Stable synergistic composition based on asiaticoside and ergothioneine

Abstract

This invention discloses a stable synergistic composition based on asiaticoside and ergothioneine, which mainly comprises asiaticoside and ergothioneine, wherein the weight ratio of asiaticoside to ergothioneine is 0.5-5:1-8, and the content of asiaticoside in the composition is 0.05-2 wt%. This invention also discloses the application of asiaticoside in inhibiting the photodegradation of ergothioneine, the antioxidant use of the composition based on asiaticoside and ergothioneine, the use of the composition based on asiaticoside and ergothioneine as an FLG gene expression promoter, or a LOR gene expression promoter, or a Claudin-1 gene expression promoter, and the use of this composition in topical skin preparations.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material technology, specifically to a stable synergistic composition based on asiaticoside and ergothionein, that is, the composition not only has excellent stability, but also has synergistic antioxidant, FLG gene expression, LOR gene expression and Claudin-1 gene expression promotion effects. Background Technology

[0002] Centella asiatica (L.) Urban, belonging to the genus Centella of the Apiaceae family, was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). According to the *Compendium of Materia Medica*, the whole plant is used medicinally. It is cold in nature, bitter and pungent in taste, and enters the liver, spleen, kidney, and stomach meridians. Its effects include promoting blood circulation, reducing swelling and relieving pain, as well as clearing heat and detoxifying, and promoting diuresis. In traditional Chinese medicine, it is often used in combination with other herbs such as safflower, angelica, and red peony to treat various injuries from falls and blows. Modern pharmacological research and applications of Centella asiatica have made significant progress. Data indicates that Centella asiatica and its extracts can be used to treat depression, skin trauma, gastric ulcers, infectious hepatitis, skin diseases, and epidemic cerebrospinal meningitis. Chemical composition analysis of the whole plant of Centella asiatica isolated and identified 16 compounds. Physicochemical and spectroscopic analyses revealed that the main components were β-sitosterol, dauco-sterol, asiatiacid, vanillic acid, and asiaticoside.

[0003] Asiaticoside is a highly effective, non-toxic, and bioactive natural ingredient with excellent anti-skin aging and scar-inhibiting functions, making it a promising and valuable ingredient for cosmetic applications. This study selected Asiaticoside, a traditional Chinese medicine ingredient with skin bioactivity and a clearly defined pharmacological mechanism, as the starting point for modern research on traditional Chinese medicine cosmetics.

[0004] Centella asiatica extract has the following main functions in the skin system:

[0005] (1) Promotes wound healing

[0006] Studies have confirmed that asiaticoside can promote the synthesis of collagen and fibronectin, accelerate cell proliferation, and promote wound healing during wound healing. Zhang Tao et al. studied the effects of asiaticoside on the expression of cyclin B1, cyclin C, and proliferating cell nuclear antigen (PCNA) during burn wound healing. Immunohistochemical analysis showed that asiaticoside can effectively promote the expression of cyclin B1 and PCNA, significantly advancing the S+G2 phase of the cell cycle, thereby accelerating cell proliferation and promoting wound healing.

[0007] (2) Inhibit the formation of skin scars

[0008] It is generally believed that the formation mechanism of scars is mainly related to repair cells, extracellular matrix, cytokines, and the interaction among these three, with fibroblasts (FB), collagen, and transforming growth factor β (TGF-β) playing the most significant roles. Asiaticoside can prevent scar formation by blocking fibroblast proliferation and inhibiting their ability to synthesize and secrete collagen. Using a burn hypertrophic scar transplantation model in nude mice, it was found that Asiaticoside inhibited scar fibroblast proliferation and significantly reduced collagen synthesis in a dose-dependent manner. Immune cells, such as T lymphocytes and macrophages, release interleukins, fibroblast growth factor, and TGF-β1, which can promote fibroblast proliferation and extracellular matrix synthesis, leading to scar formation. Therefore, Asiaticoside inhibits scar formation by suppressing the excessive infiltration of T lymphocytes and macrophages in burn scar tissue and reducing the secretion of TGF-β1, etc. Asiaticoside can inhibit scar fibroblast proliferation. When the drug concentration is between 0.1 and 1 mg / mL, fibroblast proliferation is significantly inhibited, confirming that asiaticoside can reduce granulation tissue synthesis by inhibiting fibroblasts, thereby reducing scar formation. When the drug concentration is 1 mg / mL, asiaticoside has an inhibitory effect on the mRNA expression levels of type I and type III collagen and TGF-β in scar fibroblasts, which confirms that asiaticoside reduces the synthesis of large amounts of collagen by fibroblasts by reducing TGF-β expression, thereby alleviating scar hyperplasia. At the same time, it reduces the deposition of extracellular matrix, thereby interfering with scar formation.

[0009] Given the above-mentioned medicinal effects, if asiaticoside is applied to cosmetics, it can promote fibroblast proliferation and collagen synthesis, thereby delaying skin aging. Its main mechanism of action is to continuously activate epithelial tissue, accelerating wound healing, controlling collagen synthesis, inhibiting excessive proliferation of connective tissue matrix and fibrous components, improving skin keratinization, and repairing inflammatory buildup, thus treating scars.

[0010] asiaticoside is a large-molecule, hygroscopic, white, needle-like crystal with no particular odor. Due to its structural characteristics, it is unstable in the external environment, easily undergoing oxidation and degradation reactions, and has limited ability to penetrate the stratum corneum, resulting in low bioavailability. These are the limiting factors for the application of asiaticoside.

[0011] Ergothioneine (abbreviated EGT), scientific name 2-mercapto-histidine-trimethyl inner salt, also known as ergothioneine or ergot sulfur base, CAS 497-30-3, molecular formula C9H 16 N3O2S +With a molecular weight of 230.10, ergothione is a natural super antioxidant, a histidine derivative containing a thiol group. The pure form is a white crystalline solid, readily soluble in water, with a melting point of 275-277℃. Ergothione is named after ergot, from which it was isolated in 1909. It has been found to be synthesized in most fungi, some mushrooms, streptococci, mycobacteria, and other microorganisms, and can be absorbed and accumulated by plants and animals.

[0012] Currently, ergothioneine is obtained through biosynthesis, exhibiting good safety, high fermentation efficiency, and high purity. Its antioxidant properties are manifested in its targeting of various skin oxidation pathways, such as skin oxidation reactions involving singlet oxygen, superoxide ions, and oxygen free radicals, as well as metabolic pathways, and show a dose-response relationship. Literature reports that as the content of ergothioneine increases, its antioxidant activity also increases. Figure 2 The relationship between the concentration of ergothionein and different oxides is shown.

[0013] Furthermore, compared to various reported natural antioxidants, EGT-containing cells show a 16% reduction in cellular peroxides, while idebenone-containing cells show only a 5% reduction. Similarly, EGT exhibits excellent antioxidant properties even at extremely low concentrations, whereas antioxidants like coenzyme Q10 cannot achieve the same effect at low concentrations. The primary energy source for human cells is adenine triphosphate (ATP), which is produced in the mitochondria through fatty acid conversion. This energy production inevitably leads to the generation of free radicals. Ergothioneine, as a super antioxidant, is the only antioxidant with a clearly defined mechanism capable of repairing mitochondria. The human body cannot synthesize ergothioneine itself and must obtain it from external sources. Ergothioneine is a water-soluble amino acid molecule, transported to the required sites by a unique transport protein in the human body, OCTN1.

[0014] In aqueous solution, ergothione exists as a tautomer of thiols and thioketones.

[0015] Different pH conditions and concentrations can alter the configuration of ergothionein in the application system, leading to problems such as odor and spoilage, inevitably posing challenges to its application.

