Compositions based on glycyrrhizin that inhibit the photodegradation of ergothioneine

Abstract

This invention discloses a composition based on glycyrrhizin for inhibiting the photodegradation of ergothioneine, comprising glycyrrhizin and ergothioneine in a weight ratio of 0.5-5:1-8, wherein the content of glycyrrhizin in the composition is 0.05-0.5 wt%. This invention also discloses the application of glycyrrhizin in inhibiting the photodegradation of ergothioneine, the antioxidant or anti-glycation use of the composition based on glycyrrhizin 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 glycyrrhizin that inhibits the photodegradation of ergothioneine. This composition not only has excellent stability but also synergistic antioxidant and anti-glycation effects, providing a technical method for stabilizing the decomposition of ergothioneine under light and high temperature conditions. Background Technology

[0002] Among various varieties of licorice, glycyrrhizin is found only in the roots and rhizomes of *Glycyrrhiza glabra*. Glycyrrhizin is a species-specific marker component of *Glycyrrhiza glabra*, a flavonoid bioactive substance with the molecular formula C2. 20 H 20 O4, glycyrrhizin accounts for only 0.1%-0.3% of its total content; in actual production, only 100g of high-purity glycyrrhizin can be obtained from 1000kg of licorice root. It is soluble in ethanol, ethyl acetate, butylene glycol, isononyl isononanoate, etc., but insoluble in water. Because glycyrrhizin has a very high inhibitory effect on tyrosinase activity, it exhibits significant whitening activity even at extremely low concentrations, hence its reputation as "whitening gold."

[0003] Glycyrrhizin (CAS No. 59870-68-7, molecular weight 324.26) has the following structural formula:

[0004]

[0005] The structural formula of glycyrrhizin reveals that the known pharmacophore in red contains a carbon 3 group and a stereocenter of a B ring. It has been demonstrated that the free hydroxyl groups at C-2' and C-4' of the B ring are essential for its antioxidant activity. This B ring also participates in the inhibition of cytochrome P450 enzymes such as CYP3A4 and tyrosinases involved in melanin production. The 8-isoprenated moiety highlighted in blue contributes to the hydrophobicity of glycyrrhizin, facilitating its ability to penetrate cell membranes. The skin-whitening effect of glycyrrhizin is achieved through hydrogen-bonded complexation with tyrosinase.

[0006] Studies have shown that glycyrrhizin has the lowest IC50 for inhibiting tyrosinase, approximately 0.43 μmol / L. Furthermore, glycyrrhizin also exhibits excellent anti-inflammatory effects, achieving its anti-inflammatory activity through the dual inhibition of cyclooxygenase and lipoxygenase.

[0007] Ergothioneine (EGT), also known as thiohistidine betaine, is a rare amino acid found in nature. Its scientific name is 2-mercapto-histidine-trimethyl inner salt, with a molecular weight of 229.3. The pure form is a white crystalline solid, readily soluble in water. It possesses excellent antioxidant, anti-inflammatory, and anti-photoaging physiological activities, showing promising application prospects in the food, cosmetics, and pharmaceutical industries.

[0008] Organic cation transporter 1 (OCTN1) in human cells is the main transporter of ergothioneine. It can bind to the free electrons of hydroxyl radicals (·OH), pernitrite (ONOO-), and hypochlorite (CLO-) and scavenge free radicals through high redox potential.

[0009] Ergothione exists as a tautomer of thiols and thioketones, with a solubility of 0.9 mol / L at room temperature. Because the thiol group is less stable than the thiocarbonyl group, ergothione mainly exists as a thioketone under high pH or strongly alkaline conditions. Due to its thioether-like structure, its potential is -0.06 V under standard redox conditions, exhibiting good antioxidant properties.

[0010] Different pH conditions and concentrations can alter the configuration of ergothioneine in application systems, leading to problems such as odor and spoilage. Inhibiting photodegradation (improving photostability), odor, and discoloration of ergothioneine have always been difficult problems and challenges in the industry's application of this ingredient.

