Soothing type oat anthramide multi-dimensional co-assembly as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine and beauty makeup personal care health, and provides a soothing type avenanthramide multi-dimensional co-assembly and a preparation method and application thereof.The preparation method comprises the following steps that S1, small molecule saccharide derivatives are added into an alcohol-water mixed solution and mixed to be uniform, avenanthramide and cyclodextrin are added, and full mixing is conducted; and S2, cooling the mixture obtained in the step S1 to room temperature, centrifuging, filtering, collecting filtrate, performing rotary evaporation until the filtrate is pasty, performing vacuum drying, grinding, sealing and storing, thereby obtaining the oat anthramide multi-dimensional co-assembly. Compared with similar products sold in the market, the avenanthramide, the micromolecular saccharide derivative and the cyclodextrin are organically combined, the water solubility of the avenanthramide is improved by more than 1100 times through co-assembly, and the stability of the system is enhanced by utilizing the inclusion effect of the cyclodextrin; meanwhile, by means of secondary interaction among the components, the affinity between the oat alkali and cyclodextrin is improved, a more stable system is achieved, and the product requirements of mitochondrial energy skin care are comprehensively met.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, beauty and personal care, and health, and particularly to a soothing oat anthraquinone multidimensional co-assembly, its preparation method and application. Background Technology

[0002] Mitochondria, as the cell's energy factories, are responsible for producing the energy (ATP) needed by the cell and participate in processes such as cell metabolism, signal transduction, and apoptosis, playing a crucial role in maintaining skin health and delaying aging. In recent years, mitochondrial skincare has become a research hotspot in the cosmetics field, with scientists dedicated to developing skincare ingredients that can improve mitochondrial function and enhance cellular energy metabolism. These studies not only focus on traditional skincare mechanisms such as antioxidation and anti-inflammation but also delve into new areas such as mitochondrial protection, energy metabolism promotion, and cell repair.

[0003] Small molecule sugar derivatives (XTYs) are a class of compounds obtained by chemically modifying natural sugar molecules or their structural units. These include BSY (Boswellia syl ...

[0004] Amaranthamides (YMJ) are a class of phenolic acid derivatives with antioxidant and anti-inflammatory properties, showing significant potential in mitochondrial skincare. They can reduce oxidative stress, alleviate skin damage caused by external stimuli, and possess antihistamine properties, helping to relieve itching and redness in sensitive skin. Because the activity of the phenolic hydroxyl group is higher than that of the alcohol hydroxyl group, the antioxidant and anti-inflammatory capabilities of amaranthamides are significantly superior to those of small-molecule sugar derivatives, offering certain immediate skincare benefits. However, the solubility of amaranthamides in water is very low, <30 μ / mL, limiting their widespread application in skincare products. Existing solutions, such as using organic solvents or surfactants to improve solubility, often suffer from complex processes, high production costs, poor stability, and metabolic toxicity.

[0005] Cyclodextrin (HHJ) is a cyclic oligosaccharide with a unique structure. Its hydrophobic interior and hydrophilic exterior, strong physical shielding, and thermal stability enable it to significantly improve the chemical stability of poorly soluble components. However, the limited cavity size of cyclodextrin imposes strict requirements on the size of guest molecules and is constrained by enthalpy-entropy compensation, resulting in a relatively limited loading capacity for poorly soluble drug molecules. Furthermore, cyclodextrin solutions typically have high viscosity, which hinders the diffusion of poorly soluble molecules, leading to low loading efficiency. Small molecule co-assembly technology is a novel and efficient drug delivery technique that primarily relies on non-covalent interactions (such as hydrogen bonds, van der Waals forces, and hydrophobic interactions) to form nanostructures. However, these interactions are relatively weak, causing the system to be thermodynamically unstable and susceptible to changes in solvent environment and temperature.

[0006] Therefore, it is of great significance to organically combine small molecule co-assembly technology with cyclodextrin inclusion complexation to provide a soothing oat anthraquinone multidimensional co-assembly (TYH) that can effectively improve the water solubility and stability of oat anthraquinone products and has excellent cosmetic and skin care effects, as well as its preparation method. Summary of the Invention

[0007] Given the existing problems of low water solubility and stability, low encapsulation efficiency, and the need for further improvement in cosmetic and skincare effects of arnaldehyde amide products, this invention provides a soothing arnaldehyde amide multidimensional co-assembly, its preparation method, and its application. It organically combines arnaldehyde amide, small molecule sugar derivatives, and cyclodextrin, improving the water solubility and diffusivity of arnaldehyde amide through co-assembly. Subsequently, the inclusion effect of cyclodextrin enhances the stability of the system. Simultaneously, secondary interactions between components further enhance the affinity between arnaldehyde amide and cyclodextrin, resulting in stronger inclusion and a more stable system. This system exhibits high water solubility, strong stability, and excellent cosmetic and skincare effects, fully meeting the product requirements of mitochondrial energy skincare and showing promising application prospects in skincare products.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing a soothing oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0010] S1. Add the small molecule sugar derivative to the alcohol-water mixture, mix well, and under continuous stirring, heat to 60°C, add avocado anthramide and cyclodextrin, and mix thoroughly;

[0011] S2. Cool the mixture obtained in S1 to room temperature, centrifuge, filter, collect the filtrate, rotary evaporate to a paste, vacuum dry, grind, seal and store to obtain the oat anthraquinone multidimensional co-assembly.

[0012] Furthermore, the mass ratio of the amaranthamide, the small molecule sugar derivative, and the cyclodextrin is 1:(1-3):(2-10).

[0013] Furthermore, the small molecule sugar derivatives mentioned in S1 are one or more of the following: bosine, rhamnose, glyceryl glucoside, fucose, xylitol, and sorbitol.

[0014] Furthermore, the small molecule sugar derivatives described in S1 are bosine and / or rhamnose.

[0015] Furthermore, in the alcohol-water mixture described in S1, the alcohol is ethanol and / or isopropanol, with a concentration of 5-15 wt.%.

[0016] Furthermore, the cyclodextrin in S1 is one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0017] Furthermore, the cyclodextrin in S1 is methyl-β-cyclodextrin and / or hydroxypropyl-β-cyclodextrin.

[0018] Furthermore, the mass ratio of the small molecule sugar derivative and the alcohol-water mixture in S1 is 1:(10-50).

[0019] Furthermore, the thorough mixing described in S1 is achieved through homogenization or ultrasonic mixing.

[0020] Furthermore, the homogenization is performed by cycling 3 times at 25,000-35,000 psi.

[0021] Furthermore, the ultrasound is performed at 10-25 kHz for 1 hour.

[0022] Furthermore, the centrifugation described in S2 is centrifugation at 3000 rpm for 10-15 min.

[0023] Furthermore, the filtration described in S2 is to filter to below 0.45 μm.

[0024] Another object of the present invention is to provide a soothing oat anthraquinone multidimensional co-assembly.

[0025] A soothing avocado anthraquinone multidimensional co-assembly is prepared according to the preparation method of the soothing avocado anthraquinone multidimensional co-assembly described in any of the preceding claims.

[0026] Another object of the present invention is to provide an application of a soothing oat anthraquinone multidimensional co-assembly.

[0027] The application of the above-mentioned soothing oat anthraquinone multidimensional co-assembly in the preparation of skin care products or topical medicines.

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

[0029] (1) The soothing oat anthraquinone multidimensional co-assembly of the present invention organically combines YMJ, XTY and HHJ. Due to its strong hydrophobicity, free YMJ can only enter HHJ in a folded conformation, resulting in large steric hindrance and weak inclusion effect (-0.93 kcal / mol), making the system unstable. However, the strong interaction between XTY and YMJ not only allows YMJ to enter the cavity of HHJ linearly, resulting in a stronger inclusion effect (<-14 kcal / mol) and improved water solubility of YMJ, but also makes the system more stable through secondary interactions around the cavity, enabling it to be widely used in various cosmetic formulations.

[0030] (2) The soothing oat anthraquinone multidimensional co-assembly of the present invention, with its rich interactions between YMJ, XTY, and HHJ, allows TYH to synergistically enhance the effects of YMJ without altering its activity, resulting in reduced cytotoxicity while maintaining its anti-inflammatory and antioxidant efficacy. This enables TYH to be used at higher concentrations, achieving comprehensive anti-inflammatory and antioxidant effects that surpass those of Deminex, including its ROS scavenging, SOD-promoting activity, and ability to regulate upstream and downstream inflammatory mediators (TNF-α and IL-8) and PGE2. This greatly enhances the application potential of YMJ in the field of mitochondrial skincare.

[0031] (3) The method of the present invention is simple and convenient to operate, and has strong practicality. The obtained soothing arnaldehyde amide multidimensional co-assembly increases the water solubility of YMJ by more than 1100 times. The synergistic effect of XTY and HHJ makes the solution stability of the soothing arnaldehyde amide multidimensional co-assembly excellent. There is no precipitation phenomenon below the saturation concentration. It also has low cytotoxicity and strong applicability to formulations, making it widely applicable to various cosmetic formulations. It is easy to store and transport, thereby significantly enhancing the market value of YMJ. Attached Figure Description

[0032] The invention will be further illustrated with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the invention. For those skilled in the art, other drawings can be obtained based on the following drawings without any creative effort.