[0016] Inhibiting the photodegradation (improving photostability), odor, and discoloration of ergothionein has always been a difficult problem and challenge for the industry in applying this ingredient.

[0017] Chinese patent application CN110327242B discloses a method for inhibiting the photodegradation of ergothioneine and its application, comprising: adding hyaluronic acid salt to a solution containing ergothioneine; wherein the content of ergothioneine is 0.0005-0.01 wt%; and the content of hyaluronic acid salt is 0.1 wt% or more. The combination of hyaluronic acid salt and ergothioneine can effectively inhibit the photodegradation of ergothioneine, and this composition can be used in food, health products, or cosmetics, exhibiting good moisturizing effects and stable efficacy.

[0018] This invention proposes to use a combination of ergothioneine and asiaticoside to improve the chemical stability of asiaticoside, facilitate the formulation of its pharmaceutical preparations and cosmetics, and maximize its biological activity.

[0019] This invention unexpectedly discovered that under different pH conditions and concentrations, the content of ergothionein in the system was significantly increased, and at the same time, it unexpectedly synergistically enhanced the antioxidant effect and the repair effect of asiaticoside. It also promoted the expression of FLG gene, LOR gene, and Claudin-1 gene, which has broad application prospects in the cosmetic field. Summary of the Invention

[0020] The purpose of this invention is to provide a highly efficient and cost-effective method for inhibiting the photodegradation and high-temperature instability of ergothioneine based on a combination of ergothioneine and asiaticoside. Unexpectedly, it significantly increases the content of ergothioneine in the system under different pH conditions and concentrations, and also unexpectedly enhances the antioxidant effects, promotes the expression of FLG gene, LOR gene, and Claudin-1 gene, and unexpectedly enhances the repair effects of asiaticoside. This invention has broad application potential in the cosmetics field.

[0021] This invention provides a stable synergistic composition based on asiaticoside and ergothioneine, which mainly comprises asiaticoside and ergothioneine, wherein the weight ratio of asiaticoside to ergothioneine is 0.5-5:1-8, and wherein the content of asiaticoside in the composition is 0.05-2wt%.

[0022] In a preferred embodiment, the weight ratio of asiaticoside to ergothionein is 0.5-3:1-2.

[0023] In a preferred embodiment, the composition further comprises ≤5 wt% of a C3-C6 diol.

[0024] Preferably, the C3-C6 diols are selected from: butanediol, propylene glycol, 1,3-propanediol, pentanediol, 1,2-hexanediol, glycerol, diglycerol, etc.

[0025] In a preferred embodiment, the composition further comprises ≤0.8 wt% of acrylamide dimethyl taurate ammonium / VP copolymer.

[0026] In a preferred embodiment, the composition further comprises a carrier acceptable in the field of topical skin agents.

[0027] Preferably, water is an acceptable carrier in the field of topical skin agents.

[0028] More preferably, the water content in the composition is 91-95 wt%.

[0029] In a preferred embodiment, the composition may further comprise hyaluronic acid with a molecular weight of 3K-10KDa, and the weight ratio of hyaluronic acid to asiaticoside is 1:1.

[0030] In a preferred embodiment, the composition further comprises a pH adjuster.

[0031] Preferably, the pH adjuster is selected from at least one of citric acid and tromethamine.

[0032] In a preferred embodiment, the pH value of the composition is 4-8.

[0033] Preferably, the pH value of the composition is 4-6.

[0034] The present invention also provides the application of asiaticoside in inhibiting the photodegradation of ergothionein.

[0035] In a preferred embodiment, the weight ratio of asiaticoside to ergothionein is 0.5-5:1-8.

[0036] Preferably, the application is performed at a pH of 4-8.

[0037] More preferably, the pH value is 4-6.

[0038] The present invention also provides the antioxidant use of compositions based on asiaticoside and ergothionein.

[0039] Preferably, the weight ratio of scotoxin to ergothionein in the compound is 0.5-5:1-8.

[0040] More preferably, the pH value of the composition is 4-6.

[0041] The present invention also provides the use of a composition based on asiaticoside and ergothioneine as an FLG gene expression promoter.

[0042] Or, based on the combination of asiaticoside and ergothioneine as a promoter of LOR gene expression,

[0043] Or the use of a combination of asiaticoside and ergothionein as a Claudin-1 gene expression promoter.

[0044] Preferably, the weight ratio of scotoxin to ergothionein in the compound is 0.5-5:1-8.

[0045] More preferably, the pH value of the composition is 4-6.

[0046] The present invention also provides the use of compositions based on asiaticoside and ergothioneine in topical skin preparations.

[0047] In a preferred embodiment, the topical skin agent is selected from: face cream, lotion, gel, toner, serum, face mask, eye cream, aerosol (cleansing foam), spray, shower gel, facial cleanser, and essence water.

[0048] In a preferred embodiment, the composition is used in a topical skin preparation at an amount of 0.0001 wt%-90 wt%.

[0049] The preferred weight percentage is 0.001wt%-10wt%.

[0050] A more preferred weight percentage is 0.001wt%-5wt%.

[0051] The beneficial effects of this invention are:

[0052] 1. This invention is the first to protect the stability of ergothioneine using asiaticoside as a component. Through the special role of hydrogen bonds, the conformational stability of ergothioneine under light and high temperature can be protected.

[0053] 2. This invention unexpectedly discovered that asiaticoside and ergothionein have a very good synergistic effect, which can improve the antioxidant efficacy by more than 100%.

[0054] 3. This invention unexpectedly discovered that asiaticoside and ergothionein have a very good synergistic effect, which can improve the skin repair ability by more than 100%, that is, it has a significant promoting effect on FLG gene expression, LOR gene expression and Claudin-1 gene expression. It can be used as an FLG gene expression promoter, LOR gene expression promoter or Claudin-1 gene expression promoter. Attached Figure Description

[0055] Figure 1 The results of ROS content detection in test example 4. Detailed Implementation

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar or equivalent to those described herein may be used to practice or test the invention, preferred methods and materials are described herein. For the purposes of this invention, the following terms are defined.

[0057] As used herein, the term "about" means a quantity, level, value, dimension, size, or amount that differs from that of a reference by as much as 30%, 20%, or 10%. Percentages used herein, unless otherwise stated, are by weight.

[0058] Throughout this specification and claims, unless otherwise required, the words “comprising” and its variations “containing” and “including” shall be understood to mean including the said whole or step, or a group of whole or steps, but not excluding any other whole or step, or other group of whole or steps.

[0059] C3-C6 diols

[0060] The C3-C6 diols described in this invention can be selected from: butanediol, propylene glycol, 1,3-propanediol, pentanediol, 1,2-hexanediol, glycerol, diglycerol, etc.

[0061] The present invention is further illustrated below with reference to specific embodiments. However, it should be understood that these embodiments are for illustrative purposes only and do not constitute a limitation on the scope of the invention. Test methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0062] The main experimental materials and reagents used in the examples are as follows:

[0063] Centella asiatica extract, purity ≥99.0%, Shanghai Kunqian Biotechnology Co., Ltd.

[0064] Ergothioneine, purity ≥99.0%, Shanghai Ergothioneine Co., Ltd.

[0065] Tromethamine, analytical grade, Sinopharm Group

[0066] Citric acid, analytical grade, Sinopharm Group

[0067] Acrylamide dimethyl taurate ammonium / VP copolymer (AVC), purity ≥99.0%, Clariant (China) Co., Ltd.

[0068] Hydroxypropyl β-cyclodextrin, purity ≥99%, Shandong Binzhou Zhiyuan Biotechnology Co., Ltd.

