the filtrate (to remove insoluble matter), and dried to obtain a soothing agar alkaloid-hyaluronic acid co-assembly.

[0025] Free hyaluronic acid (Ave) is strongly hydrophobic, allowing it to enter the hyaluronic acid crystal (CD) via a folded conformation. This results in significant steric hindrance, weak inclusion (-0.93 kcal / mol), and system instability. However, the preparation method provided in this invention utilizes a strong interaction between hyaluronic acid (HA) and Ave, leading to a synergistic effect without altering Ave activity. On one hand, co-assembly increases the water solubility of Ave, while hyaluronic acid compounds help retain moisture and promote skin metabolism. On the other hand, Ave enters the cavities of the CD linearly, resulting in stronger inclusion (-13.50 kcal / mol) and greater system stability. Therefore, through the organic combination of HA co-assembly and CD inclusion, the water solubility of Ave is increased by more than 1200 times compared to its free state.

[0026] In some embodiments, the preparation of a hyaluronic acid compound solution includes the following steps: adding a hyaluronic acid compound to an alcohol solution, stirring to induce swelling, and heating and stirring to ensure that the hyaluronic acid compound (HA) is fully dissolved, thereby obtaining a hyaluronic acid compound solution (HA solution).

[0027] In some embodiments, the alcohol solution is a mixture of an alcohol compound and water, wherein the mass percentage of the alcohol compound is 10-15%; the alcohol compound is ethanol or isopropanol.

[0028] In some embodiments, the alcohol solution is a mixture of an alcohol compound and deionized water;

[0029] In some embodiments, the alcohol solution is a mixture of ethanol and deionized water;

[0030] In some embodiments, the alcoholic compound in the alcohol solution is 10% by mass;

[0031] In some embodiments, the alcoholic compound in the alcohol solution is 15% by mass;

[0032] In some embodiments, the mass ratio of hyaluronic acid compound to alcohol solution is 1:20-100;

[0033] In some embodiments, the mass ratio of the hyaluronic acid compound to the alcohol solution is 5:100;

[0034] In some embodiments, the mass ratio of hyaluronic acid compound to alcohol solution is 20:500.

[0035] In some embodiments, the stirring swelling time is 0.5-1 h, and the stirring swelling rate is 300-600 rpm;

[0036] In some implementations, the stirring and swelling time is 0.5 hours.

[0037] In some implementations, the stirring and swelling time is 1 hour.

[0038] In some embodiments, the stirring temperature is 60-80°C, the stirring time is 1-2 hours, and the stirring rate is 300-600 rpm.

[0039] In some embodiments, the stirring temperature is 60°C and the stirring time is 2 hours.

[0040] In some embodiments, the stirring temperature is 60°C and the stirring time is 1 hour.

[0041] In some embodiments, adding dihydroagar alkaloids to a hyaluronic acid compound solution includes: heating the hyaluronic acid compound solution to 60-80°C, and adding the dihydroagar alkaloids to the hyaluronic acid compound solution in 3-5 portions under constant temperature conditions of 60-80°C.

[0042] In some embodiments, adding dihydroagar alkaloids to a hyaluronic acid compound solution includes: heating the hyaluronic acid compound solution to 60°C, and adding dihydroagar alkaloids to the hyaluronic acid compound solution in four portions at a constant temperature of 60°C.

[0043] In some embodiments, adding dihydroagar alkaloids to a hyaluronic acid compound solution includes: heating the hyaluronic acid compound solution to 80°C, and adding dihydroagar alkaloids to the hyaluronic acid compound solution in 5 portions at a constant temperature of 80°C.

[0044] In some embodiments, adding cyclodextrin compounds to the primary mixture includes the following steps: heating the primary mixture to 60-80°C, adding the cyclodextrin compounds in 3-5 portions at a constant temperature of 60-80°C, and stirring until completely dissolved.

[0045] In some embodiments, adding cyclodextrin compounds to the primary mixture includes the following steps: heating the primary mixture to 60°C, adding the cyclodextrin compounds in four portions at a constant temperature of 60°C, and stirring until completely dissolved.

[0046] In some embodiments, adding cyclodextrin compounds to the primary mixture includes the following steps: heating the primary mixture to 80°C, adding the cyclodextrin compounds in five portions at a constant temperature of 80°C, and stirring until completely dissolved.

[0047] In some embodiments, the homogenization process includes ultrasonic homogenization, high-pressure homogenization, and high-shear emulsification homogenization.

[0048] Specifically, this invention improves assembly efficiency and uniformity through homogenization treatment.

[0049] In some embodiments, the ultrasonic homogenization treatment temperature is 50-70°C, the ultrasonic homogenization treatment frequency is 20-50kHz, and the ultrasonic homogenization treatment time is 0.5-2 hours; if the ultrasonic homogenization treatment time is insufficient (less than 0.5 hours), some Ave will precipitate after cooling.

[0050] In some embodiments, the ultrasonic homogenization treatment is performed at a temperature of 60°C for 2 hours.

[0051] In some implementations, the high-pressure homogenization process is carried out at a temperature of 50-70°C, at a pressure of 15,000-30,000 psi, and for 1-5 cycles.

[0052] In some implementations, the high-pressure homogenization process is carried out at a temperature of 60°C, at a pressure of 30,000 psi, and for 3 cycles.

[0053] In some embodiments, the high-shear emulsification homogenization treatment is carried out at a temperature of 50-70°C, at a rotation speed of 5000-10000 rpm, and for a time of 3-10 minutes.

[0054] In some embodiments, the centrifugation speed for obtaining the supernatant is 3000-10000 rpm, and the centrifugation time is 10-15 minutes;

[0055] In some implementations, the centrifugation speed for obtaining the supernatant is 3000 rpm.

[0056] In some embodiments, the mixture is homogenized, cooled to room temperature, and then centrifuged.

[0057] In some implementations, the filtration method is vacuum filtration.

[0058] In some implementations, the filter membrane used for filtration has a pore size of 0.45 μm.

[0059] In some embodiments, the filtrate is first subjected to rotary evaporation until it becomes a paste before drying.

[0060] In some implementations, the drying method is vacuum drying.

[0061] In some implementations, the drying method is freeze drying.

[0062] In some embodiments, after the filtrate is dried, it is ground into powder using a pulverizer and then stored in a sealed container in a cool, dry place. Drying and pulverizing are for the convenience of storage and transportation.

[0063] According to a third aspect of the present invention, the application of the soothing oat base-hyaluronic acid co-assembly provided by the present invention in the preparation of topical pharmaceuticals or cosmetics.

[0064] The soothing oat alkaloid-hyaluronic acid co-assembly (AHC) provided by this invention exhibits excellent regulatory performance throughout the entire anti-inflammatory pathway network. Its inhibitory effects on upstream inflammatory mediators (TNF-α), midstream inflammatory mediators (NO), and downstream inflammatory mediators (PGE2) are significantly higher than those of free Ave and HA. Furthermore, AHC's mast cell degranulation inhibition rate is also significantly higher than that of free Ave and HA, demonstrating excellent anti-allergic effects. This makes AHC significantly superior to free Ave and HA in inhibiting UV-induced apoptosis, showing good potential for anti-photoaging applications. Therefore, the soothing oat alkaloid-hyaluronic acid co-assembly provided by this invention can be used in the preparation of topical pharmaceuticals or cosmetics.

[0065] Compared with the prior art, the present invention has the following beneficial effects and advantages:

[0066] (1) The AHC preparation process of the present invention is simple, practical, free of surfactants, and highly safe. The components in the system work synergistically and exhibit better biological efficacy and stability than single-component or binary assembly systems.

[0067] (2) The soothing oat alkali-hyaluronic acid co-assembly (AHC) provided by this invention, through the organic combination of HA co-assembly and CD inclusion complexation, increases the water solubility of Ave by more than 1200 times compared to its free state. Furthermore, the synergistic effect of Ave and HA makes the anti-inflammatory, anti-allergic, and anti-photoaging effects of AHC significantly superior to equimolar amounts of free Ave and HA. In addition, compared to commercially available similar products like Deminshu, the AHC of this invention exhibits lower cytotoxicity, better safety and stability when applied to skincare products, and can fully meet the product requirements of mitochondrial energy skincare; compared to precursor mixtures, its anti-redness and anti-itch effects are significantly improved.

