Composition for inhibiting photodegradation of ergothioneine based on glucosyl hesperidin

Abstract

This invention discloses a composition for inhibiting the photodegradation of ergothioneine based on glucosyl hesperidin, comprising glucosyl hesperidin and ergothioneine, wherein the weight ratio of glucosyl hesperidin to ergothioneine is 0.1-2.5:0.001-1, and wherein the content of glucosyl hesperidin in the composition is 0.001-3 wt%. This invention also discloses the application of glucosyl hesperidin in inhibiting the photodegradation of ergothioneine, the antioxidant use of the composition based on glucosyl hesperidin and ergothioneine, and the application 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 composition based on glucosyl hesperidin to inhibit the photodegradation of ergothionein, that is, to provide a technical method for stabilizing the decomposition of ergothionein under light and high temperature conditions. Background Technology

[0002] Ergothioneine (EGT) is a histidine derivative containing a thiol group, scientifically known as 2-mercapto-histidine-trimethylammonium sulfate, with a molecular weight of 229.3. The pure form is a white crystalline solid, readily soluble in water. It was first isolated in 1909 by the French scientist Charles Tanret from *Claviceps purpurea*. This rare, naturally occurring amino acid is primarily synthesized by fungi and bacteria in nature and enters animals and humans through the food chain. Currently, no animals or humans have been found to synthesize this amino acid themselves. Ergothioneine is highly water-soluble but cannot enter animal cells through free diffusion; it can only be selectively transported into cells through cell membranes expressing OCT1 (organic cation transporter 1). Numerous in vivo and in vitro experiments have demonstrated that ergothioneine possesses antioxidant properties unmatched by other biomolecules, resisting reactive oxygen species generated within cells, exhibiting anti-inflammatory effects, prolonging cell lifespan or anti-cellular aging activity, and improving nerve cell regeneration, among other physiological functions. It also demonstrates good cell protection and damage resistance effects in various disease models, including complications of Alzheimer's disease and diabetes.

[0003] In aqueous solution, ergothione exists as a tautomer of thiols and thioketones:

[0004]

[0005] Ergothioneine, CAS 497-30-3, molecular formula C9H 16 N3O2S + Molecular weight 230.10

[0006] Different pH conditions and concentrations can alter the configuration of ergothioneine in the application system, leading to problems such as odor and spoilage. Currently, industrial production of ergothioneine mainly involves chemical synthesis, natural product extraction, and bio-fermentation. Chemical synthesis methods result in low yields and purity, with impurities difficult to remove, and exhibit some biotoxicity. Natural product extraction primarily involves chemical extraction from fungal products, which is costly, has low yields, and leaves significant chemical extractant residues. Bio-fermentation production of ergothioneine has become a more popular industrialization method in recent years, offering advantages such as high purity and fewer impurities; however, it inevitably presents challenges in application.

[0007] 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.

[0008] Chinese patent application CN115944548 discloses an oil-controlling composition for improving the photostability of ergothioneine and its application. This oil-controlling composition combines ergothioneine with glucosylrutin and Vaccaria segetalis extract, which can significantly improve the photostability of ergothioneine. The composition has good oil-controlling effects and can be used to prepare cosmetics with oil-controlling effects. The mass ratio of the three components is ergothioneine: glucosylrutin: Vaccaria segetalis extract = 1:2:17.

[0009] Chinese patent application CN114681451 discloses the use of hyaluronic acid or its salts and / or trehalose in stabilizing ergothioneine, an additive for stabilizing ergothioneine, the additive comprising hyaluronic acid or its salts and / or trehalose, and a composition comprising ergothioneine, hyaluronic acid or its salts and trehalose, wherein the mass ratio of hyaluronic acid or its salts and trehalose is 1:19-1:10, and the molecular weight of hyaluronic acid or its salts is 3 kDa-30 kDa, preferably 3 kDa-10 kDa.

[0010] Chinese patent application CN110327242B discloses a method for inhibiting the photodegradation of ergothioneine and its application. The method involves adding hyaluronic acid salt to a solution containing ergothioneine. The ergothioneine content is 0.0005-0.01 wt%, and the hyaluronic acid salt content is 0.1 wt% or more. The hyaluronic acid salt is a mixture of 1-3 parts by weight of hyaluronic acid salt with a molecular weight of 1200 kDa-1600 kDa, 2-4 parts by weight of hyaluronic acid salt with a molecular weight of 300 kDa-800 kDa, and 2-5 parts by weight of hyaluronic acid salt with a molecular weight of 3 kDa-10 kDa.

[0011] Chinese patent application CN115363980 discloses a composition containing ergothioneine and persimmon fruit extract, comprising 0.005–10% ergothioneine and 0.05–5% persimmon (DIOSPYROS KAKI) fruit extract. Regarding the odor produced by ergothioneine during application, it was found that persimmon (DIOSPYROS KAKI) fruit extract can stabilize ergothioneine, reduce odor production, expand its application, and provide consumers with a better experience; the odor reduction effect is even more pronounced when a pH adjuster is used simultaneously. Furthermore, persimmon (DIOSPYROS KAKI) fruit extract has anti-wrinkle effects and can synergistically work with ergothioneine to combat aging.

[0012] Hesperidin is found in fruits such as lemons and oranges. It is a flavanol glycoside widely found in citrus fruits and possesses various biological activities. Belonging to the vitamin P family, it exhibits effects such as reducing inflammatory mediators and playing an important antioxidant role, anti-inflammatory and anti-cancer properties, stimulating lipid metabolism, and good anti-allergic activity. The main drawback of hesperidin is its poor solubility and instability in water. Therefore, glucosyl hesperidin, obtained through modified processes to improve its solubility, possesses similar skin-care and antioxidant effects.

[0013]

[0014] Hesperidin, CAS No. 520-26-3, molecular weight 610.56,

[0015] Glucosyl hesperidin has good water solubility. It is a glycoside formed by hesperidin and rutin, and is a dihydroflavonoid derivative. It is an important component of citrus peel and pulp, and its content can reach 1.4% of the weight of young fresh citrus fruit.

