Relieving type avenin-fermented dendrobium oligosaccharide co-assembly and preparation method thereof

Abstract

The application belongs to the field of biological medicine, cosmetics, personal care and health, and provides a soothing oat alkali-Dendrobium officinale oligosaccharide co-assembly, a preparation method and application thereof, the preparation method comprising the following steps: S1. adding oligosaccharide into an alcohol-water mixed solution, uniformly mixing, adding oat alkali and cyclodextrin, and ultrasonically mixing; S2. reducing the mixture to room temperature, centrifuging, filtering, collecting the filtrate, vacuum drying after rotary evaporation to a paste, grinding, and sealing storage, to obtain the soothing oat alkali-Dendrobium officinale oligosaccharide co-assembly. Compared with the same products on the market, the solubility of oat alkali is increased by more than 1000 times through self-assembly of oat alkali, cyclodextrin and oligosaccharide, the oligosaccharide can not only play a synergistic biological effect with oat alkali, but also the good compatibility between oligosaccharide and cyclodextrin helps to improve the drug loading capacity of cyclodextrin, improve the stability, the synergistic effect of each component in the system, and better biological effect than single component or binary assembly system, fully meeting the product demand of mitochondrial energy skin care.

Description

Technical Field

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

[0002] Oat alkaloids are an active ingredient extracted from oats, possessing significant anti-inflammatory, anti-allergic, and antipruritic effects. Their mechanism of action primarily involves inhibiting the degradation of nuclear factor NF-κB-α, which is directly related to inflammation, in keratinocytes, thereby preventing the phosphorylation of the p65 protein subunit of NF-κB and thus blocking the process of cellular inflammation. As a potent non-steroidal anti-inflammatory and antipruritic natural ingredient, oat alkaloids have a potency comparable to hydrocortisone but without the side effects of steroid hormones. Furthermore, oat alkaloids have strong antioxidant properties, effectively combating free radical damage to the skin and preventing skin aging. Therefore, oat alkaloids are widely used in personal care and cosmetic products, commonly found in infant antipruritic creams, eczema and milk rash creams, sensitive skin repair masks, insect bite care products, shampoos, and shower gels. However, oat alkaloids have poor water solubility, with a solubility of less than 30 μg / mL in water at room temperature. This limits their application in water-based products and may affect their absorption efficiency on the skin, thereby reducing product efficacy. Furthermore, oat alkaloids are pH sensitive and easily degrade in acidic or alkaline environments.

[0003] To address the solubility issue of oat alkaloids, researchers have developed various techniques, such as using alcohol solvents (e.g., Deminex), nanocarriers (e.g., Nasumin), and converting them into salt forms. While alcohol solvents and salt forms significantly improve the solubility of oat alkaloids in water, they still suffer from poor stability, such as precipitation under dilution, high concentrations, or specific pH conditions. Nanocarrier technology encapsulates oat alkaloids within nanocarriers, making them more stable in aqueous systems, less prone to precipitation, and significantly improving absorption efficiency and bioavailability. However, traditional nanocarrier technologies require large amounts of surfactants, leading to complex formulations, cumbersome processes, high production costs, low drug loading, and safety concerns. Self-assembly technology, on the other hand, offers a novel and highly efficient drug delivery method. The self-assembly process primarily utilizes the physicochemical properties of the active ingredient itself, spontaneously forming nanoscale structures under specific conditions. These structures form molecular aggregates through weak non-covalent interactions such as hydrogen bonds, van der Waals forces, π-π stacking, electrostatic interactions, and coordination bonds, without altering the original molecular structure, thus having minimal impact on drug activity. Compared with traditional nanocarrier technology, self-assembly technology has advantages such as simple preparation process, low production cost, high environmental friendliness, and no need for special equipment.

[0004] Carbohydrates possess a variety of biological functions, including energy supply, cell recognition, immune regulation, antioxidation, anti-inflammation, and promotion of gut health, and are widely used in cosmetics and biomedicine. The degree of polymerization is one of the key factors affecting the biological activity of carbohydrates. Studies have shown that polysaccharides (>10 sugar units) have high steric hindrance, making them difficult to bind to receptors; monosaccharides lack the necessary spatial conformation; oligosaccharides have better biological activity, and their moderate molecular weight is conducive to transmembrane transport. Simultaneously, carbohydrates are rich in active groups such as hydroxyl and carboxyl groups, and can self-assemble with other components through non-covalent interactions such as hydrogen bonding and hydrophobic interactions to form nanostructures, thereby enhancing solubility and transdermal efficiency. For example, cyclodextrins are cyclic oligosaccharides linked by α-1,4-glycosidic bonds, possessing hydrophobic cavities and hydrophilic outer walls. They can load hydrophobic drugs through host-guest inclusion complexes and are often used in nanocarrier technology. However, cyclodextrin inclusion complexes have strict requirements on the size of the guest molecules. If the guest molecules are too large, they cannot enter the cavities of the cyclodextrin to form stable inclusion complexes. In order to encapsulate the drug within the cyclodextrin cavity as much as possible, the amount of cyclodextrin used is often much greater than the amount of drug used, resulting in a viscous system, restricted drug diffusion, insufficient encapsulation, and low drug loading.

[0005] Therefore, it is of great significance to provide a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly that can effectively improve the water solubility and stability of oat alkaloid products and has excellent activity effects, as well as its preparation method. Summary of the Invention

[0006] Given the existing problems of low water solubility and stability, low encapsulation efficiency, and the need for further improvement in the activity of oat alkaloid products, this invention provides a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly, its preparation method, and its application. This invention addresses the solubility and stability issues of oat alkaloid through the self-assembly of oat alkaloid, cyclodextrin, and oligosaccharides. The oligosaccharides not only exert synergistic biological effects with oat alkaloid, but also exhibit good compatibility with cyclodextrin, which helps to solubilize oat alkaloid, increase the drug loading of cyclodextrin, and reduce the release of oat alkaloid from cyclodextrin cavities, thus improving the stability of the cyclodextrin-oat alkaloid inclusion complex. The preparation process is simple, surfactant-free, and highly safe. The synergistic effect of the components in the system demonstrates better biological efficacy than single-component or binary assembly systems, showing promising application prospects in skincare products.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly includes the following steps:

[0009] S1. Add oligosaccharides to the alcohol-water mixture, mix well, and heat to 50-70℃ while continuously stirring. Add oat base and cyclodextrin, and sonicate for 30-60 minutes.

[0010] 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 soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly.

[0011] Furthermore, the mass ratio of oat base, oligosaccharide, and cyclodextrin in S1 is 1:(1-3):(5-20).

[0012] Furthermore, the oligosaccharide mentioned in S1 is Dendrobium officinale oligosaccharide.