[0068] (3) Currently, commercially available skincare products like Derma-Soo enhance the water solubility of Ave (Ave) through a co-solvent method. However, the co-solvent method has significant drawbacks. Specifically, when the solvent environment changes, Ave is easily precipitated, resulting in poor formula adaptability. For example, when using deionized water to prepare a Derma-Soo diluted solution with an Ave content of 1-2 wt%, obvious solid precipitation occurs. Figure 1 As shown. In contrast, the AHC provided in this embodiment of the invention exhibits excellent stability through a co-assembly inclusion method, showing no precipitation below the saturation concentration, and has strong formulation applicability, effectively avoiding precipitation problems caused by changes in the solvent environment, thereby significantly improving the stability and reliability of the formulation. Attached Figure Description

[0069] Figure 1 Photographs of the AHC co-assembled inclusion complex (soothing oat alkali-hyaluronic acid co-assembled complex) prepared in Example 1 of this invention, and oat alkali (Ave) and solutions of commercially available skin care product Deminshu at different concentrations.

[0070] Figure 2 SEM images of the Ave-HA co-assembly prepared in Comparative Example 1 and the AHC co-assembly inclusion complex (soothing oat base-hyaluronic acid co-assembly) prepared in Example 1; Figure 2 Part a is the SEM image of Comparative Example 1. Figure 2 Part b is the SEM image of Example 1;

[0071] Figure 3 The infrared spectra of the AHC co-assembled inclusion complex (soothing oat base-hyaluronic acid co-assembled complex), Ave, HA, CD, and the mixture of Ave, HA, CD prepared in Example 1 are shown.

[0072] Figure 4 TEM images of the Ave-HA co-assembly prepared in Comparative Example 1 and the AHC co-assembly inclusion complex (soothing oat base-hyaluronic acid co-assembly) prepared in Example 1; Figure 4 Part a is the TEM image of Comparative Example 1. Figure 4Part b is a TEM image of Example 1;

[0073] Figure 5 This is a diagram showing the results of simulating the co-assembly process of Ave-HA using Gromacs software in Example 3;

[0074] Figure 6 shows the results of analyzing the interaction between Ave and HA in the co-assembly using Gauss software in Example 3; where Figure 6a This is a schematic diagram of the interaction forces between Ave and HA in the Ave-HA co-assembly, where ρBCP represents the electron density at the bond critical point (BCP). Figure 6b This is a discrete point diagram showing the interaction forces between Ave and HA in the Ave-HA co-assembly.

[0075] Figure 7 The figure shows the ESP analysis results of the Ave and Ave-HA assemblies in Example 3; Figure 7 Part a is the ESP analysis results of Ave; Figure 7 Part b is the ESP analysis results of the Ave-HA assembly;

[0076] Figure 8 The graph shows the molecular dynamics simulation results of the inclusion effect of CD on Ave under different conditions in Example 4. Figure 8 Part a is a simulation diagram of the encapsulation effect of CD on free Ave; Figure 8 Part b is a structural simulation diagram of the AHC co-assembled inclusion complex (soothing oat base-hyaluronic acid co-assembled complex); Figure 8 Part c is a schematic diagram of the intermolecular interactions in the AHC co-assembled inclusion complex (soothing oat base-hyaluronic acid co-assembled complex);

[0077] Figure 9 The image shows the CCK-8 cytotoxicity test results in Test Example 1;

[0078] Figure 10 The image shows the TNF-α detection results in Test Example 2;

[0079] Figure 11 The graph shows the comparison of NO inhibition rates between AHC and equimolar amounts of Ave and HA in Test Example 3.

[0080] Figure 12 The figure shows the comparison results of the inhibition of PGE2 gene expression by AHC and equimolar amounts of Ave and HA in test example 4.

[0081] Figure 13 The figure shows the comparison of mast cell degranulation rates between AHC and equimolar amounts of Ave and HA in test example 5.

[0082] Figure 14 shows the performance comparison of AHC with equimolar amounts of Ave and HA in inhibiting UV-induced apoptosis in Test Example 6; where... Figure 14a The graph shows the apoptosis results in the NC group; Figure 14b This is a diagram showing the apoptosis results in the MC group; Figure 14c The graph shows the apoptosis results in the PC group; Figure 14d The image shows the apoptosis results in the Ave group; Figure 14e The graph shows the apoptosis results in the HA group; Figure 14f The image shows the apoptosis results in the AHC group; Figure 14g The graph shows the statistical results of apoptosis rates for each group;

[0083] Figure 15 This is a graph showing the molecular frontier orbital analysis results of the Ave and Ave-HA assemblies in Example 5; Figure 15 Part a is the analysis result diagram corresponding to Ave; Figure 15 Part b is the analysis result diagram corresponding to Ave-HA;

[0084] Figure 16 shows the results of the stability analysis in Test Example 7; Figure 16a These are photographs of the AHC solution in Test Example 7 after being stored at different temperatures for 4 weeks. Figure 16b These are photographs of the AHC powder in Test Example 7 after being stored at different temperatures for 4 weeks.

[0085] Figure 17 The graph shows the comparison of the anti-allergic effects of Deminex and AHC at the same concentration in Test Example 9. Detailed Implementation

[0086] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. It should be noted that any processes or parameters not specifically described in detail below are those that can be understood or implemented by those skilled in the art with reference to the prior art.

[0087] The weight (mass) parts used in the following examples and comparative examples are for illustrative purposes only. The weight unit can be grams, kilograms, or any other amount commonly used in the art.

[0088] Example 1: A method for preparing AHC co-assembled inclusion complex (i.e., soothing oat alkali-hyaluronic acid co-assembly, hereinafter the same), comprising the following steps:

[0089] (1) Add 20g of hyaluronic acid compound (HA, hyaluronic acid is used here) with a molecular weight of 5kDa to a mixed solution of 500g of deionized water and ethanol (ethanol mass percentage is 10%), stir at room temperature to swell for 0.5 hours at a stirring speed of 300rpm, heat to 60℃, and continue stirring for 1 hour until HA is completely dissolved (stirring speed is 300rpm) to obtain HA solution.

[0090] (2) Heat the HA solution to 80℃. Under constant temperature of 80℃, add 20g of Ave (dihydroagar alkaloid) in small amounts (4g each time, 5 times) and stir until completely dissolved. Under constant temperature of 80℃, add 100g of cyclodextrin compound (CD) in small amounts (20g each time, 5 times) and stir until completely dissolved. Cool to 60℃ and use a high-pressure microfluidic homogenizer at 30000psi for 3 cycles. Centrifuge at 3000rpm for 10 minutes. Take the supernatant and filter it (the filter membrane used for filtration has a pore size of 0.45μm). Collect the filtrate, suspend it until it becomes a paste, and then vacuum dry it. Grind it into powder using a pulverizer and store it in a cool, dry place in a sealed container to obtain the soothing agar alkaloid-hyaluronic acid co-assembly (i.e., AHC co-assembly inclusion complex, abbreviated as AHC).

[0091] Comparative Example 1: Preparation method of Ave-HA co-assembly

[0092] Same as Example 1, except that hydroxypropyl-β-cyclodextrin (HP-β-CD) is not added in step (2).

[0093] SEM Analysis: In the preparation method of the AHC co-assembled inclusion complex provided in Example 1, step (2) involves drying and grinding into powder using a pulverizer for convenient storage and transportation. To verify that the drying and pulverizing process does not damage the microstructure of the AHC co-assembled inclusion complex, SEM analysis was performed on it. Figure 2 The SEM images showed significant differences in particle morphology between Ave and HA, while the AHC co-assembled inclusion complexes after drying and pulverizing had uniform morphology and texture, although the particle sizes were different. This indicates that the drying and pulverizing process did not destroy the uniform co-assembled structure of Ave, HA and CD.