[0016] Glucosyl hesperidin is a molecule in the hesperidin family, which consists of natural bioflavonoids, particularly found in citrus fruits.

[0017] Glucosyl-hesperidin is a molecule derived from hesperidin. It is water-soluble and more stable: glucosyl-hesperidin is obtained through an enzymatic reaction involving the addition of glucose to hesperidin, which significantly increases the solubility of the compound while maintaining its initial properties. Therefore, glucosyl-hesperidin exhibits antioxidant, angiotensinogenic, anti-inflammatory, and anticancer effects, and stimulates lipid metabolism. French patent application FR3115680A1 discovered that glucosyl-hesperidin can also reduce the inflammatory response induced by LL-37 peptide in human keratinocytes. The effect of glucosyl-hesperidin on the synthesis of interleukin IL6 induced by LL-37 peptide in human keratinocytes was investigated.

[0018] The purpose of this invention is to provide a composition that can inhibit the degradation of ergothioneine, namely, a combination of ergothioneine and glucosyl hesperidin. This provides an extremely efficient and cost-effective method for inhibiting the photodegradation and high-temperature instability of ergothioneine. Unexpectedly, it significantly increases the content of ergothioneine in the system under different pH conditions and concentrations, and also unexpectedly enhances the antioxidant effect. It has great potential for application in the cosmetics field. Summary of the Invention

[0019] The purpose of this invention is to develop a composition based on glucosyl hesperidin to inhibit the photodegradation of ergothioneine, based on the use of glucosyl hesperidin. This composition has a significantly higher efficiency in inhibiting the photodegradation or high-temperature degradation of ergothioneine than existing compositions based on hyaluronic acid. Furthermore, based on the above technical solution, a topical skin agent that can effectively inhibit the degradation of ergothioneine has been developed. Details are as follows:

[0020] This invention provides a composition for inhibiting the photodegradation of ergothioneine based on glucosyl hesperidin, comprising: a water-soluble hesperidin derivative and ergothioneine, wherein the weight ratio of the water-soluble hesperidin derivative to ergothioneine is 0.05-2.5:0.001-1, and wherein the content of the water-soluble hesperidin derivative in the composition is 0.001-3 wt%.

[0021] In a preferred embodiment, the water-soluble hesperidin derivative is selected from: glucosyl hesperidin and methyl hesperidin.

[0022] In a preferred embodiment, the weight ratio of the water-soluble hesperidin derivative to ergothioneine is 0.05-0.5:0.1-1.

[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 ≤1 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, the carrier acceptable in the field of topical skin agents is water, and the water content in the composition is 90-95 wt%.

[0028] In a preferred embodiment, the composition further comprises a pH adjuster.

[0029] Preferably, the pH adjuster is selected from at least one of citric acid and tromethamine.

[0030] In a preferred embodiment, the pH value of the composition is 4-8.

[0031] Preferably, the pH value of the composition is 4-6.

[0032] The present invention also provides the antioxidant use of compositions based on glucosyl hesperidin and ergothionein.

[0033] In a preferred embodiment, the weight ratio of glucosyl hesperidin to ergothionein is 0.001-2.5:0.001-1.

[0034] This invention also provides the application of glucosyl hesperidin in inhibiting the photodegradation of ergothionein.

[0035] In a preferred embodiment, the weight ratio of glucosyl hesperidin to ergothionein is 0.001-2.5:0.001-1.

[0036] In a preferred embodiment, the application is performed at a pH of 4-8;

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

[0038] The present invention also provides the use of the described composition in topical skin preparations.

[0039] 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.

[0040] In a preferred embodiment, the composition is used in a topical skin preparation at an amount of 0.0001 wt%-90 wt%.

[0041] The preferred weight percentage is 0.001wt%-10wt%.

[0042] A more preferred weight percentage is 0.001wt%-5wt%.

[0043] The beneficial effects of this invention are:

[0044] 1. This invention is the first to protect the stability of ergothionein by using glucosyl hesperidin as a component. The special role of hydrogen bonds can protect the conformational stability of ergothionein under light and high temperature.

[0045] 2. This invention unexpectedly discovered that glucosyl hesperidin and ergothionein have a very good synergistic effect, which can improve the antioxidant efficacy by more than 80%. Attached Figure Description

[0046] Figure 1 The results of ROS content detection in test example 4. Detailed Implementation

[0047] 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.

[0048] 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.

[0049] 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.

[0050] Water-soluble hesperidin derivatives

[0051] The water-soluble hesperidin derivative described in this invention is glucosyl hesperidin. As a "water-soluble hesperidin derivative" used in this invention, examples include hesperidins whose water solubility has been improved through enzymatic or chemical treatment. Examples include glycotransfer hesperidins or methyl hesperidins formed by bonding other sugars to the sugar moiety (rutin moiety) of hesperidins such as glucosyl hesperidin. From the viewpoint of water solubility, methyl hesperidin (also known as hesperidin methyl chalcone) and glucosyl hesperidin are preferred.

[0052] α-Glucosylhesperidin (also known as α-glucosylhesperidin) is a general term for compounds in which one or more glucose molecules are added to the hydroxyl group of the rutin unit of hesperidin via an α-1,4 bond. α-Glucosylhesperidin can be composed of a single compound with this structure or a mixture of two or more. Those with only one glucose molecule are called "α-monoglucosylhesperidin," while those with two or more glucose molecules are called "α-polyglucosylhesperidin."

[0053] α-Glucosylhesperidin can be represented by the following formula:

[0054]

[0055] In the formula, n is an integer of 0 or 1 or higher, such as an integer from 1 to 19. More preferably, it is an integer from 0 to 10, even more preferably, it is an integer from 0 to 5, and particularly preferably, it is 0.

[0056] Additionally, α-glucosyl hesperidin (also known as: enzyme-treated hesperidin, glycotransfer hesperidin, water-soluble hesperidin, or glycotransfer vitamin P).

[0057] C3-C6 diols

[0058] The C3-C6 diol mentioned in this invention is butanediol, and the C3-C6 diol can be selected from: butanediol, propylene glycol, 1,3-propanediol, pentanediol, 1,2-hexanediol, glycerol, diglycerol, etc.

[0059] 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.