[0033] Figure 1 This is the infrared spectrum of the soothing oat anthraquinone multidimensional co-assembly of Experimental Example 1 of the present invention.

[0034] Figure 2 This is the infrared spectrum of the soothing oat anthraquinone multidimensional co-assembly of Experimental Example 3 of the present invention.

[0035] Figure 3These are TEM images of the samples prepared in Experimental Example 1 and Comparative Example 1 of the present invention. The left image is Comparative Example 1, and the right image is Example 1.

[0036] Figure 4 The images show SEM images of the YMJ and BSY mixture and the dried and pulverized soothing oat anthraquinone multidimensional co-assembly of Experimental Example 1 of this invention. The left image shows the YMJ and BSY mixture, and the right image shows the dried and pulverized soothing oat anthraquinone multidimensional co-assembly.

[0037] Figure 5 This is a diagram of the co-assembly process of the BSY-YMJ of the present invention.

[0038] Figure 6 This is an interaction analysis diagram of YMJ and XTY in the soothing oat anthraquinone multidimensional co-assembly of the present invention.

[0039] Figure 7 This is an ESP analysis diagram of the YMJ, BSY-YMJ co-assembly and SLT-YMJ co-assembly of the present invention.

[0040] Figure 8 This is a molecular frontier orbital analysis diagram of the YMJ, BSY-YMJ co-assemblies and SLT-YMJ co-assemblies of the present invention.

[0041] Figure 9 This is an analysis diagram of the inclusion effect of HHJ on YMJ in this invention.

[0042] Figure 10 This is an interaction diagram between the components in the soothing oat anthraquinone multidimensional co-assembly of the present invention.

[0043] Figure 11 This is a comparison chart of the soothing oat anthraquinone multidimensional co-assembly of the present invention and Deminex.

[0044] Figure 12 This is a comparison diagram of the cytotoxicity of the soothing oat anthraquinone multidimensional co-assembly of the present invention (BSY-YMJ-HHJ multidimensional co-assembly, abbreviated as BYH) with free YMJ and Deminshu.

[0045] Figure 13 This is a comparison diagram of the cytotoxicity of the soothing oat anthraquinone multidimensional co-assembly (SLT-YMJ-HHJ multidimensional co-assembly, abbreviated as SYH) of the present invention and Deminshu.

[0046] Figure 14 This is a comparison chart of the ROS scavenging capabilities of the soothing oat anthraquinone multidimensional co-assembly of the present invention and Deminshu.

[0047] Figure 15This is a comparison diagram of the SOD-promoting activity of the soothing oat anthraquinone multidimensional co-assembly of the present invention and Deminshu.

[0048] Figure 16 This is a comparison diagram of the soothing oat anthraquinone multidimensional co-assembly of the present invention and the inhibition of mid-to-upstream inflammatory gene expression by Deminshu.

[0049] Figure 17 This is a comparison diagram of the soothing oat anthraquinone multidimensional co-assembly of the present invention and Deminshu's inhibition of downstream inflammatory mediators.

[0050] Figure 18 This is a stability test diagram of the soothing oat anthraquinone multidimensional co-assembly of the present invention.

[0051] Figure 19 This is a grading chart of skin reactions in a closed patch test.

[0052] Figure 20 This is a comparison diagram of the anti-allergic effects of the soothing oat anthraquinone multidimensional co-assembly of the present invention and Deminshu.

[0053] Figure 21 This is a diagram illustrating the repair efficacy of the soothing oat anthraquinone multidimensional co-assembly of the present invention. Detailed Implementation

[0054] To better illustrate the objectives, technical solutions, and advantages of this invention, the following embodiments are provided. Obviously, the following embodiments are only a part of the embodiments of this invention, and not all of them; it should be understood that the embodiments of this invention are only used to illustrate the technical effects of this invention, and not to limit the scope of protection of this invention.

[0055] All raw materials used in the examples are commercially available; unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field.

[0056] Example 1

[0057] A method for preparing a soothing oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0058] S1. Add 20g of Boseine to 500mL of isopropanol aqueous solution (isopropanol concentration is 10wt.%), mix well, stir at room temperature until Boseine is completely dissolved, and under continuous stirring, heat to 60℃, add 20g of arnethamide in 4 portions, 5g each time, and stir until completely dissolved. Add 80g of hydroxypropyl-β-cyclodextrin in 8 portions, 10g each time, and stir until completely dissolved. Circulate 3 times using a high-pressure microfluidic homogenizer at 30000psi.

[0059] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 10 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the soothing oat anthraquinone multidimensional co-assembly.

[0060] Example 2

[0061] A method for preparing a soothing oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0062] S1. Add 5g of Bosein to 100mL of isopropanol aqueous solution (isopropanol concentration is 5wt.%), mix well, stir at room temperature until Bosein is completely dissolved, and under continuous stirring, heat to 60℃, add 2g of alanine in 2 portions, 1g each time, and stir until completely dissolved. Add 15g of methyl-β-cyclodextrin in 3 portions, 5g each time, and stir until completely dissolved. Sonicate at 15kHz for 1 hour at 60℃.

[0063] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 15 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the soothing oat anthraquinone multidimensional co-assembly.

[0064] Example 3

[0065] A method for preparing a soothing oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0066] S1. Add 20g rhamnose to 500mL of isopropanol aqueous solution (isopropanol concentration is 10wt.%), mix well, stir at room temperature until the rhamnose is completely dissolved, and under continuous stirring, heat to 60℃, add 20g of arnethamide in 4 portions, 5g each time, and stir until completely dissolved. Add 80g of hydroxypropyl-β-cyclodextrin in 8 portions, 10g each time, and stir until completely dissolved. Circulate 3 times using a high-pressure microfluidic homogenizer at 30000psi.

[0067] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 10 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the soothing oat anthraquinone multidimensional co-assembly.

[0068] Example 4

[0069] A method for preparing a soothing oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0070] S1. Add 5g of rhamnose to 100mL of isopropanol aqueous solution (isopropanol concentration is 5wt.%), mix well, stir at room temperature until the rhamnose is completely dissolved, and under continuous stirring, heat to 60℃, add 2g of ammoniac amide in 2 portions, 1g each time, and stir until completely dissolved. Add 15g of methyl-β-cyclodextrin in 3 portions, 5g each time, and stir until completely dissolved. Sonicate at 15kHz for 1 hour at 60℃.

[0071] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 15 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the soothing oat anthraquinone multidimensional co-assembly.

[0072] Example 5

[0073] A method for preparing a soothing oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0074] S1. Add 5g of Bosein to 100mL of ethanol aqueous solution (ethanol concentration is 10wt.%), mix well, stir at room temperature until Bosein is completely dissolved, and under continuous stirring, heat to 60℃, add 2g of alanine in two portions, 1g each time, and stir until completely dissolved. Add 10g of methyl-β-cyclodextrin in two portions, 5g each time, and stir until completely dissolved. Sonicate at 20kHz for 1 hour at 60℃.

[0075] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 15 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the soothing oat anthraquinone multidimensional co-assembly.

[0076] Comparative Example 1

[0077] A method for preparing an oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0078] S1. Add 20g of Bosein to 500mL of isopropanol aqueous solution (isopropanol concentration is 10wt.%), mix well, stir at room temperature until Bosein is completely dissolved, and under continuous stirring, heat to 60℃, add 20g of oat anthracene amide in 4 portions, 5g each time, stir until completely dissolved, and circulate 3 times using a high-pressure microfluidic homogenizer at 30000psi.

[0079] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 10 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the oat anthraquinone multidimensional co-assembly.

[0080] The main difference between this comparative example and Example 1 is that no cyclodextrin was added.

[0081] Comparative Example 2

[0082] A method for preparing an oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0083] S1. Add 5g of Bosein to 100mL of isopropanol aqueous solution (isopropanol concentration is 5wt.%), mix well, stir at room temperature until Bosein is completely dissolved, and under continuous stirring, heat to 60℃, add 2g of oat anthracene amide in two portions, 1g each time, stir until completely dissolved, and sonicate at 15kHz for 1 hour at 60℃.

[0084] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 15 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the oat anthraquinone multidimensional co-assembly.

[0085] The main difference between this comparative example and Example 2 is that no cyclodextrin was added.

[0086] Comparative Example 3

[0087] A method for preparing an oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0088] S1. Add 20g rhamnose to 500mL of isopropanol aqueous solution (isopropanol concentration is 10wt.%), mix well, stir at room temperature until the rhamnose is completely dissolved, and under continuous stirring, heat to 60℃, add 20g of arnethamide in 4 portions, 5g each time, and stir until completely dissolved. Circulate 3 times using a high-pressure microfluidic homogenizer at 30000psi.

[0089] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 10 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the oat anthraquinone multidimensional co-assembly.

[0090] The main difference between this comparative example and Example 3 is that no cyclodextrin was added.