[0094] Infrared spectroscopy detection: Infrared spectroscopy was performed on the AHC co-assembled inclusion complex, Ave (dihydroagar alkaloid), HA (hyaluronic acid), CD (hydroxypropyl-β-cyclodextrin), and a mixture of Ave, HA, and CD (i.e., a solid mixture of Ave, HA, and CD, with the same type and proportion of Ave, HA, and CD as in Example 1). The results are as follows: Figure 3As shown, Figure 3 In this context, Ave+HA+CD represents a mixture of Ave, HA, and CD, and AHC represents the AHC co-assembled inclusion complex prepared in Example 1. Figure 3 It can be seen that, compared with the infrared spectrum results of AHC, the peaks of the mixture of Ave, HA, and CD are more disordered, which is a result of simple stacking of the peaks of the three substances. In the infrared spectrum results of AHC, Ave and HA are in the 1600-1700 cm⁻¹ range. -1 The weakening and shift of the nearby C=O stretching vibration peaks to lower wavenumbers indicates that Ave and HA participate in strong hydrogen bonding in the assembly. Meanwhile, the AHC co-assembled inclusion complex prepared in Example 1 showed peak values ​​at 3267.28 and 2936.98 cm⁻¹. -1 The presence of two strong peaks at the position corresponds to the stretching vibrations of OH and CH, respectively. The two peaks partially overlap and there are no other impurity peaks, indicating that oat base, hyaluronic acid, and cyclodextrin form a uniform co-assembled inclusion complex through strong intermolecular interactions.

[0095] Ave solubility analysis: Solubility experiments revealed that the solubility of free Ave in water is less than 30 μg / mL, while the solubility of AHC provided in Example 1 in water is 25 wt%, meaning that...

[0096] Ave has a solubility of 3.61 wt%, which is more than 1200 times higher than its water solubility.

[0097] Example 2: A method for preparing AHC co-assembled inclusion complex (soothing oat base-hyaluronic acid co-assembly), comprising the following steps:

[0098] (1) Add 5g of HA hyaluronic acid compound with a molecular weight of 5kDa (HA, hyaluronic acid is selected here) to 100mL of a mixed solution of deionized water and ethanol (ethanol volume percentage is 15%), stir at room temperature to swell for 1 hour at a stirring rate of 300rpm, heat to 60℃, and continue stirring for 2 hours until HA is completely dissolved (stirring rate is 300rpm) to obtain HA solution.

[0099] (2) Under constant temperature of 60℃, add 2g Ave in small amounts (0.5g each time in 4 additions) and stir until completely dissolved. Add 10g cyclodextrin compound (CD) in small amounts (2.5g each time in 4 additions) and stir until completely dissolved. After sonicating at 60℃ for 2 hours, cool to room temperature (ultrasonic frequency of 50kHz). Centrifuge at 3000rpm for 15 minutes. Take the supernatant and filter (the filter membrane used for filtration has a pore size of 0.45μm). Collect the filtrate, suspend it until it becomes a paste, and then vacuum dry it. Grind it into powder using a pulverizer and store it in a cool, dry place in a sealed container.

[0100] Comparative Example 2: Preparation method of Ave-HA co-assembly

[0101] Same as Example 2, except that methyl-β-cyclodextrin (M-β-CD) is not added in step (2).

[0102] In the preparation method provided by this invention, the inclusion of cyclodextrin compounds (CD) is to improve the stability and uniformity of the Ave-HA co-assemblies. Dynamic light scattering (DLS) was used to analyze the particle size distribution of the products prepared in Examples 1, 2, Comparative Example 1, and Comparative Example 2. The particle size distribution results of the Ave-HA co-assemblies prepared in Comparative Example 1 and Comparative Example 2, and the AHC co-assemblies prepared in Examples 1 and 2 are shown in Table 1 below. The results show that compared with the Ave-HA co-assemblies, the average particle size and polydispersity index (PDI) of the AHC co-assemblies are significantly reduced, indicating that the inclusion effect of CD significantly reduces the particle size of the co-assemblies and makes them more uniform. Figure 4 TEM images of the Ave-HA co-assembly prepared in Comparative Example 1 and the AHC co-assembly inclusion complex prepared in Example 1; as shown Figure 4 As shown, the nanoparticles of the AHC co-assembled inclusion complex prepared in Example 1 have a uniform shadow distribution, indicating that Ave, HA and CD are uniformly co-assembled and do not form a core-shell structure.

[0103] Table 1 Particle size distribution of the samples

[0104]

[0105] In addition, as shown in Table 1, compared with Comparative Example 1 and Example 1, which used a high-pressure microfluidic homogenizer for homogenization in step (2) and selected hydroxypropyl-β-cyclodextrin (HP-β-CD), the Ave-HA co-assemblies and AHC co-assemblies prepared by Comparative Example 2 and Example 2, which used ultrasonic homogenization in step (2) and selected methyl-β-cyclodextrin (M-β-CD), have larger particle sizes and wider particle size distributions.

[0106] Meanwhile, to screen for better processing conditions, this invention also conducted a series of experiments on the preparation method of AHC co-assembled inclusion complexes. The processing conditions for each group are shown in Table 2 below. The remaining processing conditions are the same as in Example 1, and can be referred to Example 1. The amount of each substance in Table 2 is in parts by mass (for example, 1 part by mass here corresponds to 20g). The temperature in Table 2 is the stirring and dissolution temperature after HA is added to the alcohol solution and swollen, and is also the temperature when Ave and CD are added. In experiments 1-6 of Table 2, hydroxypropyl-β-cyclodextrin was used as the cyclodextrin compound, while in experiments 7-12, methyl-β-cyclodextrin was used. In experiments 1-11 of Table 2, hyaluronic acid was used as the hyaluronic acid compound; in experiment 12, acetylated hyaluronic acid was used. In experiments 1-8 of Table 2, the molecular weight of hyaluronic acid was 5kDa, while in experiments 9-10, the molecular weight of hyaluronic acid was 50kDa. In the 11 experiments in Table 2, the molecular weight of hyaluronic acid was 1500 kDa. In the 12 experiments in Table 2, the molecular weight of acetylated hyaluronic acid was 1000 kDa.

[0107] Table 2

[0108]

[0109]

[0110]

[0111] As shown in Table 2, comparing experiments 1 and 2, when the number of cycles of high-pressure homogenization decreased from 3 to 2, the inclusion effect worsened, leading to a decrease in the solubility of Ave in the product; therefore, the optimal number of cycles for high-pressure homogenization is 3. Comparing experiments 1 and 3, when the temperature was adjusted from 60℃ to 50℃, the reactants could not be completely dissolved. Comparing experiments 1 and 4, when only water was added as the solvent (without ethanol), Ave could not be completely dissolved. Comparing experiments 1 and 5, when the hyaluronic acid ligand content was too high, the solubility of Ave in the product decreased. Comparing experiments 1 and 6, when the high-pressure homogenization was changed to ultrasonic homogenization, the inclusion effect worsened. The following experiments showed that when the cyclodextrin compound was changed from hydroxypropyl-β-cyclodextrin to methyl-β-cyclodextrin and the high-pressure homogenization treatment was changed to ultrasonic homogenization treatment, the inclusion effect deteriorated, leading to a decrease in the solubility of Ave in the product. Comparing experiments 7 and 8, when the content of cyclodextrin compounds was too high, they could not be completely dissolved. Comparing experiments 7, 9, 11, and 12, when the molecular weight of the ligand hyaluronic acid compound increased, the assembly performance decreased, leading to a decrease in the solubility of Ave in the product. Comparing experiments 9 and 10, when the ultrasonic time was too long, it was detrimental to the stability of the inclusion effect, leading to a decrease in the solubility of Ave in the product.

[0112] Example 3: Study on the formation mechanism of Ave-HA co-assemblies

[0113] Based on molecular dynamics, the co-assembly process of Ave (dihydrooat alkaloids) and HA (hyaluronic acid) in a 1:1 molar ratio was simulated using Gromacs software. Figure 5 As the simulation time increases, Ave and HA gradually aggregate from their initial free and dispersed state, the number of hydrogen bonds gradually increases, and the system potential energy gradually decreases, indicating that Ave and HA have excellent co-assembly properties. During the assembly process, Ave, due to its strong hydrophobicity, gradually aggregates towards the center, while HA accumulates around Ave, indicating that HA can improve the stability and solubility of Ave in water.