[0060] The main experimental materials and reagents used in the examples are as follows:

[0061] Ergothioneine, purity ≥99.0%, Shanghai Ergothioneine Co., Ltd.

[0062] Butanediol (purity ≥99.0%, Shandong Haike Xinyuan Materials Technology Co., Ltd.)

[0063] Tromethamine, analytical grade, Sinopharm Group

[0064] Citric acid, analytical grade, Sinopharm Group

[0065] Glucosyl hesperidin, purity ≥99.0%, Hayashibara Corporation, Japan

[0066] Acrylamide dimethyl taurate ammonium / VP copolymer (AVC), purity ≥99.0%, Clariant (China) Co., Ltd.

[0067] Hydroxypropyl β-cyclodextrin (purity ≥99%, Shandong Binzhou Zhiyuan Biotechnology Co., Ltd.)

[0068] Hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) (purity ≥99.0%, Bloomage Biotechnology Co., Ltd.)

[0069] Sodium hyaluronate (molecular weight 10K-100KDa) (purity ≥99.0%, Bloomage Biotechnology Co., Ltd.)

[0070] Hyaluronic acid (molecular weight 100K-1000KDa) (purity ≥99.0%, Bloomage Biotechnology Co., Ltd.)

[0071] Formic acid, purity ≥98.0%, Sigma-Aldrich (Product No. 43804)

[0072] Acetonitrile, purity ≥99.9%, Sigma-Aldrich (Product No. 34851)

[0073] Methanol, purity ≥99.9%, Sigma-Aldrich (Product No. 34885)

[0074] DCFH-DA culture medium (LMAI Bio, Shanghai Lianmai Biotechnology Co., Ltd.)

[0075] PBS, Beijing Solarbio Science & Technology Co., Ltd., Batch No. P1022

[0076] Main experimental instruments

[0077] XS205 analytical balance (METTLER TOLEDO)

[0078] Waters ARC high-performance liquid chromatograph with diode array detector (Waters)

[0079] KQ-800DE CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.)

[0080] CO2 incubator (Thermo, 150I)

[0081] Clean bench (Su Jing An Tai, SW-CJ-1F)

[0082] Flow cytometer (Beckman, CytoFLEX)

[0083] Inverted microscope (Olympus, CKX53)

[0084] UVB irradiator (Philips)

[0085] Incucell incubator (MMM GmbH, Germany)

[0086] PERCIVAL CU41L5 Illuminated Incubator (PERCIVAL Corporation, USA)

[0087] Example 1: Sample preparation of ergothioneine

[0088] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of glucosyl hesperidin 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.

[0089] Example 2: Sample preparation of ergothioneine

[0090] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of glucosyl hesperidin 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 tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0091] Example 3: Sample preparation of ergothioneine

[0092] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.3g of glucosyl hesperidin and stir well. 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.

[0093] Example 4: Sample preparation of ergothioneine

[0094] Accurately weigh 0.5g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of glucosyl hesperidin 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.

[0095] Example 5: Sample preparation of ergothioneine

[0096] Accurately weigh 1.0g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of glucosylhesperidin 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.

[0097] Example 6: Sample preparation of ergothioneine

[0098] Accurately weigh 0.5g of ergothioneine and dissolve it in 5g of butanediol. Add 2.5g of glucosyl hesperidin 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.

[0099] Example 7: Sample preparation of ergothioneine

[0100] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of glucosylhesperidin 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 tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0101] Example 8: Sample preparation of ergothioneine

[0102] 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.

[0103] Example 9: Sample preparation of ergothioneine

[0104] 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.

[0105] Example 10: Sample preparation of ergothioneine

[0106] 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.

[0107] Example 11: Sample preparation of ergothioneine

[0108] 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 glucosyl hesperidin, 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. Transfer the solution to a volumetric flask for later use.

[0109] Example 12: Sample preparation of ergothioneine

[0110] 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.

[0111] Example 13: Sample preparation of ergothioneine

[0112] 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.

[0113] Example 14: Sample preparation of ergothioneine

[0114] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of glucosylhesperidin 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.

[0115] Example 15: Sample preparation of ergothioneine

[0116] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of glucosyl hesperidin 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.

[0117] Example 16: Sample preparation of ergothioneine

[0118] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.3g of glucosyl hesperidin 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.

[0119] Example 17: Sample preparation of ergothioneine

[0120] Accurately weigh 0.5g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of glucosyl hesperidin 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.

[0121] Example 18: Sample preparation of ergothioneine

[0122] Accurately weigh 1.0g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of glucosylhesperidin 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.

[0123] Example 19: Sample preparation of ergothioneine

[0124] Accurately weigh 0.5g of ergothioneine and dissolve it in 5g of butanediol. Add 2.5g of glucosyl hesperidin and stir well. Then add 0.8g of AVC and disperse it thoroughly until uniform. Add water to adjust the total amount 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.

[0125] Example 20: Sample preparation of ergothioneine

[0126] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of glucosyl hesperidin 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.

[0127] Example 21: Sample preparation of ergothioneine

[0128] 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.

[0129] Example 22: Sample preparation of ergothioneine

[0130] 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.

[0131] Example 23: Sample preparation of ergothioneine

[0132] 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.

[0133] Example 24: Sample preparation of ergothioneine

[0134] 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 glucosyl hesperidin, 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. Transfer the solution to a volumetric flask for later use.

[0135] Example 25: Sample preparation of ergothioneine

[0136] 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.

[0137] Example 26: Sample preparation of ergothioneine

[0138] 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.

[0139] Example 27: Sample preparation of ergothioneine

[0140] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of glucosyl hesperidin 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.

[0141] Example 28: Sample preparation of ergothioneine

[0142] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of glucosyl hesperidin 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.

[0143] Example 29: Sample preparation of ergothioneine

[0144] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.3g of glucosyl hesperidin 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.

[0145] Example 30: Sample preparation of ergothioneine

[0146] Accurately weigh 0.5g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of glucosyl hesperidin 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.

[0147] Example 31: Sample preparation of ergothioneine

[0148] Accurately weigh 1.0g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of glucosyl hesperidin 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.