[0069] Hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) (purity ≥99.0%, Bloomage Biotechnology Co., Ltd.)

[0070] Sodium hyaluronate (molecular weight 10K-100KDa) (purity ≥99.0%, Bloomage Biotechnology Co., Ltd.)

[0071] Hyaluronic acid (molecular weight 100K-1000KDa) (purity ≥99.0%, Bloomage Biotechnology Co., Ltd.)

[0072] Formic acid, purity ≥98.0%, Sigma-Aldrich (Product No. 43804)

[0073] Acetonitrile, purity ≥99.9%, Sigma-Aldrich (Product No. 34851)

[0074] Methanol, purity ≥99.9%, Sigma-Aldrich (Product No. 34885)

[0075] DCFH-DA culture medium (LMAI Bio, Shanghai Lianmai Biotechnology Co., Ltd.)

[0076] Pancreatic enzyme, Beijing Solarbio Science & Technology Co., Ltd., product batch number T8151

[0077] Main experimental instruments

[0078] XS205 analytical balance (METTLER TOLEDO)

[0079] Incucell incubator (MMM GmbH, Germany)

[0080] Waters ARC high-performance liquid chromatograph with diode array detector (Waters)

[0081] KQ-800DE CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.)

[0082] CO2 incubator (Thermo, 150I)

[0083] Clean bench (Su Jing An Tai, SW-CJ-1F)

[0084] Flow cytometer (Beckman, CytoFLEX)

[0085] Inverted microscope (Olympus, CKX53)

[0086] UVB irradiator (Philips).

[0087] PERCIVAL CU41L5 Illuminated Incubator (PERCIVAL Corporation, USA)

[0088] Example 1:

[0089] Accurately weigh 0.8g of ergothioneine and dissolve it in 5g of butanediol. Add 1.0g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0090] Example 2:

[0091] Accurately weigh 0.3g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0092] Example 3:

[0093] Accurately weigh 0.6g of ergothioneine and dissolve it in 5g of butanediol. Add 2.0g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0094] Example 4:

[0095] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0096] Example 5:

[0097] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0098] Example 6:

[0099] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0100] Example 7:

[0101] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0102] Example 8:

[0103] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.3g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0104] Example 9:

[0105] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of hydroxypropyl β-cyclodextrin and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0106] Example 10:

[0107] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), stir well, add 0.8g of AVC and disperse thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethamine. Pour into a volumetric flask and set aside for later use.

[0108] Example 11:

[0109] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of sodium hyaluronate (molecular weight 10K-100KDa) and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethamine. Pour into a volumetric flask and set aside for later use.

[0110] Example 12:

[0111] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of hyaluronic acid (molecular weight 3K-10KDa) and 0.1g of asiaticoside, and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethamine. Pour into a volumetric flask and set aside for later use.

[0112] Example 13:

[0113] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of hyaluronic acid (molecular weight 100K-1000KDa) and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0114] Example 14:

[0115] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Stir until homogeneous and clear. Add 0.8g of AVC and disperse thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 4.0 using citric acid and tromethamine. Transfer the solution to a volumetric flask for later use.

[0116] Example 15:

[0117] Accurately weigh 0.8g of ergothioneine and dissolve it in 5g of butanediol. Add 1.0g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0118] Example 16:

[0119] Accurately weigh 0.3g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0120] Example 17:

[0121] Accurately weigh 0.6g of ergothioneine and dissolve it in 5g of butanediol. Add 2.0g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0122] Example 18:

[0123] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0124] Example 19:

[0125] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0126] Example 20:

[0127] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0128] Example 21:

[0129] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0130] Example 22:

[0131] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.3g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0132] Example 23:

[0133] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of hydroxypropyl β-cyclodextrin and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0134] Example 24:

[0135] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), stir well, then add 0.8g of AVC and disperse thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethamine. Pour into a volumetric flask and set aside for later use.

[0136] Example 25:

[0137] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of sodium hyaluronate (molecular weight 10K-100KDa) and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0138] Example 26:

[0139] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butylene glycol. Add 0.1g of hyaluronic acid (molecular weight 3K-10KDa) and 0.1g of asiaticoside, and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethamine. Pour into a volumetric flask and set aside for later use.

[0140] Example 27:

[0141] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of hyaluronic acid (molecular weight 100K-1000KDa) and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0142] Example 28:

[0143] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Stir until homogeneous and clear. Add 0.8g of AVC and disperse thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethamine. Transfer the solution to a volumetric flask for later use.

[0144] Example 29:

[0145] Accurately weigh 0.8g of ergothioneine and dissolve it in 5g of butanediol. Add 1.0g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0146] Example 30:

[0147] Accurately weigh 0.3g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0148] Example 31:

[0149] Accurately weigh 0.6g of ergothioneine and dissolve it in 5g of butanediol. Add 2.0g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0150] Example 32:

[0151] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0152] Example 33:

[0153] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0154] Example 34:

[0155] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0156] Example 35:

[0157] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0158] Example 36:

[0159] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.3g of asiaticoside and stir until homogeneous. Then add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethorphan. Pour the solution into a volumetric flask and set aside for later use.

[0160] Example 37:

[0161] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of hydroxypropyl β-cyclodextrin and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethamine. Pour into a volumetric flask and set aside for later use.

[0162] Example 38:

[0163] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), stir well, add 0.8g of AVC and disperse thoroughly until uniform. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethamine. Pour into a volumetric flask and set aside for later use.

[0164] Example 39:

[0165] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of sodium hyaluronate (molecular weight 10K-100KDa) and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0166] Example 40:

[0167] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butylene glycol. Add 0.1g of hyaluronic acid (molecular weight 3K-10KDa) and 0.1g of asiaticoside, and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethorphan. Pour into a volumetric flask and set aside for later use.

[0168] Example 41:

[0169] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of hyaluronic acid (molecular weight 100K-1000KDa) and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0170] Example 42:

[0171] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Stir until homogeneous and clear. Add 0.8g of AVC and disperse thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethamine. Pour into a volumetric flask and set aside.

[0172] Test Example 1: Illumination Test Experiment

[0173] Test samples: Composition samples prepared in Examples 1-42

[0174] Product stability refers to the stability of a product in an inert container. In this experiment, a transparent glass bottle with a cylindrical diameter of about 5 cm was used. The composition samples prepared in Examples 1-39 were placed in the glass bottle and the cap was tightened. The bottle was continuously irradiated in a light test light box for 14 days. The test light box was equipped with a set of lamps, and the sample was about 30-40 cm away from the lamps. A suitable lamp for this purpose is POLARIT (daylight type), a 40-watt (Thorn-EMI) lamp tube with a length of 132 cm. A set of 12 lamps provides the light intensity and spectral distribution to simulate sunlight.

[0175] The aroma characteristics and intensity of the above samples were evaluated by professional fragrance tasters using direct olfaction. This evaluation method can be used to determine the stability and degree of change of the aroma. A three-level grading standard was adopted.

[0176]

[0177] The results of the composition samples prepared in Examples 1-42 after 14 days of light exposure testing are shown in Table 1:

[0178] Table 1. Results of light irradiation tests on the composition samples prepared in Examples 1-42.

[0179]

[0180] After 14 days of simulated sunlight exposure testing, under acidic pH conditions, the composition samples containing ergothioneine, butylene glycol, AVC, asiaticoside, and water prepared in Examples 4-6 and 8 showed no perceptible change in taste; the composition samples prepared in Examples 1-3 showed only slight changes in taste.

[0181] At the same pH value (pH=4), when all or part of the asiaticoside in the composition was replaced with hydroxypropyl β-cyclodextrin, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), sodium hyaluronate (molecular weight 10K-100KDa), hyaluronic acid (molecular weight 3K-10KDa), or hyaluronic acid (molecular weight 100K-1000KDa), the composition samples showed different light irradiation test performance.