[0091] Comparative Example 4

[0092] A method for preparing an oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0093] S1. Add 5g of rhamnose to 100mL of isopropanol aqueous solution (isopropanol concentration is 5wt.%), mix well, stir at room temperature until the rhamnose is completely dissolved, and under continuous stirring, heat to 60℃, add 2g of ammoniacamide in two portions, 1g each time, and stir until completely dissolved. Sonicate at 15kHz for 1 hour at 60℃.

[0094] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 15 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the oat anthraquinone multidimensional co-assembly.

[0095] The main difference between this comparative example and Example 4 is that no cyclodextrin was added.

[0096] Comparative Example 5

[0097] A method for preparing an oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0098] S1. Add 20g of Boseine to 500mL of isopropanol aqueous solution (isopropanol concentration is 10wt.%), mix well, stir at room temperature until Boseine is completely dissolved, and under continuous stirring, heat to 60℃, add 20g of arnethamide in 4 portions, 5g each time, and stir until completely dissolved. Add 80g of hydroxypropyl-β-cyclodextrin in 8 portions, 10g each time, and stir until completely dissolved. Circulate once using a high-pressure microfluidic homogenizer at 30000psi.

[0099] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 10 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the oat anthraquinone multidimensional co-assembly.

[0100] The main difference between this comparative example and Example 1 is that the mixture was not fully mixed in step S1.

[0101] Comparative Example 6

[0102] A method for preparing an oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0103] S1. Add 20g of Boseine to 500mL of isopropanol aqueous solution (isopropanol concentration is 10wt.%), mix well, stir at room temperature until Boseine is completely dissolved, and under continuous stirring, heat to 50℃, add 20g of arnethamide in 4 portions, 5g each time, and stir until completely dissolved. Add 80g of hydroxypropyl-β-cyclodextrin in 8 portions, 10g each time, and stir until completely dissolved. Circulate 3 times using a high-pressure microfluidic homogenizer at 30000psi.

[0104] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 10 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the oat anthraquinone multidimensional co-assembly.

[0105] Compared with Example 1, the main difference of this comparative example is that the temperature in step S1 is not up to standard.

[0106] Comparative Example 7

[0107] A method for preparing an oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0108] S1. Add 20g of Boseine to 500mL of aqueous solution, mix well, and stir at room temperature until Boseine is completely dissolved. Under continuous stirring, heat to 60℃, add 20g of arnethamide in 4 portions, 5g each time, and stir until completely dissolved. Add 80g of hydroxypropyl-β-cyclodextrin in 8 portions, 10g each time, and stir until completely dissolved. Circulate 3 times using a high-pressure microfluidic homogenizer at 30000psi.

[0109] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 10 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the oat anthraquinone multidimensional co-assembly.

[0110] Compared with Example 1, the main difference of this comparative example is that step S1 is alcohol-free.

[0111] Comparative Example 8

[0112] A method for preparing an oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0113] S1. Add 10g of Boseine to 500mL of isopropanol aqueous solution (isopropanol concentration is 10wt.%), mix well, stir at room temperature until Boseine is completely dissolved, and under continuous stirring, heat to 60℃, add 20g of arnethamide in 4 portions, 5g each time, and stir until completely dissolved. Add 80g of hydroxypropyl-β-cyclodextrin in 8 portions, 10g each time, and stir until completely dissolved. Circulate 3 times using a high-pressure microfluidic homogenizer at 30000psi.

[0114] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 10 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the oat anthraquinone multidimensional co-assembly.

[0115] Compared with Example 1, the main difference in this comparative example is that the content of small molecule sugar derivatives is too low.

[0116] Comparative Example 9

[0117] A method for preparing an oat anthraquinone-based multidimensional co-assembly includes the following steps:

[0118] S1. Add 5g of Bosein to 100mL of isopropanol aqueous solution (isopropanol concentration is 5wt.%), mix well, stir at room temperature until Bosein is completely dissolved, and under continuous stirring, heat to 60℃, add 2g of alanine in 2 portions, 1g each time, and stir until completely dissolved. Add 15g of methyl-β-cyclodextrin in 3 portions, 5g each time, and stir until completely dissolved. Sonicate at 15kHz for 4 hours at 60℃.

[0119] S2. Cool the mixture obtained in S1 to room temperature, centrifuge at 3000 rpm for 15 min, take the supernatant and filter it to below 0.45 μm, collect the filtrate, evaporate it to a paste and then vacuum dry it, grind it into powder with a pulverizer, and store it in a cool, dry place in a sealed container to obtain the oat anthraquinone multidimensional co-assembly.

[0120] Compared with Example 2, the main difference in this comparative example is that the ultrasound time in step S1 is too long.

[0121] Performance tests were conducted on the above embodiments and comparative examples. The experimental test methods are as follows:

[0122] Infrared spectroscopy was performed on the obtained soothing oat anthraquinone multidimensional co-assemblies. Figure 1 This is the infrared spectrum of the soothing oat anthraquinone multidimensional co-assembly of Experimental Example 1 of this invention. Figure 1As shown, compared to the soothing oat anthraquinone multidimensional co-assembly, the peaks of the mixture are more disordered, which is a result of simple stacking of the peaks of the three substances. In the infrared spectrum of the mixture, YMJ is at 1500 cm⁻¹. -1 The nearby peaks are clearly visible, attributed to the strong absorption effect of the benzene ring; however, in the infrared spectrum of the soothing arnaldehyde amide multidimensional co-assembly, the intensity is significantly weakened and shifted towards lower wavenumbers, indicating that HHJ exerts a good inclusion effect on YMJ. Furthermore, compared to the infrared spectrum of the mixture, the infrared spectrum of the soothing arnaldehyde amide multidimensional co-assembly shows a higher intensity at 1600-1700 cm⁻¹. -1 The nearby C=O stretching vibration peaks were significantly weakened and shifted to lower wavenumbers, indicating that YMJ participated in strong hydrogen bonding in the co-assembly. Furthermore, the soothing oat anthraquinone multidimensional co-assembly of Experimental Example 1 showed a significant decrease in wavenumber at 3200 cm⁻¹. -1 Nearby and 2934.80cm -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 arnearam, bosine, and cyclodextrin form a uniform self-assembled inclusion complex through strong intermolecular interactions.

[0123] Figure 2 This is the infrared spectrum of the soothing oat anthraquinone multidimensional co-assembly of Experimental Example 3 of this invention. Figure 2 As shown, compared to the soothing oat anthraquinone multidimensional co-assembly, the peaks of the mixture are more disordered, which is a result of simple stacking of the peaks of the three substances. In the infrared spectrum of the mixture, YMJ is at 1500 cm⁻¹. -1 The nearby peaks are clearly visible, attributed to the strong absorption effect of the benzene ring; however, in the infrared spectrum of the soothing arnaldehyde amide multidimensional co-assembly, the intensity is significantly weakened and shifted towards lower wavenumbers, indicating that HHJ exerts a good inclusion effect on YMJ. Furthermore, compared to the infrared spectrum of the mixture, the infrared spectrum of the soothing arnaldehyde amide multidimensional co-assembly shows a higher intensity at 1600-1700 cm⁻¹. -1 The nearby C=O stretching vibration peaks were significantly weakened and shifted to lower wavenumbers, indicating that YMJ participated in strong hydrogen bonding in the co-assembly. Furthermore, the soothing oat anthraquinone multidimensional co-assembly of Experimental Example 3 showed a peak at 3273.17 cm⁻¹. -1 Nearby and 2900cm -1 The presence of two strong peaks nearby corresponds to the stretching vibrations of OH and CH, respectively, both of which exhibit multi-peak fusion. This indicates that ammoniac, rhamnose, and cyclodextrin form a uniform self-assembled inclusion complex through strong intermolecular interactions.

[0124] The particle size distribution of samples from Examples 3-4 and Comparative Examples 3-4 was analyzed using DLS. The results are shown in Table 1 below:

[0125] Table 1. Particle size distribution of samples from Examples 3-4 and Comparative Examples 3-4

[0126]

[0127] As shown in Table 1, compared with the SLT-YMJ co-assembly, the average particle size and polydispersity index (PDI) of the soothing arnethamide multidimensional co-assembly were significantly reduced. This indicates that the inclusion effect of HHJ significantly reduced the particle size of the soothing arnethamide multidimensional co-assembly and made it more uniform, demonstrating the importance of HHJ inclusion effect for the stability of multidimensional co-assembly.

[0128] Figure 3 These are TEM images of the samples prepared in Experimental Example 1 and Comparative Example 1 of this invention. The left image is Comparative Example 1, and the right image is Example 1. Figure 3 As shown, compared with the BSY-YMJ co-assembled nanoparticles, the nanoparticles of the soothing arnaldehyde amide multidimensional co-assembled nanoparticles are smaller and more uniform in size, consistent with the SLT system, indicating that HHJ inclusion can significantly improve the assembly performance of XTY and YMJ; the XTY-YMJ-HHJ ternary system has better assembly performance than the XTY-YMJ binary system. Furthermore, the uniform shading distribution of the nanoparticles in the soothing arnaldehyde amide multidimensional co-assembled nanoparticles in the TEM image indicates that YMJ, BSY, and HHJ are uniformly co-assembled without forming a core-shell structure.