[0114] Based on density functional theory, the interaction between Ave and HA in the co-assembly was analyzed using Gauss software (Figure 6). Through the independent gradient model (IGMH) based on Hirshfeld segmentation and the atomic topology analysis (AIM) method in the molecule, van der Waals interactions and hydrogen bonding interactions exist between Ave and HA. Among these, there are multiple strong hydrogen bonds between the carboxyl groups of Ave and HA, enabling Ave and HA to form a stable co-assembly. Surface electrostatic potential (ESP) analysis (…) Figure 7The results show that Ave forms a folded conformation due to the π-π interactions between the two benzene rings, with the surface electrostatic potential mainly concentrated near 0, consistent with its strong hydrophobicity. In the Ave-HA assembly, HA does not change the folded conformation of Ave, but due to the strong interaction between Ave and HA, the surface electrostatic potential of the assembly is enhanced, indicating better water solubility. The molecular polarity index (MPI) of the Ave-HA assembly, calculated by ESP analysis, is 0.88 eV, which is much larger than the 0.62 eV of free Ave, further proving that the water solubility of the assembly is superior to that of pure Ave.

[0115] Example 4: Study on the formation mechanism of AHC co-assembled inclusion complexes

[0116] To further verify the formation mechanism of AHC co-assembled inclusion complexes, molecular dynamics simulations were used to explore the optimal conformation. DFT calculations were then combined to analyze the inclusion effect of CD (hydroxypropyl-β-cyclodextrin) on free Ave (dihydroagar alkaloids) and Ave in the AHC co-assembled inclusion complex (preparation method as described in Example 1) (results are shown below). Figure 8 (As shown). From Figure 8 As seen in part a, free Ave enters CD in a folded conformation, experiencing significant steric hindrance. Their interaction energy is only -0.93 kcal / mol, indicating weak inclusion. From... Figure 8 As seen in part b, in the AHC co-assembled inclusion complex, Ave, HA, and CD co-assembled to form a multidimensional hierarchical structure. Benefiting from the high water solubility of the Ave-HA co-assembled complex, Ave entered CD in a linear conformation, with an interaction energy of -13.50 kcal / mol, significantly enhancing the inclusion effect. IGMH (such as...) Figure 8 As shown in section c) and AIM analysis, abundant van der Waals interactions exist between Ave and CD, which is highly consistent with the hydrophobic inclusion effect of CD on Ave. In the outer region of the cyclodextrin cavity, the dark blue area shows...

[0117] The strong hydrogen bond interaction between Ave and CD helps to enhance the stability of CD's inclusion of Ave.

[0118] Test Example 1: CCK-8 Cytotoxicity Test

[0119] The following CCK-8 cytotoxicity assays were performed according to the CCK-8 kit instructions. The cells used were HaCaT cells and Raw 264.7 cells. HaCaT (human immortalized epidermal cells) were purchased from Mirror Image (Shanghai) Cell Technology Co., Ltd., and RAW 264.7 (mouse mononuclear macrophage leukemia cells) were purchased from Wuhan Pronosei Life Sciences Co., Ltd. The CCK-8 kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number C0043. For the cell cultures described below, HaCaT cells and Raw 264.7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution.

[0120] A. Experimental Procedure

[0121] 1) After reviving the cells, passage them twice to expand the culture and obtain expanded cultured cells;

[0122] 2) After the expanded culture, the cells were digested, centrifuged, resuspended, counted, and then seeded into 96-well cell culture plates, with 10,000 cells in each well.

[0123] 3) After the cells have completely adhered to the culture medium, remove the medium containing a gradient of drug (the drug here is the AHC co-assembled inclusion complex prepared in Example 1) and add it to each of the 96 wells. The volume of drug added to each well is 100 μl. Three parallel wells are set up for each concentration.

[0124] 4) Transfer the culture plate to an incubator (conditions set to 37℃, 5% CO2) and continue incubation for 48 hours.

[0125] 5) Add 10 μl of CCK-8 solution to each well, and place the culture plate in an incubator to continue incubation for 1.5 h;

[0126] 6) Remove the culture plate and read the OD value at 450nm using an ELISA reader.

[0127] B. Calculate the inhibition rate

[0128] The inhibition rate of AHC at each concentration was calculated using the following formula.

[0129] Inhibition rate = 1 - (OD of sample group - OD of blank group) / (OD of control group - OD of blank group);

[0130] Sample group OD: OD values ​​of wells containing cells, CCK-8 solution, and drug solution;

[0131] Blank group OD: OD value of wells without cells and containing CCK-8 solution;

[0132] Control group OD: OD value of the wells containing cells and CCK-8 solution.

[0133] C. Analysis software

[0134] Statistical analysis was performed using GraphPad.Prism.10.1 software, and curve fitting was performed using Log(agonist) vs. response—Variable slope(four parameters).

[0135] The cytotoxicity of AHC co-assembled inclusion complexes was tested using a CCK-8 assay kit and compared with that of commercially available similar product, Deminex. Figure 9 Under a range of Ave concentrations, AHC showed significantly lower cytotoxicity to HaCaT cells compared to Deminex. Furthermore, at an Ave concentration of 500 μg / mL, AHC exhibited almost no cytotoxicity to Raw 264.7 cells, while at Ave concentrations ≥250 μg / mL, the survival rate of Raw 264.7 cells with Deminex was close to 0. This indicates that AHC possesses higher biocompatibility and lower skin irritation compared to Deminex.

[0136] Test Example 2: TNF-α Detection

[0137] Experimental Principle

[0138] Ave (Ave) has been proven to have anti-inflammatory and soothing effects. Comparing the biological efficacy of AHC co-assembled inclusion complexes and free Ave helps to analyze the impact of co-assembly and inclusion behavior on Ave bioactivity. TNF-α is a key pro-inflammatory cytokine, typically secreted by activated immune cells (such as macrophages and monocytes) in the early stages of an inflammatory response. Its production mainly regulates TNF-α gene expression by activating transcription factors such as NF-κB. TNF-α plays an upstream regulatory role in the inflammatory response, activating multiple downstream signaling pathways and inducing the production of other inflammatory mediators (such as NO and PGE2), thereby amplifying the inflammatory response. Ultraviolet (UV) radiation is one of the key factors leading to skin inflammation and sensitivity. Both UVA (long-wave ultraviolet radiation, wavelength 320-400 nm) and UVB (medium-wave ultraviolet radiation, wavelength 280-315 nm) can penetrate the skin, triggering oxidative stress and inflammatory responses. UVB radiation can induce skin cell DNA damage, activate inflammatory signaling pathways, leading to symptoms such as erythema, edema, and pain; while UVA can further exacerbate inflammation. The synergistic effect of these two agents can activate inflammatory signaling pathways such as NF-κB, AP-1, and MAPK, promoting the large-scale expression and release of pro-inflammatory cytokines (such as TNF-α), triggering post-sunburning "burning" and "stirring" sensations. Ave has significant anti-inflammatory and soothing effects, inhibiting the activation of inflammatory signaling pathways and reducing the release of pro-inflammatory factors such as TNF-α. Therefore, this experiment used human immortalized epidermal cells (HaCaT) as model cells, and compared the TNF-α gene expression levels between the irradiation + control group and the irradiation + sample group through synergistic stimulation with UVA and UVB to evaluate the inhibitory effect of the samples on UV-induced inflammation. The lower the TNF-α gene expression, the stronger the anti-inflammatory and soothing effect of the sample.

[0139] The following experiments were conducted using HaCaT cells, which were purchased from Mirror Image (Shanghai) Cell Technology Co., Ltd. In the cell culture described below, HaCaT cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (culture conditions set at 37℃, 5% CO2).

[0140] A. Experimental Procedure

[0141] 1) After reviving the cells, passage them twice to expand the culture and obtain expanded cultured cells;

[0142] 2) After digesting, centrifuging, resuspending, and counting the expanded-cultured cells, seed them into 24-well cell culture plates, placing 2 × 10⁶ cells per well. 5 Each cell.