[0182] Specifically as follows:

[0183] Example 9 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 6 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and asiaticoside in the latter, and with the same content of the remaining components in both, the composition sample prepared in Example 6 performed significantly better than the composition sample prepared in Example 9 under light irradiation testing. This indicates that asiaticoside has a positive stabilizing effect on ergothioneine in the composition.

[0184] Example 10 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 5 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 5 performed significantly better than the composition sample prepared in Example 10 under light irradiation testing. This indicates that asiaticoside has a positive stabilizing effect on ergothioneine in the composition. Furthermore, it also demonstrates that hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) cannot inhibit the photodegradation of ergothioneine.

[0185] Example 11 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 5 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and asiaticoside in the latter, and with the same content of the other components in both, the composition sample prepared in Example 5 performed significantly better than the composition sample prepared in Example 11 under light irradiation testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. Furthermore, it also shows that sodium hyaluronate (molecular weight 10K-100KDa) does not inhibit the photodegradation of ergothioneine as disclosed in the prior art.

[0186] Example 12 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 3K-10KDa), asiaticoside, and water. Example 6 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. In the former, the hyaluronic acid (molecular weight 3K-10KDa) content was half that of the latter, and the contents of the remaining components were the same. Under light irradiation testing, the composition sample prepared in Example 6 performed better than the composition sample prepared in Example 12. This indicates that asiaticoside not only helps inhibit the photodegradation of ergothioneine in the composition, but also inhibits the photodegradation of ergothioneine when asiaticoside and hyaluronic acid (molecular weight 3K-10KDa) are used in combination.

[0187] Example 13 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 5 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 5 performed significantly better than the composition sample prepared in Example 13 under light irradiation testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. However, hyaluronic acid (molecular weight 100K-1000KDa) cannot help inhibit the photodegradation of ergothioneine.

[0188] At the same pH value (pH=6), when all or part of the asiaticoside in the composition was replaced with hydroxypropyl β-cyclodextrin, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), sodium hyaluronate (molecular weight 10K-100KDa), or hyaluronic acid (molecular weight 100K-1000KDa), the composition samples showed different light irradiation test performance.

[0189] Specifically as follows:

[0190] The composition samples prepared in Examples 17-19 and 22, which contained ergothioneine, butylene glycol, AVC, asiaticoside, and water, showed no perceptible change in taste; the composition samples prepared in Examples 15-16 and 20-21 showed only slight changes in taste.

[0191] Example 23 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 20 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and asiaticoside in the latter, and with identical contents of the remaining components, the composition sample prepared in Example 20 performed better than the composition sample prepared in Example 23 under light irradiation testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. Hydroxypropyl β-cyclodextrin is less effective than asiaticoside in inhibiting the photodegradation of ergothioneine.

[0192] Example 24 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 19 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 19 performed significantly better than the composition sample prepared in Example 24 under light irradiation testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. However, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) cannot help inhibit the photodegradation of ergothioneine.

[0193] Example 25 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 19 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and asiaticoside in the latter, and with the same content of the remaining components, the composition sample prepared in Example 19 performed significantly better than the composition sample prepared in Example 25 under light irradiation testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. Sodium hyaluronate (molecular weight 10K-100KDa), however, cannot help inhibit the photodegradation of ergothioneine.

[0194] Example 26 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, hyaluronic acid (molecular weight 3K-10KDa), and water. Example 20 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. The former contained half the amount of hyaluronic acid (molecular weight 3K-10KDa) as the latter, and with the remaining components present in the same amounts, the composition sample prepared in Example 20 performed better than the composition sample prepared in Example 26 under light irradiation testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. However, the combination of hyaluronic acid (molecular weight 3K-10KDa) and asiaticoside did not help inhibit the photodegradation of ergothioneine.

[0195] Example 27 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 19 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 19 performed significantly better than the composition sample prepared in Example 27 under light irradiation testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. However, hyaluronic acid (molecular weight 100K-1000KDa) cannot help inhibit the photodegradation of ergothioneine.

[0196] Example 28 prepared a composition sample containing ergothioneine, butylene glycol, AVC, and water, while Examples 18-21 prepared composition samples containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. The difference between the two is that Examples 18-21 contained 0.05-0.8 wt% asiaticoside. With the other components having the same content, under light irradiation testing, the composition samples prepared in Examples 18-21 performed better than those prepared in Example 28. This further clarifies that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition.

[0197] Under alkaline conditions (pH=8), the composition samples prepared in Examples 29-30, 32-35, and 37-42 all showed significant changes in taste, mainly due to the decomposition of ergothionein in the samples caused by simulated sunlight exposure. The composition sample prepared in Example 31 showed only slight changes in taste, while the composition sample prepared in Example 36 showed no perceptible changes in taste, indicating that the addition of an appropriate amount of asiaticoside in the composition resulted in very good photostability.

[0198] At the same pH value (pH=8), when all or part of the asiaticoside in the composition was replaced with hydroxypropyl β-cyclodextrin, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), sodium hyaluronate (molecular weight 10K-100KDa), hyaluronic acid (molecular weight 3K-10KDa), or hyaluronic acid (molecular weight 100K-1000KDa), the composition samples showed different light irradiation test performance.

[0199] Specifically as follows:

[0200] Example 37 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 34 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and asiaticoside in the latter, and with identical contents of the remaining components, the composition sample prepared in Example 34 performed similarly to the one prepared in Example 34 under light irradiation testing, both being unsatisfactory. This indicates that asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions. Hydroxypropyl β-cyclodextrin also cannot help inhibit the photodegradation of ergothioneine.

[0201] Example 38 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 33 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 33 performed similarly to the composition sample prepared in Example 38 under light irradiation testing, both being unsatisfactory. This indicates that asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions. Hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) also cannot help inhibit the photodegradation of ergothioneine.

[0202] Example 39 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 33 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and asiaticoside in the latter, and with identical content of the other components, the composition sample prepared in Example 33 performed similarly to that prepared in Example 39 under light irradiation testing, both being unsatisfactory. This indicates that asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions. Similarly, sodium hyaluronate (molecular weight 10K-100KDa) also cannot help inhibit the photodegradation of ergothioneine.

[0203] Example 40 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 3K-10KDa), asiaticoside, and water. Example 33 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. The former contained half the amount of hyaluronic acid (molecular weight 3K-10KDa) as the latter, and both had the same content of the remaining components. Under light irradiation, the composition sample prepared in Example 33 performed similarly to that prepared in Example 40, both being unsatisfactory. This indicates that asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions. The combination of hyaluronic acid (molecular weight 3K-10KDa) and asiaticoside also cannot help inhibit the photodegradation of ergothioneine.

[0204] Example 41 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 33 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) in the former and asiaticoside in the latter, and with identical contents of the other components, the composition sample prepared in Example 33 performed similarly to that prepared in Example 41 under light irradiation testing, both being unsatisfactory. This indicates that asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions. Hyaluronic acid (molecular weight 100K-1000KDa) also cannot help inhibit the photodegradation of ergothioneine.

[0205] Example 42 prepared a composition sample containing ergothioneine, butylene glycol, AVC, and water. Examples 32-35 prepared composition samples containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. The difference between the two is that the latter, Examples 32-35, contained 0.05-0.8 wt% asiaticoside. With the contents of the other components being the same, under light irradiation testing, the composition samples prepared in Examples 32-35 performed similarly to those prepared in Example 42, and were both unsatisfactory. This further clarifies that asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions.