[0013] Furthermore, the Dendrobium officinale oligosaccharide mentioned in S1 is an oligosaccharide extracted from the traditional Chinese medicine Dendrobium officinale.

[0014] Furthermore, in the alcohol-water mixture described in S1, the alcohol is ethanol and / or isopropanol, with a concentration of 10-15 wt.%.

[0015] Furthermore, the cyclodextrin in S1 is one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0016] Furthermore, the cyclodextrin in S1 is methyl-β-cyclodextrin and / or hydroxypropyl-β-cyclodextrin.

[0017] Furthermore, the mass ratio of the oligosaccharide and alcohol-water mixture in S1 is 1:(10-50).

[0018] Furthermore, the ultrasound mixture described in S1 is ultrasound at 10-25 kHz.

[0019] Furthermore, the centrifugation described in S2 is centrifugation at 3000 rpm for 10-15 min.

[0020] Furthermore, the filtration described in S2 is to filter to below 0.45 μm.

[0021] Another object of the present invention is to provide a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly.

[0022] A soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly is prepared according to the preparation method of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly described in any one of the preceding claims.

[0023] Another object of the present invention is to provide an application of a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly.

[0024] Application of the above-mentioned soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly in skin care products.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] (1) The soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention solves the solubility and stability problems of oat alkaloid through the self-assembly of oat alkaloid, cyclodextrin and oligosaccharide. Due to its strong hydrophobicity, free oat alkaloid can only enter cyclodextrin in a folded conformation, resulting in large steric hindrance and weak inclusion effect (-0.93kcal / mol), making the system unstable. However, the strong interaction between oligosaccharide and oat alkaloid changes the folded conformation of oat alkaloid, improving its water solubility and molecular flexibility. This allows it to enter the cavities of cyclodextrin in a linear conformation, resulting in stronger inclusion (-19.44 kcal / mol) and a more stable system. Through the organic combination of oligosaccharide solubilization and cyclodextrin inclusion, the water solubility of alfalfa is increased by approximately 700 times compared to its free state. Furthermore, the synergistic effect of alfalfa and Dendrobium officinale oligosaccharides makes the antioxidant, anti-inflammatory, repair, and anti-allergic effects of the alfalfa-Dendrobium officinale oligosaccharide-cyclodextrin assembly significantly superior to equimolar amounts of free alfalfa and Dendrobium officinale oligosaccharides. In addition, compared to commercially available similar products like Deminshu, it exhibits lower cytotoxicity, better safety and stability; and compared to precursor mixtures, its anti-redness and antipruritic effects are significantly improved.

[0027] (2) The soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention exhibits a strong interaction between oat alkaloid and oligosaccharide, resulting in synergistic effects without altering the activities of either oat alkaloid or oligosaccharide. The antioxidant (ROS scavenging and SOD activity), repair (Col-I and MMP1 expression), and anti-allergic (redness reduction and itching) effects of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly are superior to the sum of equimolar amounts of oat alkaloid and oligosaccharide monomers; the anti-inflammatory effect (TNF-α and IL-8 gene expression) is comparable to the sum of equimolar amounts of oat alkaloid and oligosaccharide monomers. This indicates that the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly can target multiple aging pathways, providing a solution for developing novel, efficient, and multifunctional anti-aging products, which greatly enhances the application potential of oat alkaloid in the skincare field.

[0028] (3) The method of the present invention is simple and convenient to operate, and has strong practicality. The obtained soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly improves the water solubility of oat alkaloid by more than 1,000 times and has excellent stability. There is no precipitation phenomenon below the saturation concentration, which can effectively avoid the precipitation problem caused by changes in the solvent environment. It also has low cytotoxicity and strong applicability to formulations, making it widely applicable to various cosmetic formulations. The solution and powder have good stability, which is convenient for storage and transportation, thereby significantly improving the market value of oat alkaloid. Attached Figure Description

[0029] 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.

[0030] Figure 1 This is the infrared spectrum of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of Experimental Example 1 of the present invention.

[0031] Figure 2 These are particle size distribution diagrams of Embodiment 1 and Comparative Example 1 of the present invention.

[0032] Figure 3 This is a SEM image of the mixture of oat alkaloid and oligosaccharide in Experimental Example 1 of the present invention and the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly after drying and pulverizing. The left image is the mixture of oat alkaloid and oligosaccharide, and the right image is the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly after drying and pulverizing.

[0033] Figure 4 This is a diagram illustrating the interaction between alfalfa and oligosaccharides in the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly of the present invention.

[0034] Figure 5 This is an ESP analysis diagram of the alfalfa and alfalfa-oligosaccharide assembly of the present invention.

[0035] Figure 6 This is a molecular frontier orbital analysis diagram of the alfalfa and alfalfa-oligosaccharide assemblies of the present invention.

[0036] Figure 7 This is an analytical diagram showing the inclusion effect of cyclodextrin on oat alkaloids in the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention.

[0037] Figure 8 This is a comparison chart of the solubility and stability of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention.

[0038] Figure 9 This is a comparative diagram of the cytotoxicity of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention.

[0039] Figure 10 This is a comparison chart of the antioxidant capacity of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention.

[0040] Figure 11 This is a comparison chart of the anti-inflammatory capabilities of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention.

[0041] Figure 12 This is a comparison chart of the repair capabilities of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly.

[0042] Figure 13 This is a long-term stability test diagram of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention.

[0043] Figure 14 This is a grading chart of skin reactions in a closed patch test.

[0044] Figure 15 This is a comparison chart of the anti-allergic effects of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention. Detailed Implementation

[0045] 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.

[0046] 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.

[0047] The oligosaccharide used in Example 1 and the comparative example was *Dendrobium officinale* pentasaccharide extracted from the traditional Chinese medicine *Dendrobium officinale*, as described in the literature "Target acquisition of anti-aging manno-oligosaccharide that triggers ECM process via TGF-β / Smads-SIRT1 signalling pathway" (Yang, L., Zi CT, et al. *Carbohydrate Polymers*, 2023, 302, 120380) doi.org / 10.1016 / j.carbpol.2022.120380. Its chemical structure consists of 3 mannose molecules and 2 glucose molecules, with a main chain structure of β-D-Manp-(1→3)-β-D-Glcp-(1→3)-α-D-Manp-(1→2)-β-D-Glcp-(1→4)-α-D-Manp. The specific structure is shown below:

[0048]

[0049] The oligosaccharide in Example 5 is *Dendrobium officinale* hexasaccharide extracted from the traditional Chinese medicine *Dendrobium officinale*, see CN113004432B. Its chemical structure consists of 2 galactose molecules, 3 mannose molecules, and 1 glucuronic acid molecule, with the main chain structure β-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Manp-(1→4)-β-D-Manp-(1→4)-β-D-Manp-(1→4)-β-D-GlcpA. The specific structure is shown below:

[0050]

[0051] Example 1

[0052] A method for preparing a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly includes the following steps:

[0053] S1. Add 10g of Dendrobium officinale oligosaccharide to 200mL of ethanol aqueous solution (ethanol concentration is 15wt.%), mix well, stir at room temperature until Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 60℃, slowly add 10g of oat alkali in small amounts several times, keep the temperature constant at 60℃, slowly add 50g of hydroxypropyl-β-cyclodextrin in small amounts several times, stir until completely dissolved, sonicate at 15kHz at 60℃ for 30 minutes, and then cool to room temperature;

[0054] 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 grinder, and store it in a cool, dry place in a sealed container to obtain the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly.