[0143] 3) Experimental grouping: After plating, the cells were divided into a negative control group (NC), a positive control group (PC), a model group (MC), and a sample group (specified drug concentration), as shown in Table 3. The sample groups were the HA group, the AHC group from Example 1, and the Ave group.

[0144] After the cells had fully adhered to the culture medium, the culture medium was aspirated, PBS buffer was added, and the cells were irradiated with a combination of UVB and UVA. The NC group was covered with aluminum foil. After irradiation for 30 minutes, the diluted drug was added to each well (500 μl). The drug concentrations for each group are shown in Table 3.

[0145] Table 3. Experimental Groups

[0146]

[0147] 4) Place the cells in an incubator (conditions set to 37℃, 5% CO2) and continue culturing for 24 hours.

[0148] 5) Wash three times with PBS buffer, add 1 mL of RNA extraction solution, pre-cool on ice for 5 min, and gently pipette.

[0149] 6) Add 400 μl of chloroform to each well, invert the centrifuge tube for 15 seconds, mix thoroughly, and let stand for 3 minutes.

[0150] 7) Centrifuge at 4℃ and 12000rpm for 10min and collect the supernatant.

[0151] 8) Transfer 400 μl of supernatant to a new centrifuge tube, add 550 μl of isopropanol, and mix by inverting.

[0152] 9) Place at a temperature of -20℃ for 15 minutes.

[0153] 10) Centrifuge at 12000 rpm for 10 min at 4℃. The white precipitate at the bottom of the tube is RNA.

[0154] 11) Remove the liquid and add 1.5 mL of 75% ethanol (by volume percentage) to wash the precipitate.

[0155] 12) Centrifuge at 4℃ and 12000rpm for 5min.

[0156] 13) Remove all liquid and place the centrifuge tube on a clean bench and blow it for 3 minutes.

[0157] 14) Add 15 μl of Water Nuclease-Free to dissolve the RNA.

[0158] 15) Use Nanodrop 2000 to detect RNA concentration and purity: After zeroing the instrument blank, take 2.5 μl of the RNA solution to be tested on the detection base, lower the sample arm, and use the software on the computer to start the absorbance detection.

[0159] The excessively high concentration of RNA was diluted appropriately to achieve a final concentration of 200 ng / μl.

[0160] The following reversal rates were determined using a kit, specifically the G3320-01 kit (2×SYBR Green qPCR Master Mix (None ROX)) manufactured by Wuhan Saiwei Biotechnology Co., Ltd.

[0161] Prepare the reverse transcription reaction system (refer to the kit instructions), mix well and centrifuge. Set the reverse transcription program and complete the reverse transcription on a standard PCR instrument. Take 0.1 mL of PCR reaction plate and prepare the following reaction system, with 3 tubes for each reverse transcription product. After spotting, seal the plate with PCR sealing film using a sealing device. Perform PCR amplification on a real-time PCR instrument. Read the Ct value from the real-time PCR instrument. The Ct value, or Cycle Threshold, represents the number of amplification cycles required for the fluorescence signal of the amplified product to reach the set fluorescence threshold.

[0162] The primers used in PCR amplification are shown below. GAPDH is an internal reference gene.

[0163]

[0164] B. Calculation Formula

[0165] 1) RNA expression level

[0166] ΔC(t)=C(t) 目的基因 -C(t) 内参基因

[0167] ΔΔC(t)=ΔC(t) 样品组 -ΔC(t) 对照组

[0168] relative RNA expression level = 2 ΔΔC(t)

[0169] 2) Rate of change detection

[0170]

[0171] Experimental results are as follows Figure 10As shown in the figure, the TNF-α gene expression level in the MC group was significantly higher than that in the NC and PC groups, indicating successful modeling. The TNF-α gene expression levels in the Ave and HA groups were significantly lower than those in the MC group, indicating that both Ave and HA have anti-inflammatory effects. The TNF-α gene expression level in the AHC group was significantly lower than that in equimolar amounts of free Ave and free HA, indicating that Ave and HA play a synergistic role in regulating the inflammatory cascade response in the AHC co-assembled inclusion complex.

[0172] Test Example 3: NO Release Inhibition Rate Detection

[0173] Experimental Principle

[0174] Nitric oxide (NO) is a key mediator in the inflammatory response, produced by inducible nitric oxide synthase (iNOS). iNOS expression is typically induced by upstream cytokines such as TNF-α. TNF-α activation of the NF-κB signaling pathway promotes iNOS gene transcription, thereby increasing NO production. NO plays a crucial role in the early stages of inflammation, primarily by regulating vasodilation and intercellular signaling, further influencing the activation of inflammatory cells and the release of inflammatory mediators. Detecting NO levels can reflect the activation state and oxidative stress level of inflammatory cells. Lipopolysaccharides (LPS) are a major component of the outer membrane of Gram-negative bacteria. Activating the Toll-like receptor 4 (TLR4) signaling pathway induces inflammation, leading to the release of various key pro-inflammatory factors from activated macrophages. This experiment used an in vitro cell model to simulate the inflammatory response of human skin after stimulation or sensitization. The mouse macrophage cell line (RAW 264.7) was selected as the model cell, and LPS was used to simulate the inflammatory stimulus. The inhibitory effect of the sample on the LPS-induced inflammatory response was evaluated by detecting the level of the inflammatory mediator NO in the cell supernatant. The higher the inhibition rate, the stronger the anti-inflammatory and soothing effect of the sample.

[0175] The following experiments were conducted using RAW 264.7 cells, which were purchased from Wuhan Pronosei Life Sciences Co., Ltd. For all cell cultures described below, RAW 264.7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (culture conditions set at 37°C and 5% CO2).

[0176] A. Experimental Procedure

[0177] 1) After reviving the cells, passage them twice to expand the culture and obtain expanded cultured cells;

[0178] 2) After expanding the culture, digest, centrifuge, resuspend, count, and seed the cells into 24-well cell culture plates, adding 2×10⁶ cells per well.5 Each cell.

[0179] 3) Experimental grouping: After plating, the cells were divided into a negative control group (NC), a positive control group (PC), a model control group (MC), and a sample group (specified drug concentration). The grouping is shown in Table 4, where the sample groups are the HA group, the AHC group of Example 1, and the Ave group.

[0180] Table 4. Experimental Groups

[0181]

[0182] 4) After the cells have completely adhered to the well, remove the culture medium and add 500 μl of the diluted drug-containing culture medium to each of the 24 wells. The drug concentrations for each group are shown in Table 2.

[0183] 5) After placing the cells in an incubator and culturing for 24 hours, replace the culture medium with a drug-free medium and incubate for another 24 hours.

[0184] 6) Centrifuge to collect the supernatant. If precipitation occurs during storage, centrifuge again to obtain the sample.

[0185] 7) Remove Griess Reagent I and II (manufactured by Shanghai Beyotime Biotechnology Co., Ltd.) from the refrigerator and allow them to return to room temperature.

[0186] 8) Dilute the standards with cell culture medium (DMEM). Set the concentrations of the standards to 0, 1, 2, 5, 10, 20, 40, 60, and 100 μM.

[0187] 9) Add the standard and sample to the 96-well plate at a rate of 50 μl / well.

[0188] 10) Add room temperature Griess Reagent I to each well at a rate of 50 μl / well.

[0189] 11) Add 50 μl of room temperature Griess Reagent II to each well.

[0190] 12) Measure the absorbance in a spectrophotometer (at a wavelength of 536 nm).

[0191] 13) Plot a standard curve and calculate the concentration of the sample.

[0192] B. Calculation Formula

[0193] 1) Use a standard sample for linear fitting (y = aX + b)

[0194] Where y represents the NaNO2 concentration of the sample (μM), and X represents the OD of the sample. 536value

[0195] 2) Formula for calculating NO release inhibition rate

[0196]

[0197] Among them, NaNO 2模型组 This represents the NaNO2 concentration in the model group. 2(样品组) This indicates the NaNO2 concentration in the sample group. 2(空白组) This indicates the NaNO2 concentration in the blank group.