[0206] In summary, asiaticoside can significantly help inhibit the photodegradation of ergothionein, and the inhibition efficiency of asiaticoside is better under acidic conditions than under alkaline conditions.

[0207] Test Example 2: High Temperature Test Experiment

[0208] Test samples: Composition samples prepared in Examples 1-42

[0209] Generally speaking, the reaction rate roughly doubles for every 10°C increase in temperature. Many important properties of products, especially cosmetics, such as appearance, color, odor, and texture, are visually measured and not easily expressed numerically. If any changes are not described visually but recorded in five levels from slight to severe, it is easier to preserve records and evaluate results.

[0210] The samples from Examples 1-42 were poured into glass bottles, the caps were tightened, and the bottles were placed in an Incucell incubator at 48°C for 14 days to observe the changes in the products.

[0211] The aroma characteristics and intensity of the above samples were evaluated by professional fragrance tasters using direct olfaction. This evaluation method can be used to determine the stability and degree of change of the aroma. A three-level grading standard was adopted.

[0212]

[0213] Table 2 High-temperature tests of the composition samples prepared in Examples 1-42

[0214]

[0215] After 14 days of high-temperature testing at 48°C, under acidic pH conditions, the composition samples containing ergothioneine, butylene glycol, AVC, asiaticoside, and water prepared in Examples 4-6 and 8 showed no perceptible change in taste; the composition samples prepared in Examples 1-3 and 7 showed slight changes in taste.

[0216] At the same pH value (pH=4), when all or part of the asiaticoside in the composition was replaced with hydroxypropyl β-cyclodextrin, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), sodium hyaluronate (molecular weight 10K-100KDa), hyaluronic acid (molecular weight 3K-10KDa), or hyaluronic acid (molecular weight 100K-1000KDa), the composition samples exhibited different high-temperature stability characteristics.

[0217] Specifically as follows:

[0218] Example 9 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 6 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and asiaticoside in the latter, and with the same content of the remaining components in both, the composition sample prepared in Example 6 showed significantly better performance than the composition sample prepared in Example 9 under high-temperature stability testing. This indicates that asiaticoside has an excellent stabilizing effect on ergothioneine in the composition.

[0219] Example 10 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 5 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 5 showed significantly better performance than the composition sample prepared in Example 10 under high-temperature stability testing. This indicates that asiaticoside has an excellent stabilizing effect on ergothioneine in the composition. Furthermore, it also shows that hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) cannot inhibit the photodegradation of ergothioneine.

[0220] Example 11 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 5 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and asiaticoside in the latter, and with the same content of the other components in both, the composition sample prepared in Example 5 showed significantly better performance than the composition sample prepared in Example 11 under high-temperature stability testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. Furthermore, it also shows that sodium hyaluronate (molecular weight 10K-100KDa) does not inhibit the photodegradation of ergothioneine as disclosed in the prior art.

[0221] Example 12 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 3K-10KDa), asiaticoside, and water. Example 6 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. In the former, the hyaluronic acid (molecular weight 3K-10KDa) content was half that of the latter, and the contents of the remaining components were the same. Under high-temperature stability testing, the composition sample prepared in Example 2 performed better than the composition sample prepared in Example 12. This indicates that asiaticoside can not only help inhibit the photodegradation of ergothioneine in the composition, but also inhibit the photodegradation of ergothioneine when asiaticoside and hyaluronic acid (molecular weight 3K-10KDa) are used in combination, although the inhibition efficiency is lower than that of asiaticoside.

[0222] Example 13 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 5 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 5 showed significantly better performance than the composition sample prepared in Example 13 under high-temperature stability testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. However, hyaluronic acid (molecular weight 100K-1000KDa) cannot help inhibit the photodegradation of ergothioneine.

[0223] At the same pH value (pH=6), when all or part of the asiaticoside in the composition was replaced with hydroxypropyl β-cyclodextrin, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), sodium hyaluronate (molecular weight 10K-100KDa), or hyaluronic acid (molecular weight 100K-1000KDa), the composition samples showed different high-temperature stability test results.

[0224] Specifically as follows:

[0225] The composition samples prepared in Examples 17-19 and 22, which contained ergothioneine, butylene glycol, AVC, asiaticoside, and water, showed no perceptible change in taste; the composition samples prepared in Examples 15-16 and 20-21 showed slight changes in taste.

[0226] Example 23 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 20 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and asiaticoside in the latter, and with identical contents of the remaining components, the composition sample prepared in Example 20 performed better than the composition sample prepared in Example 23 under high-temperature stability testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. Hydroxypropyl β-cyclodextrin, however, cannot help inhibit the photodegradation of ergothioneine.

[0227] Example 24 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 19 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 19 showed significantly better performance than the composition sample prepared in Example 24 under high-temperature stability testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. However, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) cannot help inhibit the photodegradation of ergothioneine.

[0228] Example 25 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 19 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 19 showed significantly better performance than the composition sample prepared in Example 25 under high-temperature stability testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. Sodium hyaluronate (molecular weight 10K-100KDa) cannot help inhibit the photodegradation of ergothioneine.

[0229] Example 26 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 20 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. In the former, the hydroxypropyl β-cyclodextrin content was half that of the latter, and the contents of the remaining components were the same. Under high-temperature stability testing, the composition sample prepared in Example 20 performed better than the composition sample prepared in Example 26. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. However, the combination of hyaluronic acid (molecular weight 100K-1000KDa) and asiaticoside cannot help inhibit the photodegradation of ergothioneine; its inhibition efficiency is lower than that of asiaticoside.

[0230] Example 27 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 19 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 19 showed significantly better performance than the composition sample prepared in Example 27 under high-temperature stability testing. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition. However, hyaluronic acid (molecular weight 100K-1000KDa) cannot help inhibit the photodegradation of ergothioneine.

[0231] Example 28 prepared a composition sample containing ergothioneine, butylene glycol, AVC, and water, while Examples 18-21 prepared composition samples containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. The difference between the two is that Examples 18-21 contained 0.05-0.5 wt% asiaticoside. With the contents of the other components being the same, the composition samples prepared in Examples 18-21 performed better than those prepared in Example 28 in high-temperature stability tests. This further clarifies that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition.

[0232] Under alkaline conditions (pH=8), the composition samples prepared in Examples 29-30, 32-35 and 37-42 all showed significant changes in taste; the composition samples prepared in Examples 31 and 36 showed only slight changes in taste, indicating that the addition of an appropriate amount of asiaticoside in the composition resulted in very good high-temperature stability.

[0233] At the same pH value (pH=8), when all or part of the asiaticoside in the composition was replaced with hydroxypropyl β-cyclodextrin, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), sodium hyaluronate (molecular weight 10K-100KDa), hyaluronic acid (molecular weight 3K-10KDa), or hyaluronic acid (molecular weight 100K-1000KDa), the composition samples showed different high-temperature stability test results.

[0234] Specifically as follows:

[0235] Example 37 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 34 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and asiaticoside in the latter, and with identical contents of the remaining components, the high-temperature stability test results of the composition sample prepared in Example 34 were comparable to those of the composition sample prepared in Example 37, both being unsatisfactory. This indicates that asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions. Hydroxypropyl β-cyclodextrin also cannot help inhibit the photodegradation of ergothioneine.

[0236] Example 38 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 33 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) and asiaticoside in the former and the same content of the other components in the latter, the composition sample prepared in Example 33 performed similarly to the composition sample prepared in Example 38 under high-temperature stability testing, both being unsatisfactory. This indicates that asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions. Hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) also cannot help inhibit the photodegradation of ergothioneine.