[0055] Example 2

[0056] A method for preparing a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly includes the following steps:

[0057] S1. Add 10g of Dendrobium officinale oligosaccharide to 200mL of isopropanol aqueous solution (isopropanol concentration is 15wt.%), mix well, stir at room temperature until Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 60℃, slowly add 10g of oat alkali in small amounts several times, keep the temperature constant at 60℃, slowly add 50g of methyl-β-cyclodextrin in small amounts several times, stir until completely dissolved, sonicate at 15kHz at 60℃ for 30 minutes, and then cool to room temperature;

[0058] 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 grinder, and store it in a cool, dry place in a sealed container to obtain the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly.

[0059] Example 3

[0060] A method for preparing a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly includes the following steps:

[0061] S1. Add 20g of Dendrobium officinale oligosaccharide to 200mL of ethanol aqueous solution (ethanol concentration is 10wt.%), mix well, stir at room temperature until Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 60℃, slowly add 10g of oat alkali in small amounts several times, keep the temperature constant at 60℃, slowly add 50g of hydroxypropyl-β-cyclodextrin in small amounts several times, stir until completely dissolved, sonicate at 15kHz at 60℃ for 30 minutes, and then cool to room temperature;

[0062] 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 grinder, and store it in a cool, dry place in a sealed container to obtain the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly.

[0063] Example 4

[0064] A method for preparing a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly includes the following steps:

[0065] S1. Add 10g of Dendrobium officinale oligosaccharide to 200mL of isopropanol aqueous solution (isopropanol concentration is 10wt.%), mix well, stir at room temperature until Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 60℃, slowly add 10g of oat alkali in small amounts several times, keep the temperature constant at 60℃, slowly add 100g of hydroxypropyl-β-cyclodextrin in small amounts several times, stir until completely dissolved, sonicate at 15kHz at 60℃ for 60 minutes, and then cool to room temperature;

[0066] 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 grinder, and store it in a cool, dry place in a sealed container to obtain the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly.

[0067] Example 5

[0068] A method for preparing a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly includes the following steps:

[0069] S1. Add 10g of Dendrobium officinale oligosaccharide to 200mL of ethanol aqueous solution (ethanol concentration is 15wt.%), mix well, stir at room temperature until Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 60℃, slowly add 10g of oat alkali in small amounts several times, keep the temperature constant at 60℃, slowly add 50g of hydroxypropyl-β-cyclodextrin in small amounts several times, stir until completely dissolved, sonicate at 15kHz at 60℃ for 30 minutes, and then cool to room temperature;

[0070] 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 grinder, and store it in a cool, dry place in a sealed container to obtain the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly.

[0071] Comparative Example 1

[0072] A method for preparing an oat alkaloid-oligosaccharide assembly includes the following steps:

[0073] S1. Add 10g of Dendrobium officinale oligosaccharide to 200mL of ethanol aqueous solution (ethanol concentration is 15wt.%), mix well, stir at room temperature until Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 60℃, slowly add 10g of oat alkali in small amounts several times, stir until completely dissolved, sonicate at 15kHz at 60℃ for 30 minutes, and then cool to room temperature.

[0074] 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 grinder, and store it in a cool, dry place in a sealed container to obtain the oat alkali-oligosaccharide assembly.

[0075] The main difference between this comparative example and Example 1 is that no cyclodextrin was added.

[0076] Comparative Example 2

[0077] A method for preparing an oat base-oligosaccharide-cyclodextrin assembly includes the following steps:

[0078] S1. Add 10g of Dendrobium officinale oligosaccharide to 200mL of ethanol aqueous solution (ethanol concentration is 15wt.%), mix well, stir at room temperature until Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 60℃, slowly add 10g of oat alkali in small amounts several times, keep the temperature constant at 60℃, slowly add 50g of hydroxypropyl-β-cyclodextrin in small amounts several times, stir until completely dissolved, sonicate at 15kHz at 60℃ for 10 minutes, and then cool to room temperature;

[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 grinder, and store it in a cool, dry place in a sealed container to obtain the oat base-oligosaccharide-cyclodextrin assembly.

[0080] Compared with Example 2, the main difference in this comparative example is that the ultrasound time in step S1 is too short.

[0081] Comparative Example 3

[0082] A method for preparing an oat base-oligosaccharide-cyclodextrin assembly includes the following steps:

[0083] S1. Add 10g of Dendrobium officinale oligosaccharide to 200mL of ethanol aqueous solution (ethanol concentration is 15wt.%), mix well, stir at room temperature until Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 40℃, slowly add 10g of oat alkali in small amounts several times, keep the temperature constant at 40℃, slowly add 50g of hydroxypropyl-β-cyclodextrin in small amounts several times, stir until completely dissolved, sonicate at 15kHz at 40℃ for 30 minutes, and then cool to room temperature;

[0084] 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 grinder, and store it in a cool, dry place in a sealed container to obtain the oat base-oligosaccharide-cyclodextrin assembly.

[0085] Compared with Example 3, the main difference in this comparative example is that the temperature in step S1 is too low.

[0086] Comparative Example 4

[0087] A method for preparing an oat base-oligosaccharide-cyclodextrin assembly includes the following steps:

[0088] S1. Add 10g of Dendrobium officinale oligosaccharide to 200mL of aqueous solution, mix well, stir at room temperature until the Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 60℃, slowly add 10g of oat alkali in small amounts several times, keep the temperature constant at 60℃, slowly add 50g of hydroxypropyl-β-cyclodextrin in small amounts several times, stir until completely dissolved, sonicate at 15kHz at 60℃ for 30 minutes, and then cool to room temperature;

[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 grinder, and store it in a cool, dry place in a sealed container to obtain the oat base-oligosaccharide-cyclodextrin assembly.

[0090] Compared with Example 4, the main difference of this comparative example is that step S1 is alcohol-free.