[0149] Example 32: Sample preparation of ergothioneine

[0150] Accurately weigh 0.5g of ergothioneine and dissolve it in 5g of butanediol. Add 2.5g of glucosyl hesperidin 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.

[0151] Example 33: Sample preparation of ergothioneine

[0152] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of glucosylhesperidin 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.

[0153] Example 34: Sample preparation of ergothioneine

[0154] 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.

[0155] Example 35: Sample preparation of ergothioneine

[0156] 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.

[0157] Example 36: Sample preparation of ergothioneine

[0158] 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 into a volumetric flask and set aside for later use.

[0159] Example 37: Sample preparation of ergothioneine

[0160] 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 glucosyl hesperidin, 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. Transfer the solution to a volumetric flask for later use.

[0161] Example 38: Sample preparation of ergothioneine

[0162] 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.

[0163] Example 39: Sample preparation of ergothioneine

[0164] 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.

[0165] Test Example 1: Illumination Test Experiment

[0166] Test samples: Composition samples prepared in Examples 1-39

[0167] 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.

[0168] 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.

[0169] Classification Odor and strength standards Level 1 No discernible change in taste Level 2 Slight change in taste Level 3 Significant taste change

[0170] The results of the composition samples prepared in Examples 1-39 after 14 days of light exposure testing are shown in Table 1:

[0171] Table 1. Results of light irradiation tests on the composition samples prepared in Examples 1-39.

[0172]

[0173] After 14 days of simulated sunlight exposure testing, under acidic pH conditions, the composition samples containing ergothioneine, butanediol, AVC, glucosyl hesperidin, and water prepared in Examples 1-3 and 7 showed no perceptible change in taste; the composition samples prepared in Examples 4-6 showed only slight changes in taste.

[0174] At the same pH value (pH=4), when all or part of the glucosyl hesperidin 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.

[0175] Specifically as follows:

[0176] Example 8 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 2 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and glucosyl hesperidin in the latter, and with the same content of the remaining components in both, the composition sample prepared in Example 2 performed better than the composition sample prepared in Example 8 under light irradiation testing. This indicates that glucosyl hesperidin has a certain stabilizing effect on ergothioneine in the composition.

[0177] Example 9 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 1 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) in the former and glucosyl hesperidin in the latter, and with the same content of the other components in both, the composition sample prepared in Example 1 performed significantly better than the composition sample prepared in Example 9 under light irradiation testing. This indicates that glucosyl hesperidin has a certain 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.

[0178] Example 10 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 1 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and glucosyl hesperidin in the latter, and with the same content of the other components in both, the composition sample prepared in Example 1 performed significantly better than the composition sample prepared in Example 10 under light irradiation testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. Furthermore, it is also shown that sodium hyaluronate (molecular weight 10K-100KDa) does not inhibit the photodegradation of ergothioneine as disclosed in the prior art.

[0179] Example 11 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 3K-10KDa), glucosyl hesperidin, and water. Example 2 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, 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, the composition sample prepared in Example 2 performed similarly to that prepared in Example 11. This indicates that glucosyl hesperidin not only helps inhibit the photodegradation of ergothioneine in the composition, but also inhibits the photodegradation of ergothioneine when used in combination with hyaluronic acid (molecular weight 3K-10KDa).

[0180] Example 12 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 1 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) and glucosyl hesperidin in both samples, and with identical content of the remaining components, the composition sample prepared in Example 1 performed significantly better than the composition sample prepared in Example 12 under light irradiation testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. However, hyaluronic acid (molecular weight 100K-1000KDa) cannot help inhibit the photodegradation of ergothioneine.

[0181] At the same pH value (pH=6), when all or part of the glucosyl hesperidin 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.

[0182] Specifically as follows:

[0183] The composition samples prepared in Examples 14-16 and 20, which contained ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water, showed no perceptible change in taste; the composition samples prepared in Examples 17-19 showed only slight changes in taste.

[0184] Example 21 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 15 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and glucosyl hesperidin in the latter, and with the same content of the remaining components, the composition sample prepared in Example 15 performed better than the composition sample prepared in Example 21 under light irradiation testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. Hydroxypropyl β-cyclodextrin is less effective than glucosyl hesperidin in inhibiting the photodegradation of ergothioneine.

[0185] Example 22 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 14 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) in the former and glucosyl hesperidin in the latter, and with the same content of the other components, the composition sample prepared in Example 14 performed significantly better than the composition sample prepared in Example 22 under light irradiation testing. This indicates that glucosyl hesperidin 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.

[0186] Example 23 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 14 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and glucosyl hesperidin in the latter, and with the same content of the other components, the composition sample prepared in Example 14 performed significantly better than the composition sample prepared in Example 23 under light irradiation testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. Sodium hyaluronate (molecular weight 10K-100KDa), however, cannot help inhibit the photodegradation of ergothioneine.

[0187] Example 24 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 15 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. In the former, the hyaluronic acid (molecular weight 100K-1000KDa) content was half that of the latter, and with the remaining components present in the same amounts, the composition sample prepared in Example 15 performed better than the composition sample prepared in Example 24 under light irradiation testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. The combination of hyaluronic acid (molecular weight 100K-1000KDa) and glucosyl hesperidin can help inhibit the photodegradation of ergothioneine, but its inhibition efficiency is lower than that of glucosyl hesperidin.

[0188] Example 25 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 14 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) and glucosyl hesperidin in both examples, and with the same content of the remaining components, the composition sample prepared in Example 14 performed significantly better than the composition sample prepared in Example 25 under light irradiation testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. However, hyaluronic acid (molecular weight 100K-1000KDa) cannot help inhibit the photodegradation of ergothioneine.

[0189] Example 26 prepared a composition sample containing ergothioneine, butylene glycol, AVC, and water, while Examples 14-16 prepared composition samples containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. The difference between the two is that Examples 14-16 contained 0.1-0.3 wt% glucosyl hesperidin. With the contents of the other components being the same, under light irradiation testing, the composition samples prepared in Examples 14-16 performed significantly better than those prepared in Example 26. This further clarifies that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition.