[0237] Example 39 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 33 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and asiaticoside in the latter, and with identical contents of the other components, the composition sample prepared in Example 33 performed similarly to that prepared in Example 39 under high-temperature stability testing, both being unsatisfactory. This indicates that asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions. Sodium hyaluronate (molecular weight 10K-100KDa) also cannot help inhibit the photodegradation of ergothioneine.

[0238] Example 40 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 3K-10KDa), asiaticoside, and water. Example 34 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. In the former, the hyaluronic acid (molecular weight 3K-10KDa) content was half that of the latter, and the contents of the remaining components were the same. Under high-temperature stability testing, the composition sample prepared in Example 34 performed similarly to that prepared in Example 40, both being unsatisfactory. This indicates that asiaticoside can help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions. Furthermore, the combination of hyaluronic acid (molecular weight 3K-10KDa) and asiaticoside can also help inhibit the photodegradation of ergothioneine.

[0239] Example 41 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 33 prepared a composition sample containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) in the former and asiaticoside in the latter, and with identical contents of the other components, the high-temperature stability test results of the composition sample prepared in Example 33 were comparable to those of the composition sample prepared in Example 41, both being unsatisfactory. This indicates that asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition under alkaline conditions. Hyaluronic acid (molecular weight 100K-1000KDa) also cannot help inhibit the photodegradation of ergothioneine.

[0240] Example 42 prepared a composition sample containing ergothioneine, butylene glycol, AVC, and water. Examples 32-35 prepared composition samples containing ergothioneine, butylene glycol, AVC, asiaticoside, and water. The difference between the two is that Examples 32-35 contained 0.05-0.5 wt% asiaticoside. With the contents of the other components being the same, the high-temperature stability test results of the composition samples prepared in Examples 32-35 were comparable to those of the composition samples prepared in Example 42, both being unsatisfactory. It is further clarified that under alkaline conditions, when the ergothioneine content in the composition is 0.1 wt%, asiaticoside cannot help inhibit the photodegradation of ergothioneine in the composition.

[0241] In summary, under acidic conditions, asiaticoside can significantly help inhibit the photodegradation of ergothionein, and the inhibition efficiency of asiaticoside is better than that under alkaline conditions.

[0242] Test Example 3: Detection of Ergothionein by Ultra-High Performance Liquid Chromatography

[0243] Test samples: Composition samples prepared in Examples 1-42 after the light irradiation test in Test Example 1; Composition samples prepared in Examples 1-42 after the high temperature stability test in Test Example 2.

[0244] Test standard: NY / T 3872-2021 (UPLC)

[0245] The instrument conditions were as follows: Agilent HILIC Plus 4.6×100 mm 3.5 μm, column temperature 30℃, mobile phase 0.1% formic acid acetonitrile solution-0.1% formic acid aqueous solution (80+20 V / V), flow rate 1.0 mL / min, detection wavelength 262 nm, and injection volume 10 μL.

[0246] Testing steps:

[0247] Preparation of ergothioneine standard stock solution (0.5 g / L): Weigh 50 mg of ergothioneine standard (accurate to 0.01 mg), dissolve in water, and dilute to 100 mL. Store at 4℃. Preparation of ergothioneine standard working solutions: Accurately transfer a certain amount of ergothioneine standard stock solution and serially dilute with 0.1% formic acid methanol solution to obtain a series of standard working solutions (mass concentrations of 5 μg / mL, 10 μg / L, 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively).

[0248] Standard curve plotting: The standard working solution was measured under the above chromatographic conditions, and the standard curve was plotted with the mass concentration of the standard working solution as the abscissa and the peak area as the ordinate.

[0249] Sample preparation and determination: Weigh 0.1–0.5 g (accurate to 0.1 mg) of sample into a 10 mL stoppered colorimetric tube, add 8 mL of 0.1% formic acid in methanol solution, and sonicate for 20 min. Make up to volume with 0.1% formic acid in methanol solution, shake well, and filter a portion of the liquid through a 0.22 μm filter membrane. Then, determine the sample under the chromatographic conditions described above. Quantify the sample using the standard curve. The analyte response value in the sample solution should be within the linear range of the standard curve.

[0250] Results Calculation: Quantitative analysis using the external standard method; the formula for calculating the ergothionein content in the sample is as follows:

[0251]

[0252] In the formula:

[0253] X — Mass fraction of the analyte in the sample, %

[0254] C—The concentration of ergothioneine in the sample solution obtained from the standard curve, in micrograms per milliliter (μg·mL). -1);

[0255] V—The total volume of the sample after dilution, in milliliters (mL);

[0256] m — Sample mass, in grams (g);

[0257] The ergothioneine content in the composition samples after light exposure or high temperature stability testing is shown in the table below:

[0258] Table 3. Ergothionein content in the composition samples after light or high temperature stability tests.

[0259]

[0260] The results show that, after comprehensive investigation under various light conditions (as shown in Table 1), high temperature conditions (as shown in Table 2), different pH conditions, and different ergothioneine concentrations, very clear test results were obtained as shown in the table above.

[0261] First, under alkaline conditions (pH=8), ergothioneine tends to decompose more readily. Due to its unique structure, containing hydroxyl and carbonyl groups, ergothioneine reacts chemically with hydroxide ions under alkaline conditions, resulting in changes in structure, properties, and odor. Regardless of concentration or under light or high temperature conditions, the structural integrity and presence of ergothioneine are significantly challenged, and the odor often changes dramatically, becoming noticeably foul. However, even under these stringent conditions, we unexpectedly found that the composition sample prepared in Example 36, even at an ergothioneine:asiaticoside ratio of 2:3, exhibited excellent stability due to the combined effect of ergothioneine and asiaticoside. Asiaticoside, a triterpenoid compound, has abundant hydroxyl groups on its surface that effectively protect ergothioneine through hydrogen bonding. This condition and ratio provide a very good solution and technology for the application of ergothioneine in alkaline products.

[0262] Acidic pH values ​​(especially around pH=4) are the recommended pH values ​​for the application of ergothioneine. A comparison between pH=4 and pH=6 shows that a lower pH under acidic conditions is more conducive to the stability of ergothioneine. Studies on different ratios of ergothioneine at different concentrations have unexpectedly revealed that several different combinations and concentrations have excellent protective functions.

[0263] From the composition samples prepared in Examples 14, 28, and 42, it can be seen that the content of ergothioneine was degraded by more than 30-50% over a period of 2 weeks under different pH conditions.

[0264] In contrast to existing technologies, such as the Chinese patent application CN110327242B which discloses a method for inhibiting the photodegradation of ergothioneine using hyaluronic acid salts, the composition samples prepared in Examples 10-11, 13, 24-25, 27, 38-39, and 41 of this application were used to simultaneously investigate the inhibitory effect of hyaluronic acid and its derivatives on ergothioneine in the prior art. Compared with ergothioneine under the same concentration and pH conditions, no significant improvement in photodegradation and high-temperature degradation stability was observed. On the contrary, compared with the composition samples prepared in Examples 14, 28, and 42, the content of ergothioneine decreased to a certain extent under the same conditions. It is speculated that the possible reason is that different types of hyaluronic acid, including hyaluronic acid with a relatively small molecular weight but still having a molecular weight of several thousand Daltons, did not form a complete and tight encapsulation structure, resulting in ergothioneine concentration differences and escape.

[0265] Under pH=4 conditions, the composition samples prepared in Examples 4-6 and Example 8 unexpectedly achieved excellent protective effects against ergothioneine. After 14 days of light exposure or 14 days of high temperature stability testing, the ergothioneine content in the samples remained above 81%.