[0129] The soothing alanine anthraquinone multidimensional co-assemblies were dried and pulverized for convenient storage and transportation. To verify that the drying and pulverizing process did not damage the microstructure of the soothing alanine anthraquinone multidimensional co-assemblies, SEM analysis was performed. Figure 4 These are SEM images of the YMJ and BSY mixture and the dried and pulverized soothing alanine anthraquinone multidimensional co-assembly of Experimental Example 1 of this invention. The left image shows the YMJ and BSY mixture, and the right image shows the dried and pulverized soothing alanine anthraquinone multidimensional co-assembly. Figure 4 As shown, the mixture of YMJ and BSY consists of rods of varying sizes with clear edges and a clear growth trend, indicating that it is in a crystalline state. In contrast, the soothing oat anthraquinone multidimensional co-assemblies, after drying and pulverizing, have different particle sizes but exhibit an amorphous morphology and uniform texture, suggesting that the drying and pulverizing process did not disrupt the inclusion state of HHJ.

[0130] Dissolution tests were conducted on Examples 1, 2, and 5, and Comparative Examples 5-9. The specific test results are shown in Table 2 below:

[0131] Table 2. Dissolution test results of samples from Examples 1, 2, 5 and Comparative Examples 5-9.

[0132] Serial Number Dissolved state YMJ solubility (%) Example 1 Completely dissolved 3.38 Example 2 Completely dissolved 2.01 Example 3 Completely dissolved 3.36 Example 4 Completely dissolved 2.03 Example 5 Completely dissolved 2.54 Comparative Example 5 Cannot be completely dissolved 2.94 Comparative Example 6 Cannot be completely dissolved 2.56 Comparative Example 7 Cannot be completely dissolved 1.87 Comparative Example 8 Cannot be completely dissolved 2.39 Comparative Example 9 Cannot be completely dissolved 1.72

[0133] As shown in Table 2, the soothing ammonia amide multidimensional co-assemblies of Examples 1, 2, and 5 of this invention all exhibit excellent water solubility. Example 1 (Bosene group) showed the best performance, with a YMJ solubility as high as 3.38 wt.%, followed by Example 3 (rhamnose group), with a YMJ solubility also reaching 3.36 wt.%. The solubility of free YMJ in water is less than 30 μg / mL, while the soothing ammonia amide multidimensional co-assemblies after drying and reconstitution show YMJ solubility reaching 3.38 wt.% and 3.36 wt.%, respectively, representing an improvement of more than 1100 times. This indicates that the soothing ammonia amide multidimensional co-assemblies of this invention effectively improve the water solubility of YMJ.

[0134] In Comparative Example 5, insufficient mixing and homogenization time resulted in solid precipitation upon cooling; in Comparative Example 6, the temperature was not up to standard, and the reactants could not be completely dissolved; in Comparative Example 7, the lack of alcohol assistance resulted in incomplete dissolution of amaranthamide; in Comparative Example 8, the content of small molecule sugar derivatives was too low, resulting in incomplete dissolution of amaranthamide; and in Comparative Example 9, the ultrasonic time was too long, which destroyed the co-assembled particles, leading to solid precipitation upon cooling. Consequently, the samples obtained from Comparative Examples 5-9 could not be completely dissolved, and their water solubility could not be compared with that of the examples.

[0135] Based on molecular dynamics, the co-assembly process of BSY-YMJ with a molar ratio of 1:1 was simulated using Gromacs software. Figure 5 This is a diagram illustrating the co-assembly process of the BSY-YMJ of this invention. (See diagram below.) Figure 5 As shown, when the simulation time is 20 ns, BSY and YMJ gradually aggregate from their initial free-scattering state, with an increase in the number of hydrogen bonds and a decrease in system potential energy, indicating that BSY and YMJ have excellent co-assembly properties. When the simulation time is 60-100 ns, the number of hydrogen bonds and system potential energy gradually stabilize, indicating that the co-assembly process of BSY and YMJ is complete. However, the fluctuations in the number of hydrogen bonds and system potential energy indicate that the BSY-YMJ co-assembly is in a dynamic equilibrium state and is easily affected by the external environment. Therefore, it is necessary to introduce HHJ inclusion to improve its stability. During the assembly process, YMJ, due to its strong hydrophobicity, gradually aggregates towards the center, while BSY accumulates around YMJ. This indicates that BSY can improve the stability and solubility of YMJ in water.

[0136] Based on density functional theory, the interaction between YMJ and XTY in the co-assembly was analyzed using Gauss software. Figure 6 This is an interaction analysis diagram of YMJ and XTY in the soothing oat anthraquinone multidimensional co-assembly of the present invention. Figure 6As shown, using the Independent Gradient Model (IGMH) based on Hirshfeld segmentation and the Atomic In-Molecular Topological Analysis (AIM) method, it can be seen that van der Waals interactions and hydrogen bonding interactions exist between YMJ and BSY. Specifically, the hydroxyl and carbonyl groups of YMJ form multiple hydrogen bonds with the hydroxyl groups of BSY, allowing them to form a dynamically balanced co-assembled structure. In molecular dynamics simulations, the radial distribution function g(r) reflects the spatial distribution of the particles. A higher G(r) peak indicates stronger interactions within the system. The peak indicates a hydrogen bond. The peaks indicate van der Waals interactions. g(r) analysis of the BSY-YMJ co-assemblies ( Figure 6 b) shows that the g(r) between the hydroxyl group of YMJ and the hydroxyl group of BSY, and between the carbonyl group of YMJ and the hydroxyl group of BSY, are both in The presence of sharp, strong peaks nearby indicates that the results of quantum chemical calculations agree well with molecular dynamics simulations.

[0137] Figure 7 This is an ESP analysis diagram of the YMJ, BSY-YMJ co-assembly, and SLT-YMJ co-assembly of the present invention. (See diagram for reference.) Figure 7 As shown, surface electrostatic potential (ESP) analysis ( Figure 7 a) The results show that YMJ 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 BSY-YMJ co-assembly, the folded conformation of YMJ is disrupted due to the strong interaction between BSY and YMJ, indicating better water solubility. Compared with free YMJ, the positive and negative ESP difference of the BSY-YMJ co-assembly is larger, indicating enhanced molecular polarity, which helps improve water solubility. DFT calculations show that there are three strong hydrogen bond interactions between the carboxyl and hydroxyl groups of YMJ and the hydroxyl group of SLT, indicating that they can also form a stable co-assembly. However, SLT did not disrupt the folded conformation of YMJ, which may be because SLT is more hydrophilic than BSY and has a weaker affinity for the benzene ring of YMJ. ESP analysis also shows that compared with free YMJ, the positive and negative ESP difference of the SLT-YMJ co-assembly is larger, indicating enhanced molecular polarity, which helps improve water solubility.

[0138] 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). By analyzing the energy, distribution, and interactions of HOMOs and LUMOs, we can gain a deeper understanding of the electronic structure and chemical behavior of molecules. Figure 8This is a molecular frontier orbital analysis diagram of the YMJ, BSY-YMJ co-assemblies, and SLT-YMJ co-assemblies of this invention. (See diagram for reference.) Figure 8 As shown, the HOMO of YMJ is mainly distributed on the benzene ring linked to the hydroxyl group, while the LUMO is mainly distributed on the benzene ring linked to the carboxyl group, indicating that they are the electron-losing and electron-gaining active sites, respectively. Both the HOMO and LUMO of the BSY-YMJ co-assembly are located on YMJ, indicating that YMJ is the active site of the BSY-YMJ co-assembly. Furthermore, the structural distribution and symmetry of the HOMO and LUMO of the BSY-YMJ co-assembly are the same as those of free YMJ, suggesting that the BSY-YMJ co-assembly has the same biological activity as YMJ. Simultaneously, the lower LUMO and smaller band gap (ΔE) indicate that the BSY-YMJ co-assembly is more likely to gain electrons, resulting in superior reactivity compared to free YMJ. In contrast, the HOMO, LUMO, and ΔE of the SLT-YMJ co-assembly are very similar to those of free YMJ, indicating that the reactivity of the SLT-YMJ co-assembly is comparable to that of free YMJ. These results demonstrate that co-assembly with XTY does not impair the biological activity of YMJ.

[0139] To further verify the formation mechanism of soothing oat anthraquinone multidimensional coassemblies, molecular dynamics simulations were used to explore the optimal conformation, and DFT calculations were combined to analyze the inclusion effect of HHJ on free YMJ and YMJ in TYH. Figure 9 This is an analysis diagram of the inclusion effect of HHJ on YMJ according to the present invention. For example... Figure 9 As shown, free YMJ enters HHJ in a folded conformation, resulting in significant steric hindrance and a weak inclusion interaction energy of only -0.93 kcal / mol. In TYH, however, YMJ, XTY, and HHJ co-assemble; benefiting from the high water solubility of the YMJ-XTY co-assembly, YMJ enters HHJ in a linear conformation, significantly increasing the interaction energy. In BYH and SYH, the interaction energies between YMJ and HHJ are -14.87 and -14.21 kcal / mol, respectively, indicating that the introduction of BSY and SLT greatly enhances the inclusion interaction of HHJ with YMJ.