[0198] Experimental results are as follows Figure 11 As shown in the figure, the NO level in the MC group was significantly higher than that in the NC and PC groups, indicating successful modeling. The NO levels in the Ave and HA groups were significantly lower than those in the MC group, indicating that both Ave and HA have anti-inflammatory effects. The NO level in the AHC group was significantly lower than that in equimolar amounts of free Ave and free HA, indicating that Ave and HA played a synergistic role in inhibiting inflammatory cell activation in the AHC co-assembled inclusion complex.

[0199] Test Example 4: PGE2 Detection

[0200] Experimental Principle

[0201] Prostaglandin PGE2 plays a crucial role in physiological and pathological processes such as inflammation, pain perception, fever, and vasodilation, and is one of the important indicators for assessing the intensity of inflammatory responses. PGE2 is an inflammatory mediator generated from arachidonic acid catalyzed by cyclooxygenase (COX), and its synthesis is regulated by multiple inflammatory signaling pathways. Upstream inflammatory mediators such as TNF-α and NO can promote PGE2 production by activating COX-2 expression. PGE2 plays a downstream role in the inflammatory response, regulating the function of inflammatory cells and the expression of inflammatory factors by acting on its receptor (EP receptor), further promoting the persistence and exacerbation of the inflammatory response. PGE2 production is generally associated with the chronicity of the inflammatory response and the tissue damage repair phase. In this experiment, mouse macrophage cell line (RAW 264.7) was used as a model cell, and LPS was used to simulate inflammatory stimulation. The level of the inflammatory mediator PGE2 in the cell supernatant was detected to evaluate the inhibitory effect of the sample on the LPS-induced inflammatory response. A higher inhibition rate indicates a stronger anti-inflammatory and soothing effect of the sample.

[0202] The following experiments were conducted using Raw 264.7 cells, which were purchased from Wuhan Pronosei Biotechnology Co., Ltd. For all cell cultures described below, Raw 264.7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (culture conditions set at 37℃, 5% CO2). The following experiments were performed using an ELISA kit. Specific procedures were performed according to the kit's instructions. The ELISA kit was manufactured by Wuhan Saipei Biotechnology Co., Ltd., catalog number SP14070.

[0203] Experimental steps

[0204] 1) After reviving the cells, passage them twice to expand the culture and obtain expanded cultured cells;

[0205] 2) After expanding the culture, digest, centrifuge, resuspend, count, and seed the cells into 24-well cell culture plates, adding 2×10⁶ cells per well. 5 Each cell.

[0206] 3) Experimental grouping: After plating, the cells were divided into a negative control group (NC), a positive control group (PC), a model control group (MC), and a sample group (specified drug concentration), as shown in Table 5. The sample groups were the HA group, the AHC group from Example 1, and the Ave group.

[0207] Table 5. Experimental Groups

[0208]

[0209] 4) After the cells have completely adhered to the wall, remove the culture medium and add 500 μl of culture medium containing diluted drug to each of the 24 wells.

[0210] 5) After placing the cells in an incubator and culturing for 24 hours, replace the culture medium with a drug-free medium and incubate for another 24 hours.

[0211] 6) Collect the supernatant by centrifugation. If precipitation occurs during storage, centrifuge again.

[0212] 7) Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent; all other steps are the same) and sample wells. First, add sample diluent to the sample wells on the enzyme-labeled plate, then add the sample to be tested. Add the sample to the bottom of the well, avoiding contact with the well walls, and gently shake to mix.

[0213] 8) Incubation: After sealing the plate with sealing film, incubate at 37℃ for 30 minutes.

[0214] 9) Solution preparation: Dilute the concentrated washing solution with distilled water and set aside for later use.

[0215] 10) Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each hole with washing liquid, let stand for 30 seconds and then discard. Repeat this 5 times, then pat dry.

[0216] 11) Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for blank wells.

[0217] 12) Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0218] 13) Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each hole with washing liquid, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry.

[0219] 14) Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0220] 15) Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0221] 16) Measurement: Zero the instrument using the blank well and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0222] 17) Calculation: Use software (ELISA Calc software) to fit the standard curve and calculate the measured value based on the absorbance.

[0223] B. Calculation Formula

[0224] 1) Concentration calculation formula

[0225] y = (AD) / [1 + (x / C)] B ]+D

[0226] x represents the OD450 of the sample; y represents the PGE2 concentration of the sample (pg / mL); A: Upper asymptote of the curve, representing the upper limit of the response value; B: Curve slope, determining the steepness of the S-curve; C: Half-effective concentration, i.e., the value of the independent variable corresponding to the average of the upper and lower limits of the response value, reflecting the sensitivity of the system; D: Lower asymptote of the curve, representing the lower limit of the response value.

[0227] 2) Formula for calculating the rate of change

[0228]

[0229] PGE2 ( Model group ) This represents the PGE2 expression level in the model control group. ( Sample group ) This indicates the PGE2 expression level in the sample group.

[0230] C. Analysis software

[0231] 1) Statistical analysis was performed using GraphPad.Prism.10.1 software. This experiment employed independent samples t-tests between two groups and one-way ANOVA for statistical analysis among multiple groups. The statistical results were considered statistically significant with α = 0.05 as the test limit and p < 0.05 (compared to the model control group).

[0232] 2) Use ELISA Calc software to calculate data, and use Logistic curve fitting 2 (four parameters) to perform curve fitting and calculate sample concentration.

[0233] Experimental results are as follows Figure 12 As shown in the figure, the PGE2 expression level in the MC group was significantly higher than that in the NC and PC groups, indicating successful modeling. The PGE2 expression levels in the Ave and HA groups were significantly lower than those in the MC group, indicating that both Ave and HA have anti-inflammatory effects. The PGE2 expression level in the AHC group was significantly lower than that in equimolar amounts of free Ave and free HA, indicating that Ave and HA play a synergistic role in regulating the intensity of the inflammatory response in the AHC co-assembled inclusion complex.

[0234] Test Example 5: Detection of Mast Cell Degranulation

[0235] Experimental Principle

[0236] Mast cell degranulation is a crucial step in the immune response, triggering inflammatory and immediate hypersensitivity reactions by releasing various active substances such as β-hexosaminidase and histamine. Calcium iontophores (such as Ionomycin) are commonly used mast cell degranulation inducers, mimicking in vivo inflammatory triggering mechanisms to induce mast cells to release granular contents. The rat basophilic leukemia cell line (RBL-2H3) was selected as a model cell due to its good stability and operability, making it suitable for studying anti-inflammatory and soothing active ingredients. By stimulating mast cell degranulation with calcium iontophores and detecting the cell degranulation rate, the inhibitory effect of the sample on mast cell degranulation can be directly assessed. A higher inhibition rate indicates greater potential in alleviating immediate hypersensitivity reactions (soothing) and inhibiting inflammatory responses.

[0237] The following experiments were conducted using RBL-2H3 rat basophilic leukemia cells (purchased from Mirror Cell Technology (Shanghai) Co., Ltd., model iCell-r027). In the cell culture described below, Raw264.7 cells were cultured in MEM basal medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (culture conditions set at 37℃, 5% CO2).

[0238] A. Experimental Procedure

[0239] 1) After reviving the cells, passage them twice to expand the culture and obtain expanded cultured cells.

[0240] 2) After digesting, centrifuging, resuspending, and counting the cells from the expanded culture, seed them into 24-well cell culture plates, adding 2×10⁶ cells per well. 5 Each cell.

[0241] 3) Experimental grouping: After plating, the cells were divided into a negative control group (NC), a positive control group (PC), a model control group (MC), and a sample group (specified drug concentration). The grouping is shown in Table 6. The sample groups were the HA group, the AHC group from Example 1, and the Ave group.

[0242] Table 6 Experimental Groups

[0243]

[0244]

[0245] 4) After the cells have completely adhered to the wall, remove the culture medium and add 500 μl of the culture medium containing the diluted drug to each of the 24 wells.

[0246] 5) After culturing the cells in an incubator for 8 hours, replace the culture medium with a drug-free medium and incubate for 40 hours.

[0247] 6) Count degranulated and non-degranulated cells under a microscope.