[0266] Under pH=6 conditions, the compositions prepared in Examples 17-19 and Example 22 also unexpectedly exhibited excellent protective effects against ergothioneine. After 14 days of light exposure or 14 days of high-temperature stability testing, the ergothioneine content in the samples remained above 79.9%.

[0267] In summary, when the pH value is acidic, the asiaticoside molecule in the composition sample has abundant hydroxyl groups. The abundant hydroxyl groups on the surface provide excellent protection for ergothioneine through hydrogen bonding. There are many binding sites for both, and this binding is a spontaneous reaction dominated by hydrophobic forces. The molecular docking report indicates that the addition of asiaticoside may have caused changes in the internal water transport environment of ergothioneine, making the binding between the two more stable.

[0268] The protective effect is optimal when the weight ratio of ergothioneine to asiaticoside is 1-2:1-3. In Examples 12, 26, and 40, which used a combination of asiaticoside and hyaluronic acid, the protective efficiency of ergothioneine in the samples decreased continuously as the pH was adjusted from 4 to 6 or 8. Even at pH 4, the composition sample prepared in Example 12 showed excellent performance in both light and high-temperature stability tests, and after 14 days of light exposure or 14 days of high-temperature stability testing, the ergothioneine content in the sample remained above 83%. This indicates that only under acidic conditions can the combination of asiaticoside and hyaluronic acid inhibit the degradation of ergothioneine.

[0269] Test Example 4: Evaluation of Antioxidant Performance

[0270] Test samples: Composition samples prepared in Examples 4-6, 8, 12, 14, 18-20, 22, 28, 36, and 42.

[0271] Experimental methods:

[0272] 1) Cell seeding: at 2.2 × 10⁻⁶ 5 Seed keratinocytes at a seeding density of cells / well into 6-well plates and incubate overnight in an incubator (37°C, 5% CO2).

[0273] 2) Drug administration: According to the test group, when the cell deposition rate in the 6-well plate reaches 30%-50%, the drugs are administered to the groups (the compositions of different embodiments are added), 2 mL of sample is added to each well, and each group has 3 replicates. After drug administration, the 6-well plate is placed in an incubator (37℃, 5% CO2) for 24 h.

[0274] 3) UVB irradiation: According to the test groups, the groups exposed to UVB irradiation were subjected to 300mJ / cm² irradiation. 2 UVB irradiation.

[0275] 4) ROS content test: After irradiation, wash each well of cells three times with PBS, add 1 mL of 10 μM DCFH-DA probe to each well, incubate in an incubator (37℃, 5% CO2) for 30 min, discard the culture medium containing DCFH-DA, wash three times with PBS, digest the cells with trypsin (0.25%), wash the cells once with PBS, add a certain amount of fresh PBS, and perform flow cytometry detection.

[0276] 5) Statistical Analysis of Results: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0277] Table 4. Evaluation results of antioxidant performance of some examples

[0278]

[0279] Note: Mean fluorescence intensity (MFI) reflects the ROS content. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0280] For the preferred combination of embodiments (mainly in terms of photodegradation stability and high temperature stability), the antioxidant properties were tested. The antioxidant test adopted a more reasonable and scientific detection method, which is based on the content of reactive oxygen species (ROS) produced by keratinocytes under UVB stimulation. This method is a more accurate way to evaluate the skin's oxidation mechanism and antioxidant mechanism.

[0281] Comparing the antioxidant properties of ergothioneine under different pH conditions, it was found that commonly used vitamin E (as a control group) exhibited superior antioxidant performance. Vitamin E is a substance with strong antioxidant activity and is a crucial antioxidant in the human body, blocking free radical chain reactions. It plays an indispensable role in stabilizing and protecting cell membranes and in normal human metabolism. Vitamin E plays an important role in inhibiting inflammatory responses, fighting free radicals, anti-aging, and anti-cancer processes by influencing inflammatory factors and regulating cell signal transduction. Vitamin E has been widely used to treat various skin diseases, including photoaging, inflammatory skin diseases, connective tissue diseases, viral skin diseases, and herpes. Regarding ergothioneine itself, the compositions prepared in Examples 14, 28, and 42 also significantly improved antioxidant properties compared to vitamin E.

[0282] Comparing the combinations of ergothioneine and asiaticoside under different pH conditions, a significant increase in antioxidant capacity was unexpectedly found in all cases, with the antioxidant capacity increase exceeding 79% in most of the embodiments. Figure 1 As shown.

[0283] The composition samples prepared in Example 8, Example 22, and Example 36 differed only in their pH values, and their antioxidant capacity improvements were 94.37%, 78.58%, and 71.55%, respectively.

[0284] The composition samples prepared in Example 6 and Example 20 differed only in their pH values, and their antioxidant capacity was improved by 72.71% and 79.54%, respectively.

[0285] The composition samples prepared in Example 5 and Example 19 differed only in their pH values, and their antioxidant capacity was improved by 76.48% and 82.82%, respectively.

[0286] The composition samples prepared in Example 4 and Example 18 differed only in their pH values, and their antioxidant capacity was improved by 68.53% and 87.38%, respectively.

[0287] The composition samples prepared in Examples 14, 28 and 42 are identical to those prepared in Examples 4-6 and 18-20, except for the content of asiaticoside.

[0288] When the amount of asiaticoside in the sample was 0.05 wt% and the pH was 4, the antioxidant capacity of the composition sample prepared in Example 4 increased from 44.22% to 68.53% compared with the composition sample prepared in Example 14 (without the addition of asiaticoside), representing an increase of 54.98%.

[0289] When the amount of asiaticoside in the sample was 0.05 wt% and the pH was 6, the antioxidant capacity of the composition sample prepared in Example 18 increased from 42.79% to 87.38% compared with the composition sample prepared in Example 28 (without the addition of asiaticoside), representing an increase of 104.21%.

[0290] When the amount of asiaticoside in the sample was 0.1 wt% and the pH was 4, the antioxidant capacity of the composition sample prepared in Example 5 increased from 44.22% to 76.48% compared with the composition sample prepared in Example 14 (without the addition of asiaticoside), representing an increase of 72.95%.

[0291] When the amount of asiaticoside in the sample was 0.1 wt% and the pH was 6, the antioxidant capacity of the composition sample prepared in Example 19 increased from 42.79% to 82.82% compared with the composition sample prepared in Example 28 (without the addition of asiaticoside), an increase of 93.55%.

[0292] When the amount of asiaticoside in the sample was 0.2 wt% and the pH was 4, the antioxidant capacity of the composition sample prepared in Example 6 increased from 44.22% to 72.71% compared with the composition sample prepared in Example 14 (without the addition of asiaticoside), an increase of 64.43%.

[0293] When the amount of asiaticoside in the sample was 0.2 wt% and the pH was 6, the antioxidant capacity of the composition sample prepared in Example 20 increased from 42.79% to 79.54% compared with the composition sample prepared in Example 28 (without the addition of asiaticoside), representing an increase of 85.88%.

[0294] In conclusion, with the help of asiaticoside, the antioxidant capacity of ergothioneine was increased by at least 54%.

[0295] Ergothioneine regulates intracellular redox reactions and participates in intracellular energy regulation, acting as a physiological protectant for cells. It is safe and stable in both biological and pharmacological properties. Simultaneously, it maintains the stability of other components and promotes the absorption of other active ingredients by cells, exhibiting excellent synergistic effects, achieving a synergistic effect greater than the sum of its parts (1+1>2). Not only does it exert a powerful antioxidant effect, but it also enhances the efficacy and activity of other substances.