[0011] Currently, a Chinese patent application discloses an anti-aging composition and its application in cosmetic skincare. The anti-aging composition comprises: 0.1-5 parts of an antioxidant, 0.0001-0.05 parts of a complex polypeptide, 1-10 parts of panthenol, and 0.3-2 parts of a glycoside compound. The antioxidant is selected from at least one of arbutin, ergothioneine, grape seed extract, olive leaf extract, glycyrrhizin, retinol and its derivatives, and ascorbic acid and its derivatives. The complex polypeptide is selected from at least one of arginine / lysine polypeptide, acetyl octapeptide-3, acetyl hexapeptide-8, pentapeptide-3, tripeptide-1, and acetyl hexapeptide-1. The glycoside compound is selected from at least one of glyceryl glucoside, pyranoside, dextran, and sodium hyaluronate. Cosmetics made from this anti-aging composition, when used in conjunction with a radiofrequency beauty device, can promote dermal collagen regeneration, fill and reduce fine lines, achieving deep anti-aging, long-lasting moisturizing, anti-wrinkle, and skin-nourishing effects. Although the application mentions the simultaneous use of ergothioneine and glycyrrhizin as antioxidants, it does not reveal that glycyrrhizin can help inhibit the photodegradation of ergothioneine, nor does it show that the two have synergistic antioxidant or anti-glycation effects.

[0012] 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 content of the hyaluronic acid salt in the solution is not less than 0.1 wt%. The content of the ergothioneine in the solution is 0.0005-0.01 wt%. The molecular weight of the hyaluronic acid salt is 3 kDa-1800 kDa. 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.

[0013] The purpose of this invention is to provide a highly efficient and cost-effective method for inhibiting the photodecomposition and high-temperature instability of ergothioneine based on a combination of glycyrrhizin and ergothioneine. Unexpectedly, it significantly increases the content of ergothioneine in the system under different pH conditions and concentrations, and also unexpectedly enhances the antioxidant and anti-glycation effects in both directions, which has great application potential in the cosmetics field. Summary of the Invention

[0014] The purpose of this invention is to provide an ergothioneine composition, namely, a combination of ergothioneine and glycyrrhizin, which provides an extremely efficient and cost-effective method for inhibiting the photodecomposition and high-temperature instability of ergothioneine. Unexpectedly, under different pH conditions and concentrations, it significantly increases the content of ergothioneine in the system, and at the same time, it unexpectedly enhances the antioxidant and anti-glycation effects in both directions, which has great application potential in the cosmetics field.

[0015] The present invention provides a composition for inhibiting the photodegradation of ergothioneine based on glycyrrhizin, comprising: glycyrrhizin and ergothioneine, wherein the weight ratio of glycyrrhizin to ergothioneine is 0.5-5:1-8, and wherein the content of glycyrrhizin in the composition is 0.05-0.5 wt%.

[0016] In a preferred embodiment, the weight ratio of glycyrrhizin to ergothioneine is 0.5-4:1-8.

[0017] In a preferred embodiment, the composition further comprises ≤5 wt% of a C3-C6 diol.

[0018] In a preferred embodiment, the C3-C6 diol is selected from: butanediol, propylene glycol, 1,3-propanediol, pentanediol, 1,2-hexanediol, glycerol, diglycerol, etc.

[0019] In a preferred embodiment, the composition further comprises ≤0.8 wt% of acrylamide dimethyl taurate ammonium / VP copolymer.

[0020] In a preferred embodiment, the composition further comprises 0.05-0.8 wt% of hydroxypropyl β-cyclodextrin.

[0021] In a preferred embodiment, the composition further comprises a carrier acceptable in the field of topical skin agents.

[0022] Preferably, water is an acceptable carrier in the field of topical skin agents.

[0023] Preferably, the water content in the composition is 90-95 wt%.

[0024] In a preferred embodiment, the composition may further comprise hyaluronic acid.

[0025] Preferably, the hyaluronic acid has a molecular weight of 3K-10KDa.

[0026] Preferably, the weight ratio of hyaluronic acid to glycyrrhizin is 1:1.