[0091] Comparative Example 5

[0092] A method for preparing an oat base-oligosaccharide-cyclodextrin assembly includes the following steps:

[0093] S1. Add 10g of Dendrobium officinale oligosaccharide to 200mL of ethanol aqueous solution (ethanol concentration is 15wt.%), mix well, stir at room temperature until Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 60℃, slowly add 10g of oat alkali in small amounts several times, keep the temperature constant at 60℃, slowly add 50g of methyl-β-cyclodextrin in small amounts several times, stir until completely dissolved, sonicate at 15kHz for 120 minutes at 60℃, and then cool to room temperature;

[0094] 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 grinder, and store it in a cool, dry place in a sealed container to obtain the oat base-oligosaccharide-cyclodextrin assembly.

[0095] Compared with Example 1, the main difference in this comparative example is that the ultrasound time in step S1 is too long.

[0096] Comparative Example 6

[0097] A method for preparing an oat base-oligosaccharide-cyclodextrin assembly includes the following steps:

[0098] S1. Add 5g of Dendrobium officinale oligosaccharide to 200mL of ethanol aqueous solution (ethanol concentration is 15wt.%), mix well, stir at room temperature until Dendrobium officinale oligosaccharide is completely dissolved, and under continuous stirring, heat to 60℃, slowly add 10g of oat alkali in small amounts several times, keep the temperature constant at 60℃, slowly add 50g of methyl-β-cyclodextrin in small amounts several times, stir until completely dissolved, sonicate at 15kHz at 60℃ for 30 minutes, and then cool to room temperature;

[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 grinder, and store it in a cool, dry place in a sealed container to obtain the oat base-oligosaccharide-cyclodextrin assembly.

[0100] Compared with Example 1, the main difference of this comparative example is that the oligosaccharide content is too low.

[0101] Performance tests were conducted on the above embodiments and comparative examples. The experimental test methods are as follows:

[0102] Infrared spectroscopy was performed on the obtained soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly. Figure 1 This is the infrared spectrum of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly and the equimolar precursor mixture of Experimental Example 1 of this invention. Figure 1As shown, compared with the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly, the precursor mixture exhibits more and more disordered peaks in its infrared spectrum, which is due to the superposition of infrared absorptions from multiple components. The infrared spectrum of the precursor mixture at 1950 cm⁻¹... -1 The nearby peak corresponds to the bending vibration of the CH group of the benzene ring in alfalfa, while this peak weakens to almost invisible in the infrared spectrum of the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly, indicating that cyclodextrin has a good inclusion effect on alfalfa. Meanwhile, compared with the precursor mixture, the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly shows a higher concentration at 3200 cm⁻¹. -1 A broadened OH stretching vibration peak appears around the periphery, at 1650 cm⁻¹. -1 The intensity of the nearby C=O stretching vibration peak decreased and shifted to lower wavenumbers, which is highly consistent with the formation of intermolecular hydrogen bonds. Furthermore, the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly was observed at 3192.06 cm⁻¹. -1 and 2935.51cm -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 alkaloids, oligosaccharides, and cyclodextrin form a uniform self-assembled inclusion complex through strong intermolecular interactions.

[0103] Oligosaccharides can improve the water solubility of oat base by co-assembling with oat base, while cyclodextrin inclusion is used to improve the stability and uniformity of the oat base-oligosaccharide assembly. The particle size distribution of the samples from Example 1 and Comparative Example 1 was analyzed using DLS. Figure 2 These are particle size distribution diagrams of Embodiment 1 and Comparative Example 1 of the present invention, as shown below. Figure 2 As shown, both the alfalfa-oligosaccharide assembly and the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly are nanoscale structures. The alfalfa-oligosaccharide assembly has a larger average particle size (328.2 nm) and polydispersity index (29.7%), while the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly has a significantly smaller average particle size (181.5 nm) and polydispersity index (10.2%). This indicates that the ternary assembly performance of alfalfa-oligosaccharide-cyclodextrin is significantly better than that of the binary assembly performance of alfalfa-oligosaccharide.

[0104] The soothing avocadoin-Dendrobium officinale oligosaccharide co-assembly was dried and pulverized for convenient storage and transportation. To verify that the drying and pulverizing process did not damage the microstructure of the soothing avocadoin-Dendrobium officinale oligosaccharide co-assembly, SEM analysis was performed. Figure 3 This is a SEM image of the mixture of alfalfa and oligosaccharides from Experimental Example 1 of this invention, and the dried and pulverized soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly. The left image shows the mixture of alfalfa and oligosaccharides, and the right image shows the dried and pulverized soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly. Figure 3As shown, the mixture of oat alkaloids and oligosaccharides has two different morphologies: rod-shaped and spherical. Although the dry and pulverized soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assemblies have different particle sizes, they all have amorphous morphology, uniform texture, and smooth edges, indicating that the drying and pulverizing process did not destroy the inclusion state of cyclodextrin.

[0105] Dissolution tests were conducted on Examples 1-4 and Comparative Examples 2-6, and the specific test results are shown in Table 1 below:

[0106] Table 1. Dissolution test results of samples from Examples 1-4 and Comparative Examples 2-6

[0107] Serial Number Dissolved state Solubility (wt.%) Example 1 Completely dissolved 3.44 Example 2 Completely dissolved 2.87 Example 3 Completely dissolved 3.25 Example 4 Completely dissolved 2.69 Example 5 Completely dissolved 3.42 Comparative Example 2 Cannot be completely dissolved 3.12 Comparative Example 3 Cannot be completely dissolved 2.51 Comparative Example 4 Cannot be completely dissolved 1.93 Comparative Example 5 Cannot be completely dissolved 2.08 Comparative Example 6 Cannot be completely dissolved 1.76

[0108] As shown in Table 1, the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assemblies of Examples 1-4 of the present invention all exhibit excellent water solubility. Example 1 showed the best performance, with an oat alkaloid solubility as high as 3.44 wt.%, followed by Example 5, which also showed a high oat alkaloid solubility of 3.42 wt.%. The solubility of free oat alkaloid in water is less than 30 μg / mL, while the soothing oat alkaloid soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assemblies after drying and reconstitution can achieve solubility of 3.44 wt.% and 3.42 wt.%, representing an improvement of more than 1000 times in water solubility. This indicates that the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assemblies of the present invention effectively improve the water solubility of oat alkaloid.

[0109] In Comparative Example 2, the sonication time was too short, the mixing time was insufficient, and the assembly was incomplete, resulting in the precipitation of some oat alkali after cooling. In Comparative Example 3, the temperature was not up to standard, and the reactants could not be completely dissolved. In Comparative Example 4, without alcohol assistance, the oat alkali could not be completely dissolved. In Comparative Example 5, the sonication time was too long, the assembly was destroyed, and solids precipitated after cooling. In Comparative Example 6, the oligosaccharide content was too low, and the oat alkali could not be completely dissolved, resulting in the samples obtained from Comparative Examples 2-6 not being completely soluble, and their water solubility could not be compared with that of the examples.