[0296] Test Example 5: Repair Performance Evaluation

[0297] Test examples: Composition samples prepared in Examples 8 and 18-19

[0298] Experimental methods:

[0299] 1) The cells used in the test were keratinocytes (batch number Ep220707, provided by Guangdong Boxi Biotechnology Co., Ltd.), and the inoculation was at a rate of 2.5 x 10⁻⁶ cells / year. 5 Seed cells at a density of cells / well into 6-well plates and incubate overnight in an incubator (37°C, 5% CO2).

[0300] 2) The solution was prepared according to WY14643 (Sigma) as the positive control (PC), the drug concentration was 50 μM, the sample group was the composition sample prepared in Example 8, Example 18 and Example 19, the detection index was FLG, LOR, Claudin-1, and the detection method was qRT-PCR.

[0301] 3) Drug administration: When the cell deposition rate in the 6-well plate reaches 40-60%, administer the drug, with 2 mL per well. Set up 3 replicates per group and incubate in an incubator (37℃, 5% CO2) for 24 h.

[0302] 4) Collect cells, culture for 24 hours, discard the old solution, wash twice with PBS (Solepro), add 1 mL of RNAisoPlus (Aikerui Biotechnology) to each well, lyse the cells by pipetting, and collect the sample.

[0303] 5) Gene expression detection: RNA was extracted, reverse transcribed into cDNA (reverse transcription kit purchased from Aikerui Biotechnology), and then detected by real-time PCR. The results were calculated using the 2-ΔΔCT method.

[0304] 6) Statistical analysis of results: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0305] Table 5. Results of FLG (Fluorescent Gene) Detection in Some Examples

[0306]

[0307] Note: When performing statistical analysis using the t-test method, significance compared to group BC is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0308] Table 6. Results of LOR detection for repair genes in some examples.

[0309]

[0310] Note: When performing statistical analysis using the t-test method, significance compared to group BC is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0311] Table 7 shows the detection results of the repair gene Claudin-1 in some examples.

[0312]

[0313] Note: When performing statistical analysis using the t-test method, significance compared to group BC is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0314] Compared with the PC group, it was unexpectedly found that the combination of ergothionein and asiaticoside significantly and synergistically enhanced the expression of asiaticoside repair genes. Among the examples under different pH conditions, the synergistic improvement effect was better under acidic conditions.

[0315] Based on keratinocytes, the FLG (filaggrin, FLG) gene translation and expression product is an acidic or neutral protein (composed of 324 amino acids, approximately 38 kDa in size) found in human epidermal keratinocytes. It participates in the formation of intercellular cross-connection membranes during terminal differentiation of keratinocytes and can be broken down into a large number of natural moisturizing factors. Therefore, FLG plays a crucial role in skin barrier function and keratinocyte apoptosis, as it is responsible for initiating the transformation of keratinocytes from nucleated granular cells to enucleated keratinocytes. It is closely related to various skin diseases; studies have shown that abnormalities in the FLG gene are associated with the occurrence of ichthyosis, atopic dermatitis, psoriasis, eczema, and other diseases.

[0316] As shown in Table 5, the experimental results indicate that asiaticoside has a good effect on promoting FLG gene expression. However, it was unexpectedly found that ergothionein has an excellent synergistic effect. For example, the composition sample prepared in Example 8 can improve the gene improvement effect by 141.79%.

[0317] Loricrin (LOR) is expressed at the end of granular layer differentiation and is a specific differentiation protein that accounts for 85% of the keratinocyte capsule. It catalyzes the formation of a strong protein network of keratin, filaggrin, and other proteins through transglutaminase catalysis. Abnormal expression of Loricrin is associated with common palmar-to-palmar keratosis and hyperkeratotic skin diseases in betta fish.

[0318] As shown in Table 6, the experimental results show that asiaticoside also has a good ability to promote the expression of the acetoxin gene. However, it was unexpectedly found that ergothionein can significantly enhance the effect of asiaticoside. For example, the composition sample prepared in Example 8 increased the expression level of acetoxin by 90%.

[0319] Claudin-1 is a key component of tight junctions in skin epidermal cells and forms the last line of defense for the skin barrier function. Together with desmosomes, it is responsible for sealing the intercellular spaces of the epidermal layer and acts as a barrier against water and solutes. Claudin-1 deficiency in the skin leads to significantly impaired healing and repair of scratches, delayed migration, and reduced proliferation.

[0320] As shown in Table 7, the experimental results show that asiaticoside also has a good ability to promote the expression of Claudin-1 gene. However, it was unexpectedly found that ergothionein can significantly enhance the effect of asiaticoside. For example, the composition sample prepared in Example 8 increased the expression level of Claudin-1 by 77.14%.

[0321] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

[0322] The composition samples prepared in Examples 1-8, 15-22, 31, and 36 were used for the preparation of a topical skin agent. The topical skin agent is preferably a cosmetic composition, such as a lotion, serum, or cream. The composition samples prepared in Examples 1-8, 15-22, 31, and 36 have a weight percentage of 0.0001 wt%-90 wt% in the topical skin agent. A preferred weight percentage is 0.001 wt%-10 wt%. A more preferred weight percentage is 0.001 wt%-5 wt%.

[0323] The following are examples of the specific applications of the compositions prepared in Examples 1-8, 15-22, 31, and 36 in topical skin preparations, along with the formulations and preparation methods of these dosage forms. In the following tables, "-" indicates no additives.

[0324] Application Example 1: Preparation of Face Cream

[0325]

[0326] Application Example 2: Emulsion Preparation

[0327]

[0328] Application Example 3: Preparation of Gel

[0329]

[0330] Application Example 4: Preparation of Toner

[0331]

[0332] Application Example 5: Preparation of Serum

[0333]

[0334] Application Example 6: Preparation of Facial Masks

[0335]

[0336] Application Example 7: Preparation of Eye Cream

[0337]

[0338] Application Example 8: Preparation of Spray

[0339]

[0340] Application Example 9: Preparation of Shower Gel

[0341]

[0342] Application Example 10: Preparation of Facial Cleanser

[0343]

[0344] Application Example 11: Preparation of Essence Water

[0345]

[0346] In the examples of specific applications of all the above topical skin agents, the composition samples of Examples 1-8, Examples 15-22, Examples 31 and 36 can be used directly in the formulation, either partially or completely, to replace deionized water, or can be used directly as a formulation.

Claims

1. A stable synergistic composition based on asiaticoside and ergothioneine, comprising asiaticoside, ergothioneine, butanediol, acrylamide dimethyl taurate ammonium / VP copolymer, and water, wherein, The content of asiaticoside is 0.05-0.3 wt%. The ergothioneine content is 0.1-0.2 wt%. The weight ratio of asiaticoside and ergothioneine is 0.05-0.3:0.1-0.

2. The content of butanediol is 5 wt%. The content of the acrylamide dimethyl taurate ammonium / VP copolymer is 0.8 wt%. The pH value of the composition is 4-6.

2. The application of asiaticoside in inhibiting the photodegradation and high-temperature degradation of ergothioneine, wherein the weight ratio of asiaticoside to ergothioneine is 0.05-0.3:0.1-0.2, the content of asiaticoside is 0.05-0.3wt%, the content of ergothioneine is 0.1-0.2wt%, and the pH value of the application is 4-6.

3. Antioxidant use of the composition as claimed in claim 1 for non-therapeutic purposes.

4. Use of the composition of claim 1 for non-therapeutic purposes to repair the skin barrier.

5. The use of the composition of claim 1 in a non-therapeutic topical skin preparation.

6. The application as described in claim 5, characterized in that, The topical skin agents are selected from: face creams, lotions, gels, toners, face masks, eye creams, sprays, shower gels, and facial cleansers.

7. The application as described in claim 5, wherein the composition is used in a topical skin preparation in an amount of 0.0001 wt% to 90 wt%.

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