[0140] Reduced density gradient (RGD) analysis identifies and visualizes regions of weak intermolecular interactions by calculating the ratio of the electron density gradient to the electron density. In RGD isosurface plots, different colors represent different types of interactions: blue areas indicate strong attractive interactions, such as hydrogen bonds or other strong dipole-dipole interactions; green areas indicate weak nonbonded interactions, such as π-π packing and van der Waals forces; and red areas indicate predominantly repulsive interactions, such as repulsive forces generated when atoms are too close together. Figure 10 This is a diagram showing the interactions between the components in the soothing oat anthraquinone multidimensional co-assembly of the present invention. For example... Figure 10As shown, abundant van der Waals interactions exist between the cavities of YMJ and HHJ in TYH, which is highly consistent with the hydrophobic inclusion effect of HHJ on YMJ. Furthermore, abundant hydrogen bonding interactions (indicated by dark arrows) exist between YMJ, XTY, and HHJ in the outer region of the cyclodextrin cavities. This helps enhance the stability of the inclusion effect of HHJ on YMJ, prevents YMJ from escaping from the HHJ cavities, and improves the adaptability of TYH to different solvent environments.

[0141] Deminshu is a commercially available water-soluble YMJ product that exhibits better anti-inflammatory and antipruritic effects than pure YMJ in practical applications. Considering the very low solubility of pure YMJ, which hinders efficacy testing, this invention compares the solubility, stability, and efficacy of a soothing arnaldehyde amide multidimensional co-assembly with Deminshu to comprehensively evaluate the application potential of the soothing arnaldehyde amide multidimensional co-assembly in the cosmetics field.

[0142] (1) Comparison of the solubility and stability of TYH

[0143] Deminex is composed of butylene glycol, pentylene glycol, and YMJ. It exists as a transparent, colorless, and odorless liquid, readily soluble in alcohol, glycerin, surfactants, and commonly used cosmetic oils, exhibiting good formulation compatibility. Because YMJ is a highly effective anti-irritant, antihistamine, anti-allergic, and anti-itch ingredient, Deminex is widely used in products for sensitive skin care, sun protection, and after-sun care. However, Deminex employs a co-solvent solubilization strategy, which places high demands on the solvent environment. Its stability in water-based cosmetics is poor, severely limiting its application.

[0144] Prepare an aqueous solution of monomer with a YMJ content of 0.1 wt.%; prepare aqueous solutions of Deminshu with YMJ contents of 1 wt.% and 2 wt.%; prepare an aqueous solution of soothing oat anthraquinone multidimensional co-assembly with a YMJ content of 2 wt.%, store at room temperature for two weeks, and observe its morphological changes.

[0145] Figure 11 This is a comparison chart of the soothing oat anthraquinone multidimensional co-assembly of the present invention with the soothing properties and stability of YMJ monomer and Deminex. Figure 11 As shown, the monomer aqueous solution with a YMJ content of 0.1 wt.% could not be completely dissolved; when Deminshu was diluted to a YMJ content of 1 wt.% and 2 wt.%, a large amount of solid precipitated, indicating that it is not suitable for water-based cosmetic formulations. However, when the soothing oat anthraquinone multidimensional co-assembly was prepared into an aqueous solution with a YMJ content of 2 wt.%, it remained clear and transparent after being stored at room temperature for two weeks, without precipitation or turbidity, indicating that the stability of the soothing oat anthraquinone multidimensional co-assembly was significantly better than that of Deminshu.

[0146] (2) CCK-8 cytotoxicity assay (BYH)

[0147] The cytotoxicity of soothing arnethamide multidimensional coassemblies was tested using a CCK-8 assay kit and compared with that of commercially available similar product, Deminex.

[0148] A. Experimental Procedure

[0149] 1) After cell resuscitation, passage twice to expand the culture.

[0150] 2) After digesting, centrifuging and resuspending the expanded cultured cells, count them and then seed them into 96-well cell culture plates, with 10,000 cells per well.

[0151] 3) After the cells have completely adhered to the wall, remove the culture medium and add the diluted drug to each of the 96 wells at a rate of 100 μL.

[0152] 4) Place the cells in an incubator and continue culturing for 48 hours.

[0153] 5) Add 10 μL of CCK-8 solution to each well, and place the culture plate in an incubator for 1.5 h.

[0154] 6) Remove the culture plate and read the OD value at 450nm using an ELISA reader.

[0155] 7) Perform the experiment in parallel three times.

[0156] B. Calculation Formula

[0157]

[0158] Sample group OD: OD values ​​of wells containing cells, CCK-8 solution, and drug solution.

[0159] OD value of blank group: cell-free, OD value of CCK-8 solution present.

[0160] Control group OD: OD values ​​of cells in the CCK-8 solution wells.

[0161] C. Analysis software

[0162] Statistical analysis was performed using GrapYMJd.Prism.10.1 software, and curve fitting was performed using Log(agonist) vs. response-variable slope(four parameters).

[0163] Figure 12 This is a comparison of the cytotoxicity of the soothing oat anthraquinone multidimensional co-assembly (BYH) of Example 1 of this invention with free YMJ and Deminex. Figure 12As shown, at various YMJ concentrations, BYH exhibited significantly lower cytotoxicity to HFF-1 compared to Dermacloprid and free YMJ. When the YMJ concentration in Dermacloprid exceeded 100 ppm, the cell viability of HFF-1 rapidly decreased to around 50%, demonstrating extremely strong cytotoxicity. However, with increasing free YMJ concentration, the cell viability of HFF-1 gradually decreased. This is because free YMJ has limited solubility; when its solubility is exceeded, YMJ precipitates in solid form and cannot be absorbed by cells. Even when the YMJ content in BYH reached 500 ppm, the cell viability of HFF-1 remained close to 100%, indicating that the multidimensional assembly strategy significantly improved the biocompatibility of YMJ, suggesting lower skin irritation.

[0164] (3) CCK-8 cytotoxicity assay (SYH)

[0165] The cytotoxicity of soothing arnethamide multidimensional coassemblies was tested using a CCK-8 assay kit and compared with that of commercially available similar product, Deminex.

[0166] A. Experimental Procedure

[0167] 1) After cell resuscitation, passage twice to expand the culture.

[0168] 2) After digesting, centrifuging and resuspending the expanded cultured cells, count them and then seed them into 96-well cell culture plates, with 10,000 cells per well.

[0169] 3) After the cells have completely adhered to the wall, remove the culture medium and add the diluted drug to each of the 96 wells at a rate of 100 μL.

[0170] 4) Place the cells in an incubator and continue culturing for 48 hours.

[0171] 5) Add 10 μL of CCK-8 solution to each well, and place the culture plate in an incubator for 1.5 h.

[0172] 6) Remove the culture plate and read the OD value at 450nm using an ELISA reader.

[0173] 7) Six parallel experiments were conducted.

[0174] B. Calculation Formula

[0175]

[0176] Sample group OD: OD values ​​of wells containing cells, CCK-8 solution, and drug solution.

[0177] OD value of blank group: cell-free, OD value of CCK-8 solution present.

[0178] Control group OD: OD values ​​of cells in the CCK-8 solution wells.

[0179] C. Analysis software

[0180] Statistical analysis was performed using GrapYMJd.Prism.10.1 software, and curve fitting was performed using Log(agonist) vs. response-variable slope(four parameters).

[0181] Figure 13 This is a comparison diagram of the cytotoxicity of the soothing oat anthraquinone multidimensional co-assembly (SYH) of Example 3 of the present invention and Deminex. Figure 13 As shown, at a range of YMJ concentrations, SYH exhibited significantly lower cytotoxicity to HaCaT cells compared to Deminshu. At a YMJ concentration of 250 ppm, the HaCaT cell survival rate with SYH was 81.65%, while that with Deminshu was only 20.81%. Furthermore, at 500 ppm YMJ, the cell survival rate of Raw 264.7 cells treated with SYH approached 100%, while the survival rate of Raw 264.7 cells treated with Deminshu was close to 0% at a YMJ concentration of 250 ppm. This indicates that SYH also demonstrates higher biocompatibility and lower skin irritation compared to Deminshu.

[0182] (4) ROS detection

[0183] Antioxidant activity is a crucial strategy for mitochondrial skincare. YMJ contains phenolic hydroxyl groups, which endow it with excellent antioxidant effects. Mitochondria, as the cell's energy factories, produce reactive oxygen species (ROS) during normal physiological function. However, excessive ROS can lead to oxidative stress, damaging cellular components, including mitochondrial DNA, proteins, and lipids, thereby causing cellular dysfunction and aging. UVA (long-wave ultraviolet radiation, wavelength 320-400nm) can penetrate the dermis, generating oxidative stress, inducing mitochondrial damage and dysfunction, and thus promoting skin cell aging. UVB (medium-wave ultraviolet radiation, wavelength 280-315nm) mainly acts on the epidermis, exacerbating oxidative stress and damage to skin cells by activating inflammatory signaling pathways and increasing ROS production. Therefore, this experiment established a HaCaT stress model of immortalized human epidermal cells through the synergistic effect of UVA and UVB to simulate skin aging caused by mitochondrial damage and dysfunction. Lower ROS levels indicate stronger antioxidant capacity in the sample.