[0110] Based on molecular dynamics, the co-assembly processes of oat base and oligosaccharide in a 1:1 molar ratio and oat base, oligosaccharide, and cyclodextrin in a 1:1:1 molar ratio were simulated using XTB software to screen for the lowest-energy conformation. Based on density functional theory, the interactions of oat base, oligosaccharide, and cyclodextrin in the co-assemblies were analyzed using Gauss software through an independent gradient model based on Hirshfeld partitioning (IGMH) and atomic topological analysis (AIM). In the IGMH diagram, different colors represent the strength and nature of intermolecular interactions: blue represents attractive interactions such as hydrogen bonds, green represents van der Waals forces, and red represents repulsive interactions. In the AIM analysis, the orange line represents the path of interatomic electron density, and the points on the line represent bond critical points (BCPs) along the interatomic electron density path. The electron density at the BCP (ρ) is... BCPA higher value indicates the presence of strong hydrogen bond interactions. Figure 4 This is an interaction analysis diagram of alfalfa and oligosaccharides in the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly of the present invention, as shown in the figure. Figure 4 As shown, there are abundant van der Waals interactions and hydrogen bonding interactions between oat alkaloids and oligosaccharides. Their binding free energy is -12 kcal / mol. Furthermore, the carboxyl H and amide C=O groups of oat alkaloids form two medium-strength hydrogen bonds with the ether and hydroxyl groups of oligosaccharides, respectively, allowing them to spontaneously form assemblies.

[0111] Figure 5 This is an ESP analysis diagram of the oat alkaloid and oat alkaloid-oligosaccharide assembly of the present invention. Figure 5 As shown, surface electrostatic potential (ESP) analysis reveals that oat alkaloids form 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 oat alkaloid-oligosaccharide assembly, the oligosaccharide alters the folded conformation of oat alkaloids, weakening its π-π interactions and contributing to improved water solubility and molecular flexibility. The molecular polarity index (MPI) in the ESP analysis is an indicator of the overall polarity of the molecule, reflecting the non-uniformity of the charge distribution on the molecular surface. Due to the abundant interactions between oat alkaloids and oligosaccharides, the molecular polarity index (MPI) of the oat alkaloid-oligosaccharide assembly is 0.64 eV, and the polar surface area (ESP > 10 kcal / mol) accounts for 57.32%, both greater than the 0.54 eV and 45.17% of free oat alkaloids, further demonstrating that the water solubility of the assembly is superior to that of pure oat alkaloids.

[0112] 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 6 This is a molecular frontier orbital analysis diagram of the oat alkaloid and oat alkaloid-oligosaccharide assembly of the present invention, as shown below. Figure 6As shown, the HOMO of agar is mainly distributed on the benzene ring linking hydroxyl groups, while the LUMO is mainly distributed on the benzene ring linking carboxyl groups, indicating that they are electron-losing and electron-gaining active sites, respectively. Both the HOMO and LUMO of the agar-oligosaccharide co-assembly are located on agar, indicating that agar is the active site of the agar-oligosaccharide assembly. Furthermore, the structural distribution and symmetry of the HOMO and LUMO of the agar-oligosaccharide assembly are very similar to those of free agar, suggesting that the agar-oligosaccharide assembly has the same biological activity as agar. Simultaneously, the higher HOMO, lower LUMO, and smaller band gap (ΔE) indicate that the activity of the agar-oligosaccharide assembly is superior to that of free agar.

[0113] Figure 7 This is an analytical diagram showing the inclusion effect of cyclodextrin on oat alkaloids in the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of this invention. For comparison, the inclusion effect of cyclodextrin on free oat alkaloids was also studied using molecular dynamics and density functional theory. It can be seen that free oat alkaloids enter the cyclodextrin in a folded conformation, resulting in significant steric hindrance; the binding free energy between the two is only -0.93 kcal / mol, indicating a weak inclusion effect. Figure 7 a). In the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly, oat alkaloid, oligosaccharide, and cyclodextrin co-assembled; thanks to the high water solubility of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly, oat alkaloid entered cyclodextrin in a linear conformation, and the binding free energy of the two was -19.44 kcal / mol, significantly enhancing the inclusion effect. Figure 7 (b) IGMH analysis revealed abundant van der Waals and hydrogen bonding interactions among the three components in the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly. For ease of differentiation, the interaction between oat alkaloid and cyclodextrin was labeled based on AIM analysis. It can be seen that van der Waals interactions (green) are predominantly present within the cavities of oat alkaloid and cyclodextrin, which is highly consistent with the hydrophobic inclusion complex of cyclodextrin with oat alkaloid. At the outer edge of the cyclodextrin cavity, strong hydrogen bonding exists among oat alkaloid, oligosaccharide, and cyclodextrin (blue), consistent with the good hydrophilicity of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly. Simultaneously, the abundant interactions among the three components in the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly help enhance the stability of the inclusion complex of cyclodextrin with oat alkaloid and reduce the thermodynamic probability of oat alkaloid dissociating from the inclusion system.

[0114] Deminsu is a commercially available water-soluble oat base product composed of butylene glycol, pentanediol, and oat base. It exists as a transparent, colorless, and odorless liquid, readily soluble in alcohol, glycerin, surfactants, and commonly used cosmetic oils. In practical applications, it exhibits better anti-inflammatory and antipruritic effects than pure oat base. However, Deminsu 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.

[0115] This invention comprehensively evaluates the application potential of the soothing avocado-Dendrobium officinale oligosaccharide co-assembly in the cosmetics field by comparing it with Deminshu, and by comparing its bioefficacy with that of precursor monomers / mixtures.

[0116] (1) Study on the solubility and stability of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly

[0117] Prepare aqueous solutions with 0.1 wt% oat alkaloid content, aqueous solutions with 1 wt.% and 2 wt.% oat alkaloid content, and aqueous solutions of soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly with 2 wt.% oat alkaloid content. Observe their appearance changes.

[0118] Figure 8 This is a comparison chart of the solubility and stability of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention. Figure 8 As shown, free alfalfa precipitates at a concentration of 0.1 wt.%, while Deminshu precipitates a large amount of solids when diluted to 1 wt.% and 2 wt.% of alfalfa alkalfa content, indicating that it is not suitable for water-based cosmetic formulations. When the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly is prepared into an aqueous solution with an alfalfa alkalfa content of 2 wt.%, it remains clear and transparent after two weeks of storage at room temperature, without precipitation or turbidity, indicating that the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly has good solubility and stability, making it suitable for water-based cosmetic formulations.

[0119] (2) CCK-8 cytotoxicity test

[0120] The cytotoxicity of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly was tested using human immortalized epidermal cells (HaCaT) and compared with that of Deminshu containing an equimolar concentration of oat alkaloid.