[0027] In a preferred embodiment, the composition further comprises a pH adjuster.

[0028] Preferably, the pH adjuster is selected from at least one of citric acid and tromethamine.

[0029] In a preferred embodiment, the pH value of the composition is 4-8.

[0030] Preferably, the pH value of the composition is 4-6.

[0031] This invention also provides the application of glycyrrhizin in inhibiting the photodegradation of ergothionein.

[0032] In a preferred embodiment, the weight ratio of glycyrrhizin to ergothioneine is 0.5-4:1-8.

[0033] In a preferred embodiment, the application is performed at a pH of 4-8.

[0034] Preferably, the application is performed at a pH of 4-6.

[0035] The present invention also provides the use of compositions based on glycyrrhizin and ergothioneine for antioxidant or anti-glycation purposes.

[0036] In a preferred embodiment, the weight ratio of glycyrrhizin to ergothionein in the composition is 0.5-4:1-8, and the pH value of the composition is 4-6.

[0037] The present invention also provides the use of the above-mentioned composition based on glycyrrhizin and ergothioneine in topical skin preparations.

[0038] 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.

[0039] In a preferred embodiment, the composition is used in a topical skin preparation at an amount of 0.0001 wt%-90 wt%.

[0040] The preferred weight percentage is 0.001wt%-10wt%.

[0041] A more preferred weight percentage is 0.001wt%-5wt%.

[0042] The beneficial effects of this invention are:

[0043] 1. This invention is the first to protect the stability of ergothioneine using glycyrrhizin as a component. Through the special role of hydrogen bonds, the conformational stability of ergothioneine under light and high temperature can be protected.

[0044] 2. This invention unexpectedly discovered that glycyrrhizin and ergothioneine have a very good synergistic effect, which can improve the antioxidant efficacy by more than 100%.

[0045] 3. This invention unexpectedly discovered that ergothioneine and glycyrrhizin have a very good synergistic effect, which can significantly improve efficacy in anti-glycation. 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] C3-C6 diols

[0051] 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.

[0052] 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.

[0053] The main experimental materials and reagents used in the examples are as follows:

[0054] 1. Ergothioneine, purity ≥99.0%, Shanghai Ergothioneine Co., Ltd.

[0055] 2. Tromethamine, analytical grade, Sinopharm Group

[0056] 3. Citric acid, analytical grade, Sinopharm Group

[0057] 4. Glycyrrhizin, purity ≥99.0%, Gansu Fanzhi Biotechnology Co., Ltd.

[0058] 5. Acrylamide dimethyl taurate ammonium / VP copolymer (AVC), purity ≥99.0%, Clariant (China) Co., Ltd.

[0059] 6. Hydroxypropyl β-cyclodextrin, purity ≥99%, Shandong Binzhou Zhiyuan Biotechnology Co., Ltd.

[0060] 7. Hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) (purity ≥99.0%, Bloomage Biotechnology Co., Ltd.)

[0061] 8. Sodium hyaluronate (molecular weight 10K-100KDa) (purity ≥99.0%, Bloomage Biotechnology Co., Ltd.)

[0062] 9. Hyaluronic acid (molecular weight 100K-1000KDa) (purity ≥99.0%, Bloomage Biotechnology Co., Ltd.)

[0063] 10. Formic acid, purity ≥ 98.0%, Sigma-Aldrich (Product No. 43804)

[0064] 11. Acetonitrile, purity ≥ 99.9%, Sigma-Aldrich (Product No. 34851)

[0065] 12. Methanol, purity ≥ 99.9%, Sigma-Aldrich (Product No. 34885)

[0066] 13. PBS (Beijing Solarbio Science & Technology Co., Ltd., batch number P1022)

[0067] 14. DCFH-DA culture medium, LMAI Bio (Shanghai Lianmai Biotechnology Co., Ltd.)