[0190] Under alkaline conditions (pH=8), the composition samples prepared in Examples 28 and 30-39 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 27 showed only slight changes in taste, while the composition sample prepared in Example 29 showed no perceptible changes in taste. This indicates that the addition of an appropriate amount of glucosyl hesperidin to the composition resulted in very good photostability.

[0191] At the same pH value (pH=8), when all or part of the glucosyl hesperidin 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.

[0192] Specifically as follows:

[0193] Example 34 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 28 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and glucosyl hesperidin in the latter, and with identical contents of the remaining components, the composition sample prepared in Example 28 performed significantly better than the composition sample prepared in Example 34 under light irradiation testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. Hydroxypropyl β-cyclodextrin, however, cannot help inhibit the photodegradation of ergothioneine.

[0194] Example 35 prepared a composition containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 27 prepared a composition containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) in the former and glucosyl hesperidin in the latter, and with identical contents of the other components, the composition prepared in Example 27 performed significantly better than the composition prepared in Example 35 under light irradiation. This indicates that glucosyl hesperidin 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.

[0195] Example 36 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 27 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and glucosyl hesperidin in the latter, and with the same content of the other components, the composition sample prepared in Example 27 performed better than the composition sample prepared in Example 36 under light irradiation testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. Sodium hyaluronate (molecular weight 10K-100KDa), however, cannot help inhibit the photodegradation of ergothioneine.

[0196] Example 37 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 3K-10KDa), glucosyl hesperidin, and water. Example 28 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. In the former, the hyaluronic acid (molecular weight 3K-10KDa) content was half that of the glucosyl hesperidin content in the latter, and with the contents of the remaining components being the same, the composition sample prepared in Example 28 performed significantly better than the composition sample prepared in Example 37 under light irradiation testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. While the combination of hyaluronic acid (molecular weight 3K-10KDa) and glucosyl hesperidin can help inhibit the photodegradation of ergothioneine, its inhibition efficiency is lower than that of glucosyl hesperidin.

[0197] Example 38 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 27 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) in the former and glucosyl hesperidin in the latter, and with the same content of the other components, the composition sample prepared in Example 27 performed better than the composition sample prepared in Example 38 under light irradiation testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. However, hyaluronic acid (molecular weight 100K-1000KDa) cannot help inhibit the photodegradation of ergothioneine.

[0198] Example 39 prepared a composition sample containing ergothioneine, butylene glycol, AVC, and water, while Example 27 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. The difference between the two is that Example 27 contained 0.1 wt% glucosyl hesperidin. With the contents of the other components being the same, under light irradiation testing, the composition sample prepared in Example 27 performed better than the composition sample prepared in Example 39. This further clarifies that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition.

[0199] In summary, glucosyl hesperidin can significantly help inhibit the photodegradation of ergothionein, and its inhibitory efficiency is better under acidic conditions than under alkaline conditions. In addition, the combination of glucosyl hesperidin with hyaluronic acid (molecular weight 3K-10KDa) and hyaluronic acid (molecular weight 100K-1000KDa) can also help inhibit the photodegradation of ergothionein.

[0200] Test Example 2: High Temperature Test Experiment

[0201] Test samples: Composition samples prepared in Examples 1-39

[0202] 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.

[0203] Samples from Examples 1-39 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 changes in the products.

[0204] 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.

[0205] Classification Odor and strength standards Level 1 No discernible change in taste Level 2 Slight change in taste Level 3 Significant taste change

[0206] Table 2 High-temperature tests of the composition samples prepared in Examples 1-39

[0207]

[0208] After 14 days of high-temperature testing at 48°C, under acidic pH conditions, the composition samples containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water prepared in Examples 1-3 and 7 showed no perceptible change in taste; the composition samples prepared in Examples 4-6 showed only slight changes in taste.

[0209] At the same pH value (pH=4), when all or part of the glucosyl hesperidin 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.

[0210] Specifically as follows:

[0211] Example 8 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 2 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. Given that the hydroxypropyl β-cyclodextrin content in the former and the glucosyl hesperidin content in the latter were the same, and the contents of the remaining components were also the same, the composition sample prepared in Example 2 showed better performance than the composition sample prepared in Example 8 under high-temperature stability testing. This indicates that glucosyl hesperidin has a certain stabilizing effect on ergothioneine in the composition.

[0212] Example 9 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 1 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) in the former and glucosyl hesperidin in the latter, and with the same content of the other components in both, the composition sample prepared in Example 1 showed significantly better performance than the composition sample prepared in Example 9 under high-temperature stability testing. This indicates that glucosyl hesperidin has a certain 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.

[0213] Example 10 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 1 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and glucosyl hesperidin in the latter, and with the same content of the other components in both, the composition sample prepared in Example 1 showed significantly better performance than the composition sample prepared in Example 10 under high-temperature stability testing. This indicates that glucosyl hesperidin 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.

[0214] Example 11 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 3K-10KDa), glucosyl hesperidin, and water. Example 2 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, 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 comparable to that prepared in Example 11. This indicates that glucosyl hesperidin not only helps inhibit the photodegradation of ergothioneine in the composition, but also inhibits the photodegradation of ergothioneine when used in combination with hyaluronic acid (molecular weight 3K-10KDa).

[0215] Example 12 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 1 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) in the former and glucosyl hesperidin in the latter, and with the same content of the other components, the composition sample prepared in Example 1 showed significantly better performance than the composition sample prepared in Example 12 under high-temperature stability testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. However, hyaluronic acid (molecular weight 100K-1000KDa) cannot help inhibit the photodegradation of ergothioneine.

[0216] At the same pH value (pH=6), when all or part of the glucosyl hesperidin 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.

[0217] Specifically as follows:

[0218] The composition samples prepared in Examples 14-16 and 20, which contained ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water, showed no perceptible change in taste; the composition samples prepared in Examples 17-19 showed only slight changes in taste.

[0219] Example 21 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 15 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and glucosyl hesperidin in the latter, and with identical contents of the remaining components, the composition sample prepared in Example 15 performed better than the composition sample prepared in Example 21 under high-temperature stability testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. Hydroxypropyl β-cyclodextrin is less effective than glucosyl hesperidin in inhibiting the photodegradation of ergothioneine.