[0121] A. Experimental Procedure

[0122] 1) After cell resuscitation, passage twice to expand the culture.

[0123] 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.

[0124] 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.

[0125] 4) Place the cells in an incubator and continue culturing for 48 hours.

[0126] 5) Add 10 μL of CCK-8 solution to each well, and place the culture plate in an incubator for 1.5 h.

[0127] 6) Remove the culture plate and read the OD value at 450nm using an ELISA reader.

[0128] 7) Perform the experiment in parallel three times.

[0129] B. Calculation Formula

[0130]

[0131] Sample group OD: OD values ​​of wells containing cells, CCK-8 solution, and drug solution.

[0132] OD value of blank group: cell-free, OD value of CCK-8 solution present.

[0133] Control group OD: OD values ​​of cells in the CCK-8 solution wells.

[0134] C. Analysis software

[0135] Statistical analysis was performed using Grap oat alkali d.Prism.10.1 software, and curve fitting was performed using Log(agonist) vs. response-variable slope(four parameters).

[0136] Figure 9 This is a comparative diagram of the cytotoxicity of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention. Figure 9As shown, under a range of oat alkaloid concentrations, the cytotoxicity of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly to human immortalized epidermal cells was significantly lower than that of Deminshu. When the oat alkaloid concentration was 125 ppm, the HaCaT cell survival rate of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly was higher than 90%, while the HaCaT cell survival rate of Deminshu was lower than 80%. When the oat alkaloid concentration was 500 ppm, the HaCaT cell survival rate of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly was higher than 80%, while the HaCaT cell survival rate of Deminshu was close to 0%. Furthermore, under oat alkaloid concentrations ≤500 ppm, the HFF-1 cell survival rate after treatment with the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly was higher than 90%, while the HFF-1 cell survival rate of Deminshu was significantly lower than 80% at an oat alkaloid concentration of 200 ppm. This means that, compared to Deminex, the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly has higher biocompatibility and lower skin irritation.

[0137] (3) Antioxidant performance testing

[0138] Ultraviolet (UV) radiation is a key factor leading to skin inflammation and photoaging. UVA (long-wave ultraviolet radiation, 320-400nm) can penetrate the dermis, causing photoaging, oxidative stress, and inflammatory damage. UVB (medium-wave ultraviolet radiation, 280-315nm) can induce DNA damage in skin cells, accumulate intracellular reactive oxygen species (ROS), activate inflammatory signaling pathways, and lead to collagen degradation, thereby causing symptoms such as erythema, edema, and pain. ROS levels directly reflect the degree of oxidative stress induced by UV radiation, and as a scavenger of O2... - The activity of superoxide dismutase (SOD), a key antioxidant enzyme, represents the cell's own antioxidant defense capacity. Therefore, this invention constructs an oxidative stress model of human immortalized epidermal cells (HaCaT) through synergistic stimulation by UVA and UVB to evaluate the antioxidant capacity of a soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly and compare it with equimolar concentrations of alfalfa and oligosaccharides.

[0139] A. Experimental Procedure

[0140] 1) After cell resuscitation, passage twice to expand the culture.

[0141] 2) After digesting, centrifuging, resuspending, and counting the expanded cells, the cells were seeded into 24-well cell culture plates.

[0142] 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 2 below.

[0143] Table 2 Experimental Groups

[0144]

[0145] 4) ROS Assay: After cells have fully adhered, aspirate the culture medium and add 500 μL of the diluted drug to each well of a 24-well culture. Continue culturing for 48 h. Aspirate the culture medium, add a small amount of PBS, and irradiate the cells using UVB combined with UBA. For the NC group, cover with aluminum foil. After irradiation, add 500 μL of the diluted drug to each well of a 24-well culture. Perform ROS assay after 30 min of culture. Dilute DCFH-DA with complete culture medium at a ratio of 1:1000 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 sufficient to fully cover the cells. Incubate at 37°C for 20 min. Wash the cells three times with complete culture medium to thoroughly remove any DCFH-DA that has not entered the cells. Detect using a multi-mode microplate reader. Perform ROS assay in triplicate.

[0146] 5) SOD Assay: After cells have fully adhered to the culture medium, aspirate the medium and irradiate the cells with a combination of UVB and UVA. For the NC group, cover with aluminum foil. After irradiation, or add 2000 μl of diluted drug to each of the 6 wells. Perform SOD assay after 48 hours of culture. 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 approximately 12,000g for 3-5 minutes at 4°C, and collect the supernatant as the test sample. After sample preparation, determine the protein concentration using a BCA protein concentration assay kit. Based on the protein concentration and the expected protein usage, appropriately dilute the sample with the SOD detection buffer provided in the kit. Prepare the corresponding working solution according to the kit requirements and analyze the samples using a microplate reader. Perform the SOD content assay in triplicate.

[0147] B. Calculation Formula

[0148] 1) Standard curve fitting formula

[0149] y = aX 2 +bX+c

[0150] X:OD 536 y: Protein concentration (mg / mL)

[0151] 2) ROS calculation

[0152]

[0153] 3) SOD calculation

[0154]

[0155] Figure 10 This is a comparison chart of the antioxidant capacity of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention. (See figure) Figure 10 As shown in Figure a, the ROS level in the MC group was significantly higher than that in the NC and PC groups, indicating successful modeling. Treatment with oat alkaloids, oligosaccharides, and the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly all significantly reduced ROS levels compared to the MC group, indicating that all three had good ROS scavenging capabilities. The soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly reduced the ROS level in HaCaT cells by 59.5% compared to the MC group, comparable to that in the PC group, and significantly higher than the sum of oat alkaloid (26%) and oligosaccharide (21.1%). Figure 10 As shown in b, the SOD activity in the MC group was significantly lower than that in the NC and PC groups, indicating successful modeling. Treatment with oat alkaloids, oligosaccharides, and the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly significantly increased SOD activity compared to the MC group, indicating that all three significantly enhanced the antioxidant defense capacity of HaCaT cells. The soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly increased the SOD activity of HaCaT cells by 69.3% compared to the MC group, comparable to PC, and significantly higher than the sum of oat alkaloids (24.2%) and oligosaccharides (31.9%). In summary, the antioxidant capacity of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly was superior to the sum of oat alkaloids and oligosaccharides alone, indicating that oat alkaloids and oligosaccharides played a synergistic antioxidant role in the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly, consistent with the results of molecular frontier orbital analysis.