[0068] 15. Pancreatic enzyme, Beijing Solarbio Science & Technology Co., Ltd., product batch number T8151

[0069] 16. Methylglyoxal, 40% aqueous solution, Sigma-Aldrich, lot number M0252

[0070] 17. Aminoguanidine sulfate, AM, Sigma-Aldrich

[0071] Main experimental instruments

[0072] 1. Waters ARC high-performance liquid chromatograph with diode array detector (Waters)

[0073] 2. XS205 analytical balance (METTLER TOLEDO)

[0074] 3. KQ-800DE CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.)

[0075] 4. CO2 incubator (Thermo, 150I)

[0076] 5. Clean bench (Su Jing An Tai, SW-CJ-1F)

[0077] 6. Flow cytometer (Beckman, CytoFLEX)

[0078] 7. Inverted microscope (Olympus, CKX53)

[0079] 8. UVB irradiator (Philips). (Please confirm that this is a UVA irradiator.)

[0080] 9. Incucell incubator (MMM GmbH, Germany)

[0081] 10. PERCIVAL CU41L5 Illuminated Incubator (PERCIVAL Corporation, USA)

[0082] Example 1:

[0083] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of glycyrrhizin and stir until homogeneous. Then add 0.5g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0084] Example 2:

[0085] Accurately weigh 0.4g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of glycyrrhizin and stir until homogeneous. Then add 0.8g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0086] Example 3:

[0087] Accurately weigh 0.6g of ergothioneine and dissolve it in 5g of butanediol. Add 0.3g of glycyrrhizin and stir until homogeneous. Then add 0.6g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0088] Example 4:

[0089] Accurately weigh 0.8g of ergothioneine and dissolve it in 5g of butanediol. Add 0.4g of glycyrrhizin and stir until homogeneous. Then add 2.4g of hydroxypropyl β-cyclodextrin and stir thoroughly. Next, 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.

[0090] Example 5:

[0091] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of glycyrrhizin 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 4.0 using citric acid and tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0092] Example 6:

[0093] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.01g of glycyrrhizin and stir until homogeneous. Then add 0.05g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0094] Example 7:

[0095] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of glycyrrhizin and stir until homogeneous. Then add 0.2g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0096] Example 8:

[0097] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of glycyrrhizin and stir until homogeneous. Then add 0.2g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0098] Example 9:

[0099] Accurately weigh 0.4g of ergothioneine and dissolve it in 5g of butanediol. Add 0.8g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0100] Example 10:

[0101] 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.

[0102] Example 11:

[0103] 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.

[0104] Example 12:

[0105] 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 glycyrrhizin, 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.

[0106] Example 13:

[0107] 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.

[0108] Example 14:

[0109] 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.

[0110] Example 15:

[0111] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of glycyrrhizin and stir until homogeneous. Then add 0.5g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0112] Example 16:

[0113] Accurately weigh 0.4g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of glycyrrhizin and stir until homogeneous. Then add 0.8g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0114] Example 17:

[0115] Accurately weigh 0.6g of ergothioneine and dissolve it in 5g of butanediol. Add 0.3g of glycyrrhizin and stir until homogeneous. Then add 0.6g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0116] Example 18:

[0117] Accurately weigh 0.8g of ergothioneine and dissolve it in 5g of butanediol. Add 0.4g of glycyrrhizin and stir until homogeneous. Then add 2.4g of hydroxypropyl β-cyclodextrin and stir thoroughly until homogeneous. 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.

[0118] Example 19:

[0119] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of glycyrrhizin 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 tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0120] Example 20:

[0121] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.01g of glycyrrhizin and stir until homogeneous. Then add 0.05g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0122] Example 21:

[0123] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of glycyrrhizin and stir until homogeneous. Then add 0.2g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0124] Example 22:

[0125] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of glycyrrhizin and stir until homogeneous. Then add 0.2g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0126] Example 23:

[0127] Accurately weigh 0.4g of ergothioneine and dissolve it in 5g of butanediol. Add 0.8g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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.

[0128] Example 24:

[0129] 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.

[0130] Example 25:

[0131] 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.