[0220] Example 22 prepared a composition containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 14 prepared a composition containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) and glucosyl hesperidin in the former and the same content of the other components in the latter, the composition prepared in Example 14 showed significantly better performance than the composition prepared in Example 22 under high-temperature stability testing. This indicates that glucosyl hesperidin 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.

[0221] Example 23 prepared a composition containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 14 prepared a composition containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and glucosyl hesperidin in the latter, and with identical contents of the other components, the composition prepared in Example 14 showed significantly better performance than the composition prepared in Example 23 under high-temperature stability testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. Sodium hyaluronate (molecular weight 10K-100KDa), however, cannot help inhibit the photodegradation of ergothioneine.

[0222] Example 24 prepared a composition containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 15 prepared a composition containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, 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 prepared in Example 15 performed better than that prepared in Example 24. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. The combination of hyaluronic acid (molecular weight 100K-1000KDa) and glucosyl hesperidin can help inhibit the photodegradation of ergothioneine, but its inhibition efficiency is lower than that of glucosyl hesperidin.

[0223] Example 25 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 14 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) and glucosyl hesperidin in both examples, and with identical content of the remaining components, the composition sample prepared in Example 14 showed significantly better performance than the composition sample prepared in Example 25 under high-temperature stability testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. However, hyaluronic acid (molecular weight 100K-1000KDa) cannot help inhibit the photodegradation of ergothioneine.

[0224] Example 26 prepared a composition sample containing ergothioneine, butylene glycol, AVC, and water, while Examples 14-16 prepared composition samples containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. The difference between the two is that Examples 14-16 contain 0.1-0.3 wt% glucosyl hesperidin. With the contents of the other components being the same, the composition samples prepared in Examples 14-16 showed significantly better performance than those prepared in Example 26 under high-temperature stability testing. This further clarifies that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition.

[0225] Under alkaline conditions (pH=8), the composition samples prepared in Examples 28 and 30-39 all showed significant changes in taste; the composition sample prepared in Example 27 showed only slight changes in taste, while the composition sample prepared in Example 29 showed no noticeable changes in taste. This indicates that the addition of an appropriate amount of glucosyl hesperidin to the composition resulted in very good high-temperature stability.

[0226] At the same pH value (pH=8), when all or part of the glucosyl hesperidin 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.

[0227] Specifically as follows:

[0228] Example 34 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 28 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydroxypropyl β-cyclodextrin in the former and glucosyl hesperidin in the latter, and with identical contents of the remaining components, the composition sample prepared in Example 28 showed significantly better performance than the composition sample prepared in Example 34 under high-temperature stability testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. Hydroxypropyl β-cyclodextrin, however, cannot help inhibit the photodegradation of ergothioneine.

[0229] Example 35 prepared a composition containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 27 prepared a composition containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) in the former and glucosyl hesperidin in the latter, and with identical contents of the remaining components, the composition prepared in Example 27 showed significantly better performance than the composition prepared in Example 35 under high-temperature stability testing. This indicates that glucosyl hesperidin 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.

[0230] Example 36 prepared a composition containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 27 prepared a composition containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of sodium hyaluronate (molecular weight 10K-100KDa) in the former and glucosyl hesperidin in the latter, and with identical contents of the other components, the composition prepared in Example 27 performed better than the composition prepared in Example 36 under high-temperature stability testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. Sodium hyaluronate (molecular weight 10K-100KDa), however, cannot help inhibit the photodegradation of ergothioneine.

[0231] Example 37 prepared a composition containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 3K-10KDa), glucosyl hesperidin, and water. Example 28 prepared a composition containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. In the former, the hyaluronic acid (molecular weight 3K-10KDa) content was half that of the latter, and with the remaining components present in the same amounts, the composition prepared in Example 28 showed significantly better performance than the composition prepared in Example 37 under high-temperature stability testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. While the combination of hyaluronic acid (molecular weight 3K-10KDa) and glucosyl hesperidin can help inhibit the photodegradation of ergothioneine, its inhibition efficiency is lower than that of glucosyl hesperidin.

[0232] Example 38 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Example 27 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. With the same content of hyaluronic acid (molecular weight 100K-1000KDa) in the former and glucosyl hesperidin in the latter, and with the same content of the other components, the composition sample prepared in Example 27 performed better than the composition sample prepared in Example 38 under high-temperature stability testing. This indicates that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition. However, hyaluronic acid (molecular weight 100K-1000KDa) cannot help inhibit the photodegradation of ergothioneine.

[0233] Example 39 prepared a composition sample containing ergothioneine, butylene glycol, AVC, and water, while Example 27 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glucosyl hesperidin, and water. The difference between the two is that Example 27 contained 0.1 wt% glucosyl hesperidin. With the contents of the other components being the same, the composition sample prepared in Example 27 performed better than the composition sample prepared in Example 39 under high-temperature stability testing. This further clarifies that glucosyl hesperidin can help inhibit the photodegradation of ergothioneine in the composition.

[0234] In summary, glucosyl hesperidin can significantly help inhibit the photodegradation of ergothionein, and its inhibitory efficiency is better under acidic conditions than under alkaline conditions. In addition, the combination of glucosyl hesperidin with hyaluronic acid (molecular weight 3K-10KDa) and hyaluronic acid (molecular weight 100K-1000KDa) can also help inhibit the photodegradation of ergothionein.

[0235] Test Example 3: Detection of Ergothionein by Ultra-High Performance Liquid Chromatography

[0236] Test samples: Composition samples prepared in Examples 1-39 after the light exposure test in Test Example 1; Composition samples prepared in Examples 1-39 after the high temperature stability test in Test Example 2.

[0237] Test standard: NY / T 3872-2021 (UPLC)

[0238] 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.

[0239] Testing steps:

[0240] 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).

[0241] 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.

[0242] 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.