[0156] (4) Anti-inflammatory capacity test

[0157] Besides causing oxidative damage, UVA and UVB can synergistically activate key transcription factors (such as NF-κB and AP-1), leading to the expression and release of large amounts of pro-inflammatory cytokines (IL-8 and TNF-α), triggering skin inflammation (equivalent to the "burning" and "stirring" sensations after sun exposure). Simultaneously, oxidative stress and inflammation can mutually promote each other, accelerating skin aging and causing problems such as wrinkles, sagging, and pigmentation. Therefore, this invention constructs an inflammatory response model of HaCaT cells through synergistic stimulation by UVA and UVB to evaluate the anti-inflammatory capacity of a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly, and compare it with equimolar concentrations of oat alkaloid and oligosaccharide. Lower gene expression of pro-inflammatory cytokines (IL-8 and TNF-α) indicates stronger anti-inflammatory capacity of the sample.

[0158] A. Experimental Procedure

[0159] 1) After cell resuscitation, passage twice to expand the culture.

[0160] 2) After digesting, centrifuging, resuspending, and counting the expanded cells, the cells were seeded into 24-well cell culture plates.

[0161] 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 2.

[0162] 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 combined with UBA. The NC group is covered with aluminum foil. After irradiation, add the diluted drug to the 24 wells, 500 μL per well.

[0163] 5) Place the cells in an incubator and continue culturing for 24 hours.

[0164] 6) Wash three times with PBS, add 1 mL of RNA extraction buffer, pre-cool on ice for 5 min, and gently pipette.

[0165] 7) Add 400 μL of chloroform, invert the centrifuge tube for 15 seconds, mix thoroughly, and let stand for 3 minutes.

[0166] 8) Centrifuge at 12000 rpm for 10 min at 4℃.

[0167] 9) Transfer 400 μL of supernatant to a new centrifuge tube, add 550 μL of isopropanol, and mix by inverting.

[0168] 10) Place at -20°C for 15 minutes.

[0169] 11) Centrifuge at 12000 rpm for 10 min at 4℃. The white precipitate at the bottom of the tube is RNA.

[0170] 12) Remove the liquid and wash the precipitate with 1.5 mL of 75% ethanol.

[0171] 13) Centrifuge at 12000 rpm for 5 min at 4℃.

[0172] 14) Remove all liquid and place the centrifuge tube on a clean bench and blow it for 3 minutes.

[0173] 15) Add 15 μL of Water Nuclease-Free to dissolve the RNA.

[0174] 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 on the detection base, lower the sample arm, and use the software on the computer to start the absorbance detection.

[0175] 17) Dilute the excessively high concentration of RNA by an appropriate ratio to achieve a final concentration of 200 ng / μL.

[0176] 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.

[0177] 19) The experiment was conducted in parallel three times.

[0178] B. Calculation Formula

[0179] 1) RNA expression level

[0180] ΔC(t)=C(t) 目的基因 -C(t) 内参基因

[0181] ΔΔC(t)=ΔC(t) 样品组 -ΔC(t) 对照组

[0182] relative RNA expression level = 2 ΔΔΔ(Δ)

[0183] 2) Rate of change detection

[0184]

[0185] Figure 11 This is a comparative diagram showing the anti-inflammatory capabilities of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention. (See diagram for reference.) Figure 11 As shown, the expression of TNF-α and IL-8 genes in the MC group was significantly higher than that in the NC and PC groups, indicating successful modeling. Treatment with alfalfa, oligosaccharides, and the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly significantly reduced the expression of TNF-α and IL-8 genes compared to the MC group, indicating that all three have good anti-inflammatory capabilities. The anti-inflammatory ability of the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly was significantly better than that of alfalfa; simultaneously, the anti-inflammatory ability of alfalfa was significantly better than that of oligosaccharides. The alfalfa, oligosaccharides, and the soothing alfalfa-Dendrobium officinale oligosaccharide co-assembly reduced TNF-α gene expression by 41.9%, 15%, and 58.8%, respectively, compared to the MC group; and reduced IL-8 gene expression by 44.8%, 14.3%, and 57.7%, respectively, compared to the MC group. This indicates that the anti-inflammatory capacity of the soothing avocado-Dendrobium officinale oligosaccharide co-assembly is comparable to the sum of avocado and oligosaccharide, and the ternary assembly does not impair the biological activity of the precursor.

[0186] (5) Repair capability test

[0187] The synergistic effect of oxidative stress and inflammatory factors accelerates the degradation of the skin extracellular matrix and inhibits collagen synthesis, jointly driving the skin aging process. Type I collagen (Col-I), as the main structural protein in the dermis (accounting for 80-90%), shows a marked characteristic of skin aging due to reduced synthesis and abnormal degradation. UVA activates the AP-1 pathway through ROS, upregulating matrix metalloproteinase (MMP) expression and promoting collagen degradation; UVB can directly induce collagen fiber cross-linking and breakage. Therefore, this invention constructs a photoaging model of human immortalized fibroblasts (HFF-1) through synergistic stimulation by UVA and UVB. The repair capacity of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly is evaluated using Col-I and MMP1 expression levels, and compared with equimolar concentrations of oat alkaloid and oligosaccharide. Higher Col-I expression and lower MMP1 expression indicate a stronger ability of the sample to promote collagen homeostasis reconstruction.

[0188] A. Col-I Experimental Procedure

[0189] 1) After cell resuscitation, passage twice for expanded culture. After digestion, centrifugation, resuspending, and counting of the expanded cells, transfer them into 24-well cell culture plates.

[0190] 2) Experimental grouping: After plating, the cells were divided into a negative control group (NC), a model control group (MC), and a sample group (specified drug concentration). The specific experimental groupings are shown in Table 3 below:

[0191] Table 3 Experimental Groups

[0192]

[0193]

[0194] 3) Remove the old culture medium and rinse each well with a small amount of PBS. Except for the blank control group (NC), the other groups were irradiated with UVB combined with UBA. The NC group was covered with aluminum foil to protect it from light. Add the corresponding sample (final volume 500 μL) to each well and continue culturing for 48 h. Collect the supernatant for Col-I detection.

[0195] 4) 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.

[0196] 5) Washing: Absorb the liquid and wash 4 times with detergent.

[0197] 6) Add enzyme: Add 50 μL of biotinylated antibody to each well except for the blank wells, and incubate twice.

[0198] 7) 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.

[0199] 8) Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0200] 9) 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.

[0201] 10) 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.

[0202] 11) The experiment was conducted in three parallel sessions.

[0203] B.MMP1 Experimental Procedure

[0204] 1) The HFF-1 cell culture method and experimental grouping are the same as above.

[0205] 2) After incubation, the supernatant was collected for experiments. The content of MMP1 in the sample was detected using a human matrix metalloproteinase-1 (MMP1) ELISA kit, and the absorbance was detected at 450 nm using an ELISA reader.

[0206] 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.

[0207] 4) Perform three parallel experiments.