[0184] A. Experimental Procedure

[0185] 1) After cell resuscitation, passage twice to expand the culture.

[0186] 2) After digesting, centrifuging, resuspending, counting, and seeding the expanded cultured cells into 24-well cell culture plates.

[0187] 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). Details are shown in Table 3 below.

[0188] Table 3 Experimental Groups

[0189]

[0190] 4) After the cells have completely adhered to the culture medium, remove the medium and add 500 μL of the diluted drug to each of the 24 wells. Continue culturing for 48 h.

[0191] 5) Remove the culture medium, add a small amount of PBS, and irradiate the cells using a combination of UVB and UVA. Cover the NC group with aluminum foil. After irradiation, add the diluted drug to each of the 24 wells at a rate of 500 μL. Perform ROS detection after culturing for 30 min.

[0192] 6) Dilute DCFH-DA 1:1000 with complete culture medium to a final concentration of 10 μmol / L. Remove the cell culture medium and add an appropriate volume of diluted DCFH-DA. The volume added should be enough to fully cover the cells. Incubate at 37°C for 20 minutes. Wash the cells three times with complete culture medium to thoroughly remove any DCFH-DA that has not yet entered the cells.

[0193] 7) Use a multi-functional microplate reader for detection.

[0194] 8) Perform three parallel experiments.

[0195] B. Calculation Formula

[0196]

[0197] Figure 14 This is a comparison chart of the ROS scavenging capabilities of the soothing oat anthraquinone multidimensional co-assembly of this invention and Deminshu. (See figure) Figure 14 As shown, the ROS level in the MC group was significantly higher than that in the NC and PC groups, indicating successful modeling. Treatment with Deminex and the soothing alanine multidimensional co-assembly significantly reduced ROS levels compared to the MC group, indicating that both had good ROS scavenging capabilities. At a YMJ concentration of 50 ppm, the ROS scavenging capabilities of the soothing alanine multidimensional co-assembly were comparable to those of Deminex. At a YMJ concentration of 100 ppm, the ROS scavenging capability of the soothing alanine multidimensional co-assembly was significantly better than at lower concentrations, indicating a dose-dependent effect. Due to its high cytotoxicity, Deminex could not be tested at higher concentrations.

[0198] (5) SOD detection

[0199] Superoxide dismutase (SOD), as an endogenous antioxidant enzyme, catalyzes the dismutation of superoxide anion free radicals into hydrogen peroxide and oxygen, thereby effectively reducing oxidative stress. In cells, SOD activity directly reflects the cell's antioxidant defense capacity. When cells are subjected to oxidative stress, SOD activity increases accordingly to scavenge excess superoxide anion free radicals, maintaining mitochondrial function and cell health. Therefore, higher SOD activity indicates a stronger antioxidant capacity in the sample.

[0200] A. Experimental Procedure

[0201] 1) After cell resuscitation, passage twice to expand the culture.

[0202] 2) After digesting, centrifuging, resuspending, counting, and seeding the expanded cultured cells into 24-well cell culture plates.

[0203] 3) Experimental grouping: After plating, the cells were divided into negative control group (NC), positive control group (PC), model control group (MC) and sample group (specified drug concentration), as shown in Table 3.

[0204] 4) After the cells have fully adhered to the culture medium, remove the medium and irradiate the cells with a combination of UVB and UVA. For the NC group, cover with aluminum foil. After irradiation, add 2000 μL of the diluted drug to each of the 6 wells. Perform SOD detection after 48 hours of culture.

[0205] 5) Aspirate the cell culture medium, wash once with PBS at 4°C, add 100 μL of SOD sample preparation solution, and gently pipette to fully lyse the cells. Centrifuge at 12000 rpm for 3-5 minutes at 4°C, and use the supernatant as the sample to be tested.

[0206] 6) After the sample is prepared, use the BCA protein concentration assay kit to determine the protein concentration. Based on the protein concentration and the expected amount of protein to be used, dilute the sample appropriately with the SOD detection buffer provided in this kit.

[0207] 7) Prepare WST-8 / enzyme working solution, prepare reaction start-up working solution, and test samples.

[0208] 8) Use an enzyme-linked immunosorbent assay (ELISA) reader for detection.

[0209] 9) Perform three parallel experiments.

[0210] B. Calculation Formula

[0211] 1) Standard curve fitting formula

[0212] y = aX 2 +bX+c

[0213] X:OD 536 y: Protein concentration (mg / mL)

[0214] 2) Standard curve fitting formula

[0215]

[0216] Figure 15 This is a comparison diagram of the SOD-promoting activity of the soothing oat anthraquinone multidimensional co-assembly of this invention and Deminshu. Figure 15 As shown, the SOD activity in the MC group was significantly lower than that in the NC and PC groups, indicating successful modeling. Treatment with Deminex and the soothing alanine-based multidimensional co-assembly significantly increased SOD activity compared to the MC group, indicating that both have good SOD-promoting capabilities. When the YMJ concentration was 50 ppm, the SOD activity of the soothing alanine-based multidimensional co-assembly was comparable to that of HaCaT cells treated with Deminex. When the YMJ concentration was further increased, the SOD-promoting antioxidant activity of the soothing alanine-based multidimensional co-assembly significantly improved to be comparable to that of PC, indicating a dose-dependent effect, and excellent antioxidant activity at high concentrations. However, due to its high cytotoxicity, Deminex could not be tested at higher concentrations.

[0217] (6) TNF-α and IL-8 gene detection

[0218] Anti-inflammation is another important strategy in mitochondrial skincare. YMJ has been proven to have anti-inflammatory and soothing effects. TNF-α and IL-8 play important roles in inflammatory responses, and their level changes are closely related to mitochondrial dysfunction, which is of great significance in the fields of skin health and skincare. TNF-α is a major pro-inflammatory cytokine that can activate transcription factors such as NF-κB and promote the production of various inflammatory mediators (such as IL-8, IL-6, and PGE2), thereby amplifying the inflammatory response. IL-8 is a chemokine that can attract neutrophils and T cells to the site of inflammation, further exacerbating the inflammatory response. UV radiation is one of the key factors leading to skin inflammation and sensitivity. UVB radiation can induce DNA damage in skin cells, activate inflammatory signaling pathways, and lead to symptoms such as skin erythema, edema, and pain; while UVA can trigger oxidative stress, further aggravating inflammation. The synergistic effect of the two can activate inflammatory signaling pathways such as NF-κB, AP-1, and MAPK, prompting the large-scale expression and release of pro-inflammatory cytokines, causing post-sunburn "burning" and "stirring" sensations. Therefore, this experiment established a HaCaT inflammation model through synergistic stimulation by UVA and UVB to simulate skin aging caused by mitochondrial damage and dysfunction. Lower expression levels of TNF-α and IL-8 genes indicate stronger anti-inflammatory and soothing effects of the sample.

[0219] A. Experimental Procedure

[0220] 1) After cell resuscitation, passage twice to expand the culture.

[0221] 2) After digesting, centrifuging, resuspending, counting, and seeding the expanded cultured cells into 24-well cell culture plates.

[0222] 3) Experimental grouping: After plating, the cells were divided into negative control group (NC), positive control group (PC), model control group (MC) and sample group (specified drug concentration), as shown in Table 3.

[0223] 4) After the cells have completely adhered to the wall, remove the culture medium, add a small amount of PBS, and irradiate the cells with UVB and UVA combined. The NC group is covered with aluminum foil. After irradiation, add the diluted drug to the 24 wells, 500 μL per well.

[0224] 5) Place the cells in an incubator and continue culturing for 24 hours.

[0225] 6) Wash three times with PBS, add 1 mL of RNA extraction buffer, pre-cool on ice for 5 min, and gently pipette.

[0226] 7) Add 400 μL of chloroform, invert the centrifuge tube for 15 seconds, mix thoroughly, and let stand for 3 minutes.

[0227] 8) Centrifuge at 12000 rpm for 10 min at 4℃.

[0228] 9) Transfer 400 μL of supernatant to a new centrifuge tube, add 550 μL of isopropanol, and mix by inverting.

[0229] 10) Place at -20°C for 15 minutes.

[0230] 11) Centrifuge at 12000 rpm for 10 min at 4℃. The white precipitate at the bottom of the tube is RNA.

[0231] 12) Remove the liquid and wash the precipitate with 1.5 mL of 75% ethanol.

[0232] 13) Centrifuge at 12000 rpm for 5 min at 4℃.

[0233] 14) Remove the liquid completely and place the centrifuge tube on the clean bench and blow it for 3 minutes.

[0234] 15) Add 15 μL of Water Nuclease-Free to dissolve the RNA.

[0235] 16) 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 onto the detection base, lower the sample arm, and use the software on the computer to start the absorbance detection.

[0236] 17) Dilute the excessively high concentration of RNA by an appropriate ratio to achieve a final concentration of 200 ng / μL.

[0237] 18) Prepare the reverse transcription reaction system, mix gently 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, making 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.

[0238] 19) The experiment was conducted in parallel three times.