[0248] B. Calculation Formula

[0249]

[0250] Experimental results are as follows Figure 13As shown in the figure, the mast cell degranulation rate in the MC group was significantly higher than that in the NC and PC groups, indicating successful modeling. The mast cell degranulation rates in the Ave and HA groups were significantly lower than those in the MC group, indicating that both Ave and HA have anti-allergic effects. The mast cell degranulation rate in the AHC group was significantly lower than that in equimolar amounts of free Ave and free HA, indicating that Ave and HA played a synergistic role in alleviating immediate hypersensitivity reactions in the AHC co-assembled inclusion complex.

[0251] Test Example 6: Detection of Apoptosis Inhibition

[0252] Experimental Principle

[0253] Inflammation is a major contributing factor to cellular senescence. AHC's excellent anti-inflammatory and soothing effects demonstrate significant potential for anti-aging applications. UV radiation is a crucial factor leading to skin photoaging and cell damage, especially UVB (medium-wave ultraviolet radiation), which can penetrate the epidermis and induce DNA damage, oxidative stress, and apoptosis. Apoptosis is a key mechanism of skin aging; excessive apoptosis leads to a decrease in the number and function of skin cells, thus accelerating skin aging. This experiment used HaCaT cells as a model cell. Inducing HaCaT cell damage through UVB radiation simulates the skin's natural response to UV exposure, including inflammatory responses, oxidative stress, DNA damage, and apoptosis. Based on this, flow cytometry was used to detect the apoptosis rate, allowing for a direct assessment of AHC's inhibitory effect on UV-induced apoptosis. A lower apoptosis rate after sample treatment indicates a greater potential to protect cells from UV damage and delay cellular senescence.

[0254] The following experiments were conducted using HaCaT cells, which were purchased from Mirror Image (Shanghai) Cell Technology Co., Ltd. In the cell culture described below, HaCaT cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (culture conditions set at 37℃, 5% CO2).

[0255] A. Experimental Procedure

[0256] 1) After reviving the cells, passage them twice to expand the culture and obtain expanded cultured cells.

[0257] 2) After expanding the culture, digest, centrifuge, resuspend, count, and seed the cells into 6-well cell culture plates, adding 2×10⁶ cells per well. 6 Each cell.

[0258] 3) Experimental Grouping: After plating, the cells were divided into a negative control group (NC), a positive control group (PC), a model control group (MC), and a sample group (specified drug concentration). The drug concentrations for each group are shown in Table 7. The positive control group received vitamin E (VE). The sample groups were the HA group, the AHC group from Example 1, and the AVE group.

[0259] Table 7. Experimental Groups

[0260]

[0261]

[0262] 4) After the cells have fully adhered to the culture medium, remove the medium, add PBS buffer, and irradiate the cells with a combination of UVB and UVA for 30 min. The NC group was covered with aluminum foil. After irradiation, 2000 μl of culture medium containing diluted drug was added to each of the 6 wells. Apoptosis was detected after 48 h of culture.

[0263] 5) Aspirate the cell culture medium into a centrifuge tube, wash the adherent cells once with PBS buffer, and digest the cells with trypsin cell digestion solution. Incubate at room temperature until the adherent cells can be easily pipetted off, then aspirate the trypsin cell digestion solution; add the collected cell culture medium, gently pipet the cells off, transfer them to a centrifuge tube, centrifuge at 1000g for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS, and count them.

[0264] 6) Take 50,000-100,000 cells and resuspend them. Centrifuge at 1000g for 5 minutes, discard the supernatant, and gently resuspend the cells in 195μl of Annexin V-FITC binding buffer (from the Annexin V-FITC apoptosis detection kit, manufactured by Shanghai Beyotime Biotechnology Co., Ltd.). Add 5μl of Annexin V-FITC (from the Annexin V-FITC apoptosis detection kit, manufactured by Shanghai Beyotime Biotechnology Co., Ltd.) and mix well. Add 10μl of propidium iodide staining solution and mix gently. Incubate at room temperature (20-25℃) in the dark for 10-20 minutes, then place on ice. Aluminum foil can be used for light protection. Resuspend the cells 2-3 times during incubation to improve staining results.

[0265] 7) Detect the cells using a flow cytometer (Thermo Fisher Attune NxT flow cytometer).

[0266] B. Formula and p-value

[0267] 1)Formula

[0268] Total apoptosisrate(%)=Early apoptosis rate(%)+Advanced apoptosisrate(%)

[0269] 2) Significance p-value

[0270] Compared with the NC group, the statistically significant differences were: #p≤0.05; ##P≤0.01; ###P≤0.001; ####P≤0.0001.

[0271] Compared with the MC group, the statistically significant differences were: *p≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001.

[0272] The experimental results are shown in Figure 14. The apoptosis rate in the MC group was significantly higher than that in the NC and PC groups, indicating successful modeling. The apoptosis rates in the Ave and HA groups were significantly lower than those in the MC group, indicating that both Ave and HA have an inhibitory effect on apoptosis. The apoptosis rate in the AHC group was significantly lower than that in equimolar amounts of free Ave and free HA, indicating that Ave and HA play a synergistic role in inhibiting apoptosis (anti-photoaging) in the AHC co-assembled inclusion complex.

[0273] Example 5: Study on the synergistic mechanism of Ave and HA

[0274] Molecular frontier orbital analysis is an important concept in quantum chemistry, used to study the electronic structure and chemical reactivity of molecules. Frontier orbitals refer to the highest-energy occupied molecular orbitals (HOMOs) and the lowest-energy unoccupied molecular orbitals (LUMOs). Analyzing the energy, distribution, and interactions of HOMOs and LUMOs using GaussView software provides a deeper understanding of the electronic structure and chemical behavior of molecules. Figure 15 As can be seen, the HOMO of the Ave is mainly distributed on the benzene ring linking the hydroxyl group, while the LUMO is mainly distributed on the benzene ring linking the carboxyl group, indicating that they are the electron-losing and electron-gaining active sites, respectively. Both the HOMO and LUMO of the Ave-HA assembly are located on the Ave, indicating that the Ave is the active site of the Ave-HA assembly. Furthermore, the structural distribution and symmetry of the HOMO and LUMO of the Ave-HA assembly are the same as those of the free Ave, suggesting that the Ave-HA assembly has the same biological activity as the Ave. At the same time, the higher HOMO, lower LUMO, and smaller band gap (ΔE) indicate that the activity of the Ave-HA assembly is superior to that of the free Ave.

[0275] Test Example 7: Stability Test

[0276] To verify the stability of the AHC co-assembled inclusion complex (prepared in Example 1), a 2 wt% solution was prepared, and its pH, conductivity, and color changes were tested after storage at -15°C, 20°C, and 45°C for 4 weeks. Figure 16a As shown in Table 8, the pH and conductivity of the solution remained almost unchanged under different high and low temperature conditions, and the appearance remained clear and transparent. Therefore, the AHC co-assembled inclusion complex solution has good stability. The dried AHC co-assembled inclusion complex powder also showed good stability, showing no yellowing after 4 weeks of storage under different high and low temperature conditions (e.g., ...). Figure 16b (As shown). This indicates that, regardless of whether in solution or dry environment, the Ave in the AHC co-assembled inclusion complex are well encapsulated by HA and CD, exhibiting strong stability and wide applicability to various applications and storage scenarios.

[0277] Table 8. Stability of 2 wt% AHC co-assembled inclusion complex solution

[0278]

[0279] Test Example 8: Skin Irritation Test

[0280] Experimental Principle

[0281] Human patch testing is a method used to assess the potential allergic reactions and irritations of chemicals, cosmetics, and pharmaceuticals to human skin. The principle involves applying the test substance to the skin and observing the skin's reaction over a certain period to determine whether the test substance will cause skin allergies or irritation. This test is commonly used for safety assessments of cosmetics, pharmaceuticals, and personal care products and is an important tool for detecting potential allergens and irritants.

[0282] Experimental steps

[0283] A closed patch test was conducted on at least 30 healthy volunteers. Subjects were required to have no skin diseases or other serious illnesses within the past three months and to have not taken any antibiotics or hormonal medications. A 0.7 wt% AHC solution (the AHC co-assembled inclusion complex prepared in Example 1, with an Ave concentration of 0.1 wt%) was prepared. 0.020 g–0.025 g of the solution was placed in a suitable patch applicator and applied to the flexor surface of the subject's forearm using hypoallergenic adhesive tape. Deionized water was used as a control. The patch was gently pressed onto the skin with the palm of the hand to ensure even application. After 24 hours of continuous application, the patch was removed, and the indentation was allowed to disappear. Skin reactions such as erythema, infiltration, edema, papules, and vesicles were observed in the test area at 30 minutes (after the indentation disappeared), 24 hours, and 48 hours. The results were recorded according to the skin reaction grading standards (Table 9) in the *Cosmetic Safety Technical Specifications* (2015 edition).