[0132] Example 26:

[0133] 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 glycyrrhizin, and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 6.0 using citric acid and tromethamine. Pour into a volumetric flask and set aside for later use.

[0134] Example 27:

[0135] 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.

[0136] Example 28:

[0137] 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.

[0138] Example 29:

[0139] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of glycyrrhizin and stir until homogeneous. Then add 0.5g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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. Transfer the solution to a volumetric flask for later use.

[0140] Example 30:

[0141] Accurately weigh 0.4g of ergothioneine and dissolve it in 5g of butanediol. Add 0.2g of glycyrrhizin and stir until homogeneous. Then add 0.8g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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. Transfer the solution to a volumetric flask for later use.

[0142] Example 31:

[0143] Accurately weigh 0.6g of ergothioneine and dissolve it in 5g of butanediol. Add 0.3g of glycyrrhizin and stir until homogeneous. Then add 0.6g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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. Transfer the solution to a volumetric flask for later use.

[0144] Example 32:

[0145] Accurately weigh 0.8g of ergothioneine and dissolve it in 5g of butanediol. Add 0.4g of glycyrrhizin and stir until homogeneous. Then add 2.4g of hydroxypropyl β-cyclodextrin and stir thoroughly until homogeneous. 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. Transfer the solution to a volumetric flask for later use.

[0146] Example 33:

[0147] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.5g of glycyrrhizin 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 tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0148] Example 34:

[0149] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.01g of glycyrrhizin and stir until homogeneous. Then add 0.05g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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. Transfer the solution to a volumetric flask for later use.

[0150] Example 35:

[0151] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of glycyrrhizin and stir until homogeneous. Then add 0.2g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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. Transfer the solution to a volumetric flask for later use.

[0152] Example 36:

[0153] Accurately weigh 0.2g of ergothioneine and dissolve it in 5g of butanediol. Add 0.05g of glycyrrhizin and stir until homogeneous. Then add 0.2g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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. Transfer the solution to a volumetric flask for later use.

[0154] Example 37:

[0155] Accurately weigh 0.4g of ergothioneine and dissolve it in 5g of butanediol. Add 0.8g of hydroxypropyl β-cyclodextrin and stir thoroughly until clear and transparent. 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. Transfer the solution to a volumetric flask for later use.

[0156] Example 38:

[0157] 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.

[0158] Example 39:

[0159] Accurately weigh 0.1g of ergothioneine and dissolve it in 5g of butanediol. Add 0.1g of sodium hyaluronate (molecular weight 10K-100KDa) and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethamine. Pour the solution into a volumetric flask and set aside for later use.

[0160] Example 40:

[0161] 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 glycyrrhizin, and stir until homogeneous and clear. Add 0.8g of AVC and disperse it thoroughly until homogeneous. Add water to adjust the total volume to 100g of aqueous solution. Adjust the pH of the solution to 8.0 using citric acid and tromethamine. Pour into a volumetric flask and set aside for later use.

[0162] Example 41:

[0163] 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.

[0164] Example 42:

[0165] 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.

[0166] Test Example 1: Illumination Test Experiment

[0167] Test samples: Composition samples prepared in Examples 1-42

[0168] 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.

[0169] 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.

[0170] Classification Odor and strength standards Level 1 No discernible change in taste Level 2 Slight change in taste Level 3 Significant taste change

[0171] The results of the composition samples prepared in Examples 1-42 after 14 days of light exposure testing are shown in Table 1:

[0172] Table 1. Results of light irradiation tests on the composition samples prepared in Examples 1-42.

[0173]

[0174] After 14 days of simulated sunlight exposure testing, under acidic conditions (pH=4), the composition samples containing ergothioneine, butylene glycol, AVC, glycyrrhizin, and water prepared in Examples 1-4 and 6-8 showed no perceptible change in taste; the composition sample prepared in Example 5 showed only a slight change in taste.

[0175] At the same pH value (pH=4), when all or part of the glycyrrhizin in the composition was replaced with 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.