[0243] Results Calculation: Quantitative analysis using the external standard method; the formula for calculating the ergothionein content in the sample is as follows:

[0244]

[0245] In the formula:

[0246] X — Mass fraction of the analyte in the sample, %

[0247] C—The concentration of ergothioneine in the sample solution obtained from the standard curve, in micrograms per milliliter (μg·mL). -1);

[0248] V—The total volume of the sample after dilution, in milliliters (mL);

[0249] m — Sample mass, in grams (g);

[0250] The ergothioneine content in the composition samples after light exposure or high temperature stability testing is shown in the table below:

[0251] Table 3. Ergothionein content in the composition samples after light or high temperature stability tests.

[0252] sample pH value After the light test in Test Example 1 After the high-temperature stability test in Test Example 2 Example 1 4 95.1% 94.4% Example 2 4 92.3% 92.6% Example 3 4 100.0% 97.2% Example 4 4 74.7% 79.8% Example 5 4 56.9% 59.8% Example 6 4 80.2% 82.4% Example 7 4 85.3% 90.7% Example 8 4 53.6% 67.2% Example 9 4 53.8% 67.4% Example 10 4 60.0% 76.4% Example 11 4 82.3% 89.9% Example 12 4 64.2% 78.5% Example 13 4 71.7% 82.3% Example 14 6 86.2% 89.2% Example 15 6 82.7% 87.6% Example 16 6 100.0% 96.3% Example 17 6 66.5% 73.7% Example 18 6 49.7% 52.3% Example 19 6 74.1% 78.7% Example 20 6 85.3% 85.6% Example 21 6 56.7% 62.9% Example 22 6 56.9% 66.9% Example 23 6 51.0% 67.3% Example 24 6 69.6% 77.2% Example 25 6 59.2% 67.7% Example 26 6 68.7% 70.1% Example 27 8 70.1% 74.9% Example 28 8 64.5% 72.7% Example 29 8 86.7% 87.3% Example 30 8 45.2% 52.1% Example 31 8 37.7% 44.6% Example 32 8 56.9% 60.2% Example 33 8 67.3% 70.5% Example 34 8 51.3% 54.7% Example 35 8 41.6% 54.5% Example 36 8 39.4% 50.3% Example 37 8 46.8% 61.8% Example 38 8 46.8% 61.8% Example 39 8 51.6% 53.7%

[0253] 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.

[0254] First, under alkaline conditions (e.g., pH=8), the ergothioneine in the compositions prepared in Examples 28 and 30-39 was more easily decomposed. Due to the unique structure of ergothioneine, containing hydroxyl and carbonyl groups, it reacts chemically with hydroxide ions under alkaline conditions, resulting in changes in structure, properties, and odor. Regardless of the ergothioneine content in the composition, or under light and / or high temperature conditions, the structural integrity and presence of ergothioneine in the composition samples were significantly challenged, and the odor often changed markedly, becoming noticeably foul.

[0255] However, even under such stringent conditions, we were pleasantly surprised to find that the composition sample prepared in Example 29 exhibited very good stability under the combined action of ergothioneine and glucosyl hesperidin. Specifically, when the content of glucosyl hesperidin in the composition sample was ≥0.3wt%, the abundant hydroxyl groups on the surface provided excellent protection for ergothioneine through hydrogen bonding. This condition and ratio provide a very good solution and technology for the application of ergothioneine in alkaline products.

[0256] Secondly, an acidic pH is recommended for the application of ergothioneine. Comparing pH=4 and pH=6, a lower pH under acidic conditions is more conducive to the stability of ergothioneine. By adjusting the ratios of different ergothioneine concentrations and compositions, several different combinations and concentrations were unexpectedly found to have excellent protective functions. Specifically:

[0257] From the composition samples prepared in Examples 13, 26, and 39, 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.

[0258] In contrast to existing technologies, such as the Chinese patent application CN110327242B which discloses a method for inhibiting the photodegradation of ergothionein using hyaluronic acid salts, the composition samples prepared in Examples 9-10, 12, 22-23, 25, 35-36, and 38 of this application reproduce the inhibitory effect of hyaluronic acid and its derivatives on ergothionein in the prior art. Compared with ergothionein under the same concentration and pH conditions, no obvious photodegradation and high-temperature degradation stabilization effect was observed. On the contrary, the content of ergothionein under such conditions decreased to a certain extent. It is speculated that the possible reason is that different types of hyaluronic acid, including hyaluronic acid with a relatively small molecular weight of several thousand Daltons, cannot form a complete and tight encapsulation structure, resulting in ergothionein concentration differences and escape.

[0259] Under pH=4 conditions, the composition samples prepared in Examples 1-3, 7 and 11 unexpectedly obtained excellent protective effects against ergothioneine. After 14 days of light exposure or 14 days of high temperature stability test, the ergothioneine content in the samples was retained at more than 88%.

[0260] Under pH 6 conditions, the compositions prepared in Examples 14-16 and Example 20 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 85%.

[0261] In summary, when the pH value is acidic, the glucosyl hesperidin 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 glucosyl hesperidin may have caused changes in the internal water transport environment of ergothioneine, making the binding of the two more stable.

[0262] The protective effect is optimal when the weight ratio of ergothioneine to glucosyl hesperidin is 1-2:1-3. In Examples 11, 24, and 37, which used a combination of glucosyl hesperidin 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 11 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 82%. This indicates that only under acidic conditions can the combination of glucosyl hesperidin and hyaluronic acid inhibit the degradation of ergothioneine.

[0263] Test Example 4: Evaluation of Antioxidant Performance

[0264] Test samples: Composition samples prepared in Examples 1-3, 7-8, 11, 13-16, 20, 29, 26 and 39.

[0265] Experimental methods:

[0266] 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).

[0267] 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.

[0268] 3) UVB irradiation: According to the test groups, the groups exposed to UVB irradiation were subjected to 300mJ / cm² irradiation. 2 UVB irradiation.

[0269] 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.

[0270] 5) Statistical analysis of results: GrapHPad Prism plots were used, 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.