[0239] B. Calculation Formula

[0240] 1) RNA expression level

[0241] ΔC(t)=C(t) 目的基因 -C(t) 内参基因

[0242] ΔΔC(t)=ΔC(t) 样品组 -ΔC(t) 对照组

[0243] relative RNA expression level = 2 ΔΔΔ(Δ)

[0244] 2) Rate of change detection

[0245]

[0246] Figure 16 This is a comparison diagram of the soothing oat anthraquinone multidimensional co-assembly of the present invention and the inhibition of mid-to-upper-level inflammatory gene expression by Deminshu. (See diagram for reference.) Figure 16As shown, the expression levels of TNF-α and IL-8 genes in the MC group were significantly higher than those in the NC and PC groups, indicating successful modeling. Treatment with Deminshu and the soothing ammonia-anthraamide multidimensional co-assembly significantly reduced the expression levels of both TNF-α and IL-8 genes compared to the model group, indicating that both have strong inhibitory effects on upstream mediators of the inflammatory signaling pathway. When the YMJ concentration was 50 ppm, the inhibition rate of TNF-α gene expression by the soothing ammonia-anthraamide multidimensional co-assembly was higher than that of Deminshu. With increasing concentration, the inhibition rate of TNF-α gene expression by the soothing ammonia-anthraamide multidimensional co-assembly further increased, significantly better than Deminshu and not significantly different from PC. When the YMJ concentration was 50 ppm, the inhibition rate of IL-8 gene expression by the soothing ammonia-anthraamide multidimensional co-assembly was comparable to that of Deminshu. With increasing concentration, the inhibition rate of IL-8 gene expression by the soothing ammonia-anthraamide multidimensional co-assembly increased significantly to the point where it was not significantly different from PC. The above results indicate that the anti-inflammatory effect of the soothing alanine anthraquinone multidimensional co-assembly is dose-dependent, with higher concentrations exhibiting stronger inhibitory effects on inflammatory amplification and cascade reactions. When YMJ in the soothing alanine anthraquinone multidimensional co-assembly reaches a certain concentration, its anti-inflammatory effect is excellent, while Deminex, due to its high cytotoxicity, cannot be used at high concentrations.

[0247] (7) PGE2 detection

[0248] Prostaglandin PGE2 levels are closely related to mitochondrial health. PGE2 is an inflammatory mediator produced 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 IL-8 can promote PGE2 production by activating COX-2 expression. During cellular senescence, PGE2 signaling promotes glucose conversion to glycogen via its EP2 receptor, reducing glucose utilization and limiting mitochondrial respiration, leading to decreased mitochondrial function. PGE2 plays an important role in physiological and pathological processes such as inflammatory responses, pain perception, fever, and vasodilation, and is one of the important indicators for assessing the intensity of skin inflammation. Therefore, this experiment used the mouse macrophage cell line (RAW 264.7) as a model cell and used LPS to simulate inflammatory stimulation. By detecting the level of the inflammatory mediator PGE2 in the cell supernatant, the inhibitory effect of the sample on the LPS-induced inflammatory response was evaluated. A higher inhibition rate indicates a stronger anti-inflammatory and soothing effect of the sample.

[0249] A. Experimental Procedure

[0250] 1) After cell resuscitation, passage twice to expand the culture.

[0251] 2) After digesting, centrifuging, resuspending, counting, and seeding the expanded cultured cells into 24-well cell culture plates.

[0252] 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 4 below:

[0253] Table 4 Experimental Groups

[0254]

[0255] 4) After the cells have completely adhered to the wall, remove the culture medium and add the diluted drug to each of the 24 wells at a rate of 500 μL.

[0256] 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.

[0257] 6) Carefully collect the supernatant. If precipitation occurs during storage, centrifuge again.

[0258] 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.

[0259] 8) Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0260] 9) Solution preparation: Dilute the concentrated washing solution with distilled water and set aside for later use.

[0261] 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.

[0262] 11) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for blank wells.

[0263] 12) Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes.

[0264] 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.

[0265] 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.

[0266] 15) Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0267] 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.

[0268] 17) Calculation: Use software to fit the standard curve and calculate the measured value based on the absorbance.

[0269] 18) The experiment was conducted in parallel three times.

[0270] B. Calculation Formula

[0271] 1) Concentration calculation formula

[0272] y = (AD) / [1 + (x / C)] B ]+D

[0273] x: OD450; y: Sample concentration (pg / mL).

[0274] 2) Formula for calculating the rate of change

[0275]

[0276] C. Analysis software

[0277] 1) Statistical analysis was performed using GrapYMJd.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).

[0278] 2) Use ELISA Calc software to calculate data, and use Logistic curve fitting 2 (four parameters) to perform curve fitting and calculate sample concentration.

[0279] Figure 17 This is a comparison diagram of the inhibitory effects of the soothing oat anthraquinone multidimensional co-assembly of the present invention and Deminex on downstream inflammatory mediators. (See diagram for reference.) Figure 17As shown, 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 YMJ group and the soothing alanine multidimensional co-assembly group were significantly lower than those in the MC group, indicating that both YMJ and the soothing alanine multidimensional co-assembly have anti-inflammatory effects. When the YMJ concentration was 50 ppm, the inhibitory effect of the soothing alanine multidimensional co-assembly on PGE2 was comparable to that of Deminex. With increasing concentration, the inhibitory effect of the soothing alanine multidimensional co-assembly on PGE2 further increased to be comparable to that of PC, significantly better than low-concentration Deminex, indicating that the anti-inflammatory effect of the soothing alanine multidimensional co-assembly is dose-dependent, and can significantly inhibit downstream inflammatory responses at high concentrations. However, Deminex, due to its high cytotoxicity, cannot be used at high concentrations.

[0280] (8) Stability test

[0281] To verify the stability of the soothing amaranthamide multidimensional co-assembly, a solution containing 2 wt.% YMJ was prepared, and its pH, conductivity, and color changes were tested after storage at -15℃, room temperature, 45℃, and under light conditions for 4 and 12 weeks. The specific test results are shown in Table 5 below.

[0282] Table 5. Stability test results of 2 wt.% soothing oat anthraquinone multidimensional coassemblies

[0283]

[0284] Figure 18 This is a stability test diagram of the soothing oat anthraquinone multidimensional co-assembly of the present invention, as shown in Table 5 and... Figure 18 As can be seen, both the solution and powder of the soothing arnaldehyde amide multidimensional co-assembly exhibit good stability under high and low temperature conditions, with pH, ​​conductivity, and appearance remaining basically stable after 12 weeks of storage. Compared with the powder, the pH and conductivity of the solution fluctuate slightly more, which may be due to the presence of phenolic hydroxyl groups in YMJ, making it easily oxidized. In aqueous solution, the soothing arnaldehyde amide multidimensional co-assembly is in a dynamic equilibrium state, with continuous formation and dissociation of inclusion compounds, resulting in a small amount of YMJ existing in a free form, making it susceptible to environmental influences. After drying, the soothing arnaldehyde amide multidimensional co-assembly exhibits uniform co-assembly, allowing YMJ to be well encapsulated by BSY and HHJ, further improving its stability.

[0285] (9) Skin irritation test

[0286] 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.

[0287] A. Experimental Procedure

[0288] A closed patch test was conducted on at least 30 healthy volunteers. Participants had to be free of any skin diseases or other serious illnesses within the past three months and had not taken any antibiotics or hormonal medications. A soothing oat anthraquinone multivitamin assembly solution containing 0.1 wt.% YMJ was prepared. 0.020-0.025 g of the solution was placed in a suitable patch applicator and applied to the flexor surface of the participant'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 in the *Cosmetic Safety Technical Specifications* (2015 edition).

[0289] Figure 19 This is a grading chart of skin reactions in a closed patch test.

[0290] Evaluation principles

[0291] 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. Specific test results are shown in Table 6 below:

[0292] Table 6. Patch test results of soothing oat anthraquinone multidimensional coassemblies

[0293]

[0294]

[0295] As shown in Table 6, all 30 subjects in this experiment had negative reactions, indicating that the 0.6 wt.% solution of the soothing oat anthraquinone multidimensional co-assembly (YMJ concentration of 0.1 wt.%) has excellent safety and no skin irritation.

[0296] (10) Human body redness and itching relief experiment

[0297] 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.

[0298] Experimental steps

[0299] Seventeen healthy volunteers, aged 18-60 years, were selected. They had no skin diseases or other serious illnesses within the past three months and had not taken any antibiotics or hormone medications. The inner arm of the subjects was used as the test site to evaluate the anti-allergic effect of aqueous solutions of different concentrations of soothing ammoniac multidimensional co-assemblies (YMJ concentration of 0.05-0.1 wt.%), and compared with aqueous solutions containing a mixture of YMJ, XTY, and HHJ at the same concentrations. Results showed that aqueous solutions of the precursor mixture at a YMJ concentration of 0.05-0.1 wt.% could not be completely dissolved, indicating that simple physical mixing methods cannot significantly improve the water solubility of YMJ. A 3% histamine solution was applied to the inner arm of the subjects. After obvious allergic signs appeared, three equally sized allergic areas were marked. One marked area served as a blank control, while the remaining two marked areas were treated with appropriate amounts of different concentrations of the same sample. The left and right arms were treated with aqueous solutions of the soothing ammoniac multidimensional co-assemblies and aqueous solutions of the precursor mixture at equimolar concentrations, respectively, and the skin condition was compared with the blank area. Thirty minutes later, the desensitization effect of the test samples was scored and recorded (volunteer self-assessment). The scoring criteria are shown in Table 7: self-assessment score for redness reduction 0-5 (the higher the score, the better the redness reduction effect), and self-assessment score for itching relief 0-5 (the higher the score, the better the itching relief effect).