[0208] Figure 12 This is a comparison chart of the repair capabilities of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly. (Example:) Figure 12 As shown in Figure a, Col-I expression in the MC group was significantly lower than that in the NC and PC groups, indicating successful modeling. Treatment with oat alkaloids, oligosaccharides, and the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly significantly increased Col-I expression compared to the MC group, indicating that all three significantly improved the collagen homeostasis reconstitution capacity of HFF-1 cells. The soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly increased Col-I expression in HFF-1 cells by 194.5% compared to the MC group, comparable to PC, and higher than the sum of oat alkaloid (25.5%) and oligosaccharide (140.8%). Figure 12As shown in b, MMP1 expression in the MC group was significantly higher than that in the NC and PC groups, indicating successful modeling. Treatment with oat alkaloids, oligosaccharides, and the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly significantly reduced MMP1 expression compared to the MC group, indicating that all three had good inhibitory effects on collagen degradation. The soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly reduced MMP1 expression in HFF-1 cells by 51.5% compared to the MC group, comparable to PC, and higher than the sum of oat alkaloids (15.8%) and oligosaccharides (31.9%). In summary, the repair capacity of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly was superior to the sum of oat alkaloids and oligosaccharides alone, indicating that oat alkaloids and oligosaccharides played a synergistic role in the reconstruction of cellular collagen homeostasis within the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly, consistent with the results of molecular frontier orbital analysis.

[0209] (6) Long-term stability test

[0210] To verify the stability of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly, a 2 wt.% solution and powder form were prepared and stored at -15℃, room temperature, and 45℃ for 4 and 8 weeks, respectively, to test the pH, conductivity, and color changes. The specific test results are shown in Table 4 below.

[0211] Table 4. Stability test results of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assemblies

[0212]

[0213] Figure 13 This is a long-term stability test diagram of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention. (See Table 4 and...) Figure 13 As can be seen, both the solution and powder of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly exhibit good stability under both high and low temperature conditions. After 4 and 8 weeks of storage, the pH, conductivity, and appearance remained essentially stable. This indicates that, regardless of whether in solution or dry conditions, the oat alkaloid in the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly is well encapsulated by the oligosaccharides and cyclodextrins, demonstrating strong stability and wide applicability to various applications and storage scenarios.

[0214] (7) Skin irritation test

[0215] 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.

[0216] A. Experimental Procedure

[0217] 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 alkaloid-Dendrobium officinale oligosaccharide co-assembly solution containing 0.1 wt.% oat alkaloid 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).

[0218] Figure 14 This is a grading chart of skin reactions in a closed patch test.

[0219] Evaluation principles

[0220] 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 5 below:

[0221] Table 5. Patch test results of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assemblies.

[0222]

[0223]

[0224] As shown in Table 5, all 30 subjects in this experiment had negative reactions, indicating that the 0.7 wt.% solution of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly (oat alkaloid concentration of 0.1 wt.%) has excellent safety and no skin irritation.

[0225] (8) Human body redness and itching relief experiment

[0226] 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.

[0227] Experimental steps

[0228] 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 hormones, were selected. The inner forearm of the subjects was used as the test site to evaluate the anti-allergic effect of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly prepared in Example 1, and to compare it with a mixture of oat alkaloid, oligosaccharide, and cyclodextrin. First, a 0.7 wt.% aqueous solution of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly (oat alkaloid concentration 0.1 wt.%) was prepared, and an aqueous solution containing the same concentration of oat alkaloid, oligosaccharide, and cyclodextrin was also prepared. The experimental results showed that the aqueous solution of the mixture of oat alkaloid, oligosaccharide, and cyclodextrin could not be completely dissolved, indicating that oat alkaloid had poor solubility in the mixed solution. Then, two test areas of equal area were selected on the inner forearms of the subjects, and equal amounts of 3% histamine solution were applied. After obvious allergic reactions appeared, equal amounts of a soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly aqueous solution and a mixture aqueous solution (oat alkaloid 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 6: 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).

[0229] Table 6 Scoring criteria for redness reduction and itch relief in antihistamine allergy tests.

[0230]

[0231] Figure 15 This is a comparison chart showing the anti-allergic effects of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly of the present invention. (Example) Figure 15 As shown, subjects exhibited significant allergic reactions on their skin after applying a 3% histamine solution, indicating successful modeling. When the oat alkaloid content was 0.1 wt.%, the anti-redness effect of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly was significantly better than that of its precursor mixture. This may be due to the poor solubility and low bioavailability of oat alkaloid in the mixture solution.

[0232] In summary, this invention proposes an innovative soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly. By self-assembling oat alkaloid, cyclodextrin, and oligosaccharides, the solubility and stability of oat alkaloid are addressed, increasing its solubility by more than 1000 times. The oligosaccharides not only exert synergistic biological effects with oat alkaloid, but the good compatibility between oligosaccharides and cyclodextrin also helps to increase the drug loading capacity of cyclodextrin and improve stability. The synergistic effect of the components in the system exhibits better biological efficacy than single-component or binary assembly systems, fully meeting the product requirements of mitochondrial energy skincare and showing promising application prospects in skincare products.

[0233] 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 oat alkaloid-Dendrobium officinale oligosaccharide co-assembly, characterized in that, Includes the following steps: S1. Add oligosaccharides to the alcohol-water mixture, mix well, and heat to 50-70℃ while continuously stirring. Add oat base and cyclodextrin, and sonicate for 30-60 min. 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 soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly; The mass ratio of oat base, oligosaccharide, and cyclodextrin in S1 is 1:(1-3):(5-20); The oligosaccharide mentioned in S1 is Dendrobium officinale pentasaccharide and / or Dendrobium officinale hexasaccharide; The alcohol in the alcohol-water mixture described in S1 is ethanol and / or isopropanol, with a concentration of 10-15 wt%. The cyclodextrin described in S1 is methyl-β-cyclodextrin and / or hydroxypropyl-β-cyclodextrin; The structural formula of the Dendrobium officinale pentaglycosides is shown below: ； The structural formula of the Dendrobium officinale hexasaccharide is shown below: 。 2. A method for preparing the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly according to claim 1, characterized in that: The mass ratio of the oligosaccharide and alcohol-water mixture in S1 is 1:(10-50).

3. A method for preparing the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly according to claim 1, characterized in that: The ultrasound mixture described in S1 is ultrasound at 10-25 kHz.

4. A method for preparing the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly according to claim 1, characterized in that: The centrifugation described in S2 is centrifugation at 3000 rpm for 10-15 min.

5. A soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly, characterized in that, The soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly according to any one of claims 1-4 was prepared.

6. The application of the soothing oat alkaloid-Dendrobium officinale oligosaccharide co-assembly according to claim 5 in the preparation of skin care products.

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