[0284] Table 9. Grading Criteria for Skin Reactions in Closed Patch Tests

[0285]

[0286] Evaluation principles

[0287] Of the 30 subjects, no more than 5 should experience a Grade 1 skin adverse reaction, no more than 2 should experience a Grade 2 skin adverse reaction, and no more than 1 should experience a Grade 3 or higher skin adverse reaction. If these requirements are exceeded, it suggests that the test substance may cause adverse reactions on human skin. As shown in Table 10, all 30 subjects in this experiment had negative reactions, indicating that the 0.7 wt% solution of the AHC co-assembled inclusion complex (Ave concentration of 0.1 wt%) has excellent safety and no skin irritation.

[0288] Table 10 Patch test results of AHC co-assembled inclusion complexes

[0289]

[0290]

[0291] Test Example 9: Human Redness and Itch Relief Experiment

[0292] Experimental Principle

[0293] Histamine is an important inflammatory mediator. In allergic reactions, mast cells and basophils release histamine, leading to vasodilation and increased permeability, thereby causing symptoms such as erythema, edema, and itching. Antihistamines alleviate these allergic symptoms by blocking the binding of histamine to its receptors (mainly H1 receptors). Therefore, by inducing histamine release and observing the skin reaction, the anti-redness and antipruritic effects of anti-allergy products can be evaluated.

[0294] Experimental steps

[0295] More than 30 healthy volunteers, aged 18-60 years, with no skin diseases or other serious illnesses in the past three months, and who had not taken any antibiotics or hormone drugs, were selected. The inner forearm of the subjects was used as the test site to evaluate the anti-allergic effect of aqueous solutions of different concentrations of AHC co-assembled inclusion complex, and to compare it with a mixture of oat alkaloids, hyaluronic acid and cyclodextrin. First, a 0.7 wt% aqueous solution of the AHC co-assembled inclusion complex in Example 1 (Ave concentration of 0.1 wt%) was prepared, and an aqueous solution of a mixture containing the same concentration and type of Ave, HA and CD was prepared (Ave concentration was also 0.1 wt%, the preparation method can be referred to as in Example 1, but the solvent is pure water, and the operation of heating, high pressure homogenization, centrifugation, filtration, drying and powdering was not performed in step (2)). The experimental results showed that the aqueous solution of the mixture of Ave, HA and CD (precursor mixture) could not be completely dissolved, indicating that the solubility of Ave in the aqueous solution of the mixture was poor. Then, two test areas of equal area were selected on the inner forearm of the subjects, and an equal amount of 3% histamine solution was applied. After obvious allergic reactions appeared, equal amounts of AHC aqueous solution and a mixture of aqueous solutions (Ave content 0.1 wt%) were applied to two test areas on the left and right arms, respectively. The desensitization effect on the left and right arms was assessed by volunteers' self-rating after 30 minutes. The rating criteria are shown in Table 11: self-reported redness reduction score 0-5 (higher score indicates better redness reduction effect), and self-reported itching relief score 0-5 (higher score indicates better itching relief effect).

[0296] Table 11 Scoring criteria for redness reduction and itch relief in antihistamine allergy tests.

[0297]

[0298] Experimental results are as follows Figure 17 As shown, obvious allergic reactions appeared on the skin after applying a 3% histamine solution, indicating successful modeling. When the Ave content was 0.1 wt%, the anti-redness and antipruritic effects of AHC were significantly better than those of the precursor mixture. This result indicates that the AHC co-assembled inclusion complex has better anti-allergic efficacy than the precursor mixture, which may be due to the better solubility of Ave and the synergistic effect of Ave and HA in anti-inflammatory and anti-allergic signaling pathways in the AHC co-assembled inclusion complex.

[0299] In summary, the AHC preparation method provided in this invention is simple, convenient, and highly practical. This method utilizes the co-assembly of HA and Ave to solubilize Ave, and improves the stability of the system through CD inclusion. Based on the good compatibility between HA and CD, it simultaneously achieves high Ave solubility and system stability. The resulting AHC co-assembled inclusion complex increases the water solubility of Ave by more than 1200 times. Furthermore, the synergistic effect of Ave and HA makes the anti-inflammatory, anti-allergic, and anti-photoaging effects of AHC significantly superior to equimolar amounts of free Ave and HA. The strong interaction between Ave and HA allows them to synergistically enhance their effects without altering Ave activity. AHC exhibits excellent regulatory performance throughout the entire anti-inflammatory pathway network, with significantly higher inhibitory effects on upstream inflammatory mediators (TNF-α), midstream inflammatory mediators (NO), and downstream inflammatory mediators (PGE2) than free Ave and HA. In addition, the mast cell degranulation inhibition rate of AHC is also significantly higher than that of free Ave and HA, demonstrating excellent anti-allergic effects. This makes AHC significantly superior to free Ave and HA in inhibiting UV-induced apoptosis, demonstrating good potential for anti-photoaging applications. Compared with commercially available similar products like Deminshu, it has lower cytotoxicity, better safety and stability, and can fully meet the product requirements of mitochondrial energy skincare; compared with precursor mixtures, its anti-redness and anti-itch effects are significantly improved.

[0300] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a soothing oat base-hyaluronic acid co-assembly, characterized in that, Includes the following steps: Hyaluronic acid compounds are added to an alcohol solution, stirred to swell, and heated to obtain a hyaluronic acid compound solution. The hyaluronic acid compound solution is heated to 60-80°C, and dihydroagar alkaloids are added under constant temperature conditions of 60-80°C and stirred until completely dissolved to obtain a primary mixture. The primary mixture is heated to 60-80°C, and cyclodextrin compounds are added in 3-5 portions under constant temperature conditions of 60-80°C and stirred until completely dissolved to obtain a mixture. The mixture is homogenized, centrifuged to collect the supernatant, filtered to collect the filtrate, and dried to obtain the soothing agar alkaloid-hyaluronic acid co-assembly. The mass ratio of the dihydroaerol alkaloid, hyaluronic acid compounds, and cyclodextrin compounds is 1:(1-5):(5-20); The cyclodextrin compound is one or more of hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin; The hyaluronic acid compounds include one or more of hyaluronic acid and acetylated hyaluronic acid; The molecular weight of the hyaluronic acid compound is less than 1500 kDa; The alcohol solution is a mixture of an alcohol compound and water, wherein the mass percentage of the alcohol compound is 10-15%; the alcohol compound is ethanol or isopropanol; and the mass ratio of the hyaluronic acid compound to the alcohol solution is 1:20-100. The homogenization process includes ultrasonic homogenization and high-pressure homogenization; the ultrasonic homogenization process is carried out at a temperature of 60°C, a frequency of 50kHz, and a duration of 1 hour; the high-pressure homogenization process is carried out at a temperature of 60°C, a pressure of 30,000 psi, and a cycle number of 3 times.

2. The method for preparing the soothing oat base-hyaluronic acid co-assembly according to claim 1, characterized in that, The stirring and swelling time is 0.5-1h, the stirring rate is 300-600rpm, the stirring temperature is 60-80℃, the stirring time is 1-2 hours, and the stirring rate is 300-600rpm.

3. The method for preparing the soothing oat base-hyaluronic acid co-assembly according to claim 1, characterized in that, The centrifugation speed for obtaining the supernatant is 3000-10000 rpm, and the centrifugation time is 10-15 minutes; the filtration method is vacuum filtration, and the filter membrane used for filtration has a pore size of 0.45 μm.

4. The soothing oat base-hyaluronic acid co-assembly prepared by the preparation method according to any one of claims 1-3.

5. The use of the soothing oat base-hyaluronic acid co-assembly according to claim 4 in the preparation of topical medicines or cosmetics.

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