[0300] Table 7 Scoring criteria for redness reduction and itch relief in antihistamine allergy tests.

[0301]

[0302] Figure 20 This is a comparison chart showing the anti-allergic effects of the soothing oat anthraquinone multidimensional co-assembly of the present invention and its aqueous solution. (See figure below.) Figure 20As shown, the skin exhibited obvious allergic reactions after applying a 3% histamine solution, indicating successful modeling. When the YMJ content was 0.05 wt.%, the anti-redness effect of the soothing alanine-based multidimensional co-assembly was significantly better than that of the aqueous mixture, while its antipruritic effect was comparable. When the YMJ content was 0.1 wt., both the anti-redness and antipruritic effects of the soothing alanine-based multidimensional co-assembly were significantly better than those of the aqueous mixture, possibly due to the poor solubility and low bioavailability of YMJ in the aqueous mixture. Furthermore, with increasing concentration, the anti-redness and antipruritic effects of the soothing alanine-based multidimensional co-assembly showed an increasing trend, while those of the aqueous mixture showed a decreasing trend. This may be because higher concentrations of the aqueous mixture lead to increased YMJ precipitation and increased skin irritation. These results demonstrate that the soothing alanine-based multidimensional co-assembly provides a new technical pathway for the efficient utilization of alanine.

[0303] (11) Repair efficacy test

[0304] Collagen is the most abundant protein in the extracellular matrix, providing mechanical strength and support for tissues. It forms a fibrous network, providing a scaffold for cell attachment and growth. Glycosaminoglycans (GAGs) can bind to collagen fibers to form complex structures, jointly maintaining tissue homeostasis. Furthermore, GAGs regulate cell adhesion, migration, and proliferation by binding to cell surface receptors; these functions complement the structural support provided by collagen. They work synergistically in normal physiological processes, influence each other in disease states, and play a crucial role in tissue repair and regeneration. UV radiation is one of the main factors leading to photoaging of the skin, inducing oxidative stress in skin cells, which in turn damages extracellular matrix components such as GAGs and collagen, resulting in decreased skin elasticity, increased wrinkles, and other signs of aging. XTY can inhibit photoaging of the skin by promoting the generation and remodeling of GAGs and collagen in the extracellular matrix. Therefore, this project established an HFF-1 cell photoaging model through synergistic stimulation by UVA and UVB to evaluate the repair efficacy of a soothing oat anthraquinone multidimensional co-assemblies. The higher the expression of type I collagen (Col-I) and GAGs, the stronger the repair efficacy of the sample.

[0305] A. Col-I Experimental Procedure

[0306] 1) After cell resuscitation, the cells were passaged twice for expanded culture. The expanded cells were digested, centrifuged, resuspended, counted, and then seeded into 24-well cell culture plates. Experimental grouping: The seeded cells were divided into a negative control group (NC), a model control group (MC), and a sample group (specified drug concentration), as shown in Table 8.

[0307] Table 8 Experimental Groups

[0308]

[0309] 2) After the cells have fully adhered to the culture medium, remove the culture medium and irradiate the cells with a combination of UVB and UVA. The NC group was covered with aluminum foil. After irradiation, add 500 μL of the diluted drug to each of the 24 wells. Continue culturing for 48 h, and then collect the supernatant for Col-I assay.

[0310] 3) Follow the instructions for the Type I Collagen Kit (Elabscience, E-EL-H0869c / 96T): serially dilute the standards and dilute the samples appropriately; add the standards / samples to the pre-coated ELISA plate and incubate.

[0311] 4) Washing: Discard the liquid and wash 4 times with detergent.

[0312] 5) Add enzyme: Add 50 μL of biotinylated antibody to each well except for the blank wells, and incubate twice.

[0313] 6) Color development: Add streptavidin-HRP and TMB for color development, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0314] 7) Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0315] 8) Measurement: Zero the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm using an ELISA reader.

[0316] 9) Plot a standard curve on graph paper with the concentration of the standard as the x-axis and the OD value as the y-axis. Find the corresponding concentration of the sample from the standard curve based on the OD value of the sample, and then multiply it by the dilution factor to get the actual concentration of the sample.

[0317] 10) Perform three parallel experiments.

[0318] B.GAGs Experimental Procedure

[0319] 1) The HFF-1 cell culture method and experimental grouping are the same as above.

[0320] 2) After incubation, collect the supernatant and detect the glycosaminoglycan content in the sample according to the glycosaminoglycan kit. Detect the absorbance at 450 nm using an ELISA reader.

[0321] 3) Plot a standard curve on graph paper with the concentration of the standard as the x-axis and the OD value as the y-axis. Find the corresponding concentration of the sample from the standard curve based on the OD value of the sample, and then multiply it by the dilution factor to get the actual concentration of the sample.

[0322] 4) Perform three parallel experiments.

[0323] Figure 21This is a diagram illustrating the repair efficacy of the soothing oat anthraquinone multidimensional co-assembly of the present invention. (Example) Figure 21 As shown in the figure, the expression levels of Col-I and GAGs in the MC group were significantly lower than those in the NC group, indicating that the skin photoaging model was successfully established. The expression levels of Col-I and GAGs in the soothing arnaldehyde multidimensional co-assembly group were significantly higher than those in the MC group, indicating that the soothing arnaldehyde multidimensional co-assembly retains the repair efficacy of XTY and can inhibit skin photoaging by promoting the generation and remodeling of GAGs and collagen in the extracellular matrix. These results demonstrate that the soothing arnaldehyde multidimensional co-assembly can exert a synergistic and complementary effect of YMJ anti-inflammatory and XTY repair, providing better mitochondrial skincare efficacy.

[0324] In summary, this invention proposes an innovative soothing oat anthraquinone-based multidimensional co-assembly. Compared to similar commercially available products, this invention enhances the water solubility and diffusivity of YMJ through the co-assembly of XTY and YMJ, and subsequently utilizes the inclusion effect of HHJ to enhance the stability of the system. Simultaneously, by leveraging the secondary interactions between XTY and YMJ, as well as HHJ, the affinity between YMJ and HHJ is further enhanced, thereby achieving stronger inclusion and a more stable system that comprehensively meets the product requirements for mitochondrial skincare.

[0325] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a soothing avenanthramide-based polydivalent co-assemblies, characterized in that, Comprising the following steps: S1. Adding small molecule sugar derivatives into alcohol-water mixture, mixing uniformly, under the condition of continuous stirring, heating to 50-70℃, adding avenanthramide and cyclodextrin, mixing thoroughly; S2. Reducing the mixture obtained in S1 to room temperature, centrifuging, filtering, collecting the filtrate, rotary evaporation to paste, vacuum drying, grinding, sealing storage, obtaining the avenanthramide multi-dimensional co-assembly.

2. A method of preparing the soothing oat avenanthramide polydimensional co-assembly of claim 1, characterized by: The mass ratio of avenanthramide, small molecule sugar derivatives and cyclodextrin in S1 is 1:(1-3):(2-10).

3. A method of preparing the soothing oat avenanthramide polydimensional co-assembly of claim 1, characterized by: The small molecule sugar derivatives in S1 are one or more of the following: boswellic acid, rhamnose, glycerol glucoside, fucose, xylitol, sorbitol.

4. A process for the preparation of the soothing oat avenanthramide polydivalent co-assemblies of claim 1, characterized by: The alcohol in the alcohol-water mixture in S1 is ethanol and / or isopropyl alcohol, with a concentration of 5-15wt.%.

5. A method of preparing the soothing oat avenanthramide polydivalent co-assemblies of claim 1, characterized by: The cyclodextrin in S1 is one or more of the following: ɑ-cyclodextrin, β-cyclodextrin, γ-cyclodextrin.

6. A method of preparing the soothing oat avenanthramide polydivalent co-assemblies of claim 1, characterized by: The mass ratio of small molecule sugar derivatives and alcohol-water mixture in S1 is 1:(10-50).

7. A method of preparing the soothing oat avenanthramide polydimensional coassemblies of claim 1, characterized by: The thorough mixing in S1 is by homogenization or ultrasonic mixing.

8. A method of preparing the soothing oat avenanthramide polydimensional coassemblies of claim 6, characterized by: The homogenization is 3 cycles at 25000-35000 psi; the ultrasonic mixing is 1 hour at 10-25 kHz.

9. A soothing oat avenanthramide class multi-dimensional co-assemblies, characterized in that, Prepared according to the method of any one of claims 1-8.

10. The soothing avenanthramide multi-dimensional co-assembly of claim 9 for use in the manufacture of a skin care product or a medicine for external use.

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