[0271] Table 4 Antioxidant test results of the samples

[0272] Group ROS Mean (MFI) SD p-value Inhibition rate (vs NC) BC 44592.87 5181.08 / / NC (UVB stimulation) 139009.70 14578.38 <![CDATA[0.000 ## ]]> / PC (VE + UVB stimulation) 91112.23 5741.27 0.006** 34.46% Example 1 13638.35 1984.33 0.002** 90.19% Example 2 12690.73 2753.04 0.002** 90.87% Example 3 7636.94 8422.43 0.001** 94.51% Example 7 23638.35 1985.41 0.003** 83.00% Example 8 32690.73 2864.37 0.003** 76.48% Example 11 18636.94 1196.35 0.002** 86.59% Example 14 24812.92 1703.42 0.003** 82.15% Example 15 29446.68 3062.45 0.004** 78.82% Example 16 25469.05 2323.18 0.005** 81.68% Example 20 36943.64 2258.31 0.004** 73.42% Example 29 44932.77 5739.23 0.007** 67.68% Example 13 77539.21 6649.28 0.009** 44.22% Example 26 79534.37 7452.66 0.011** 42.79% Example 39 80047.89 9629.45 0.007** 42.42%

[0273] Note: Mean fluorescence intensity (MFI) reflects the ROS content. When using the t-test method for statistical analysis, 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 **).

[0274] 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.

[0275] Comparing the antioxidant properties of ergothioneine under different pH conditions, it was found to be superior to commonly used vitamin E (VE). Vitamin E is a substance with strong antioxidant activity, a very important antioxidant in the human body that blocks free radical chain reactions, plays an indispensable role in stabilizing and protecting cell membranes, and is essential for 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.

[0276] Comparing the combinations of ergothionein and glucosyl hesperidin under different pH conditions, a significant increase in antioxidant capacity was unexpectedly found in all cases, with the antioxidant capacity increase exceeding 84% in most of the embodiments. Figure 1 As shown, the composition samples prepared in Example 3, Example 16, and Example 29 differ only in their pH values, and their antioxidant capacity improvements are 94.51%, 81.68%, and 67.68%, respectively.

[0277] The composition sample prepared in Example 1 and the composition sample prepared in Example 14 differed only in their pH values, and their antioxidant capacity was improved by 90.19% and 82.15%, respectively.

[0278] The composition sample prepared in Example 2 and the composition sample prepared in Example 15 differed only in pH value, and their antioxidant capacity was improved by 90.87% and 78.82%, respectively.

[0279] The composition samples prepared in Examples 13, 16 and 39 are identical to those prepared in Examples 1-2 and 14-15, except for the content of glucosyl hesperidin.

[0280] When the glucosyl hesperidin content in the sample was 0.1 wt% and the pH was 4, the antioxidant capacity of the composition sample prepared in Example 1 increased from 44.22% to 90.19% compared to the composition sample prepared in Example 13 (without glucosyl hesperidin), representing an increase of 103.96%.

[0281] When the glucosyl hesperidin content in the sample was 0.1 wt% and the pH was 6, the antioxidant capacity of the composition sample prepared in Example 14 increased from 42.79% to 82.15% compared to the composition sample prepared in Example 26 (without glucosyl hesperidin), representing an increase of 91.98%.

[0282] When the glucosyl hesperidin content in the sample was 0.2 wt% and the pH was 4, the antioxidant capacity of the composition sample prepared in Example 2 increased from 44.22% to 90.87% compared to the composition sample prepared in Example 13 (without glucosyl hesperidin), representing an increase of 105.50%.

[0283] When the glucosyl hesperidin content in the sample was 0.2 wt% and the pH was 6, the antioxidant capacity of the composition sample prepared in Example 15 increased from 42.79% to 78.82% compared to the composition sample prepared in Example 26 (without glucosyl hesperidin), representing an increase of 84.82%.

[0284] In summary, the antioxidant capacity of ergothioneine was enhanced by at least 84.82% with the help of glucosyl hesperidin. This indicates that when glucosyl hesperidin and ergothioneine are combined, not only can the photodegradation of ergothioneine be effectively inhibited, but the antioxidant capacity of both is synergistically enhanced.

[0285] 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.

[0286] The composition samples prepared in Examples 1-3, 7, 11, 14-16, 20, and 29 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 1-3, 7, 11, 14-16, 20, and 29 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%.

[0287] The following are examples of the specific applications of the compositions prepared in Examples 1-3, 7, 11, 14-16, 20, and 29 in topical skin preparations, along with the formulations and preparation methods of these dosage forms. In the following tables, "-" indicates no additives.

[0288] Application Example 1: Preparation of Face Cream

[0289]

[0290] Application Example 2: Emulsion Preparation

[0291]

[0292] Application Example 3: Preparation of Gel

[0293]

[0294] Application Example 4: Preparation of Toner

[0295]

[0296] Application Example 5: Preparation of Serum

[0297]

[0298] Application Example 6: Preparation of Facial Masks

[0299]

[0300] Application Example 7: Preparation of Eye Cream

[0301]

[0302] Application Example 8: Preparation of Spray

[0303]

[0304] Application Example 9: Preparation of Shower Gel

[0305]

[0306] Application Example 10: Preparation of Facial Cleanser

[0307]

[0308] Application Example 11: Preparation of Essence Water

[0309]

[0310] In all the above examples of specific applications of topical skin agents, the composition samples prepared in Examples 1-3, 7, 11, 14-16, 20 and 29 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 composition based on glucosyl hesperidin inhibiting the photodegradation of ergothioneine, comprising glucosyl hesperidin, ergothioneine, butanediol, acrylamide dimethyl taurate ammonium / VP copolymer, and water, wherein, The weight ratio of glucosyl hesperidin to ergothioneine is 0.05-0.3:0.1-0.

2. The content of glucosyl hesperidin is 0.05-0.3 wt%. The ergothioneine content is 0.1-0.2 wt%. 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 glucosyl hesperidin in inhibiting the photodegradation and high-temperature degradation of ergothionein, among which, The weight ratio of glucosyl hesperidin to ergothioneine is 0.05-0.3:0.1-0.

2. The pH value of the application is 4-6. The content of glucosyl hesperidin is 0.05-0.3 wt%. The ergothioneine content is 0.1-0.2 wt%.

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

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

5. The application as described in claim 4, 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.

6. The application as described in claim 4, wherein the composition is used in a topical skin preparation in an amount of 0.0001 wt% to 90 wt%.

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