[0176] Specifically as follows:

[0177] Example 9 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, glycyrrhizin, and water. The former contained 0.2 wt% less glycyrrhizin than the latter, and with the remaining components present in the same amounts, the composition sample prepared in Example 2 performed significantly better than the composition sample prepared in Example 9 under light irradiation testing. This indicates that glycyrrhizin has a positive stabilizing effect on ergothioneine in the composition.

[0178] Example 10 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 6 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glycyrrhizin, hydroxypropyl β-cyclodextrin, and water. The former contained 0.1 wt% hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), while the latter contained 0.06 wt% glycyrrhizin and hydroxypropyl β-cyclodextrin. With the remaining components present in the same amounts, under light irradiation testing, the composition sample prepared in Example 6 showed significantly better performance than that prepared in Example 10. This indicates that glycyrrhizin and hydroxypropyl β-cyclodextrin have a positive stabilizing effect on ergothioneine in the composition. Furthermore, it also demonstrates that hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) cannot inhibit the photodegradation of ergothioneine.

[0179] Example 11 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 6 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glycyrrhizin, hydroxypropyl β-cyclodextrin, and water. The former contained 0.1 wt% sodium hyaluronate (molecular weight 10K-100KDa), while the latter contained 0.06 wt% glycyrrhizin and hydroxypropyl β-cyclodextrin. With the remaining components present in the same amounts, under light irradiation testing, the composition sample prepared in Example 6 performed significantly better than that prepared in Example 11. This indicates that glycyrrhizin and hydroxypropyl β-cyclodextrin 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.

[0180] Example 12 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 3K-10KDa), glycyrrhizin, and water. Under light irradiation, a slight change in taste was observed. This indicates that the combination of glycyrrhizin and hyaluronic acid (molecular weight 3K-10KDa) can also inhibit the photodegradation of ergothioneine.

[0181] Example 13 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Under light irradiation, a significant change in taste was observed. Hyaluronic acid (molecular weight 100K-1000KDa) did not help inhibit the photodegradation of ergothioneine.

[0182] After 14 days of simulated sunlight exposure testing, under acidic conditions (pH=6), the composition samples containing ergothioneine, butylene glycol, AVC, glycyrrhizin, and water prepared in Examples 18 and 20-22 showed no perceptible change in taste; the composition samples prepared in Examples 15-17 and 19 showed only slight changes in taste.

[0183] At the same pH value (pH=6), when all or part of the glycyrrhizin in the composition was replaced with 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.

[0184] Specifically as follows:

[0185] Example 23 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 16 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glycyrrhizin, hydroxypropyl β-cyclodextrin, and water. The difference between the former and the latter is that the latter has an additional 0.2 wt% glycyrrhizin. With the remaining component contents being the same, under light irradiation testing, the composition sample prepared in Example 16 performed better than the composition sample prepared in Example 23. This indicates that glycyrrhizin can help inhibit the photodegradation of ergothioneine in the composition. Hydroxypropyl β-cyclodextrin, however, cannot help inhibit the photodegradation of ergothioneine and is less effective than glycyrrhizin.

[0186] Example 24 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Under light irradiation, the composition sample prepared in Example 24 showed a significant change in taste. This indicates that hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) does not help inhibit the photodegradation of ergothioneine.

[0187] Example 25 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Under light irradiation, the composition sample prepared in Example 25 showed a significant change in taste. This indicates that sodium hyaluronate (molecular weight 10K-100KDa) does not help inhibit the photodegradation of ergothioneine.

[0188] Example 26 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glycyrrhizin, hyaluronic acid (molecular weight 3K-10KDa), and water. Under light irradiation, the composition sample prepared in Example 26 showed a significant change in taste. This indicates that the combination of hyaluronic acid (molecular weight 3K-10KDa) and glycyrrhizin does not help inhibit the photodegradation of ergothioneine.

[0189] Example 27 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Under light irradiation, the composition sample prepared in Example 27 showed a significant change in taste. This indicates that hyaluronic acid (molecular weight 100K-1000KDa) does not help inhibit the photodegradation of ergothioneine.

[0190] Example 28 prepared a composition sample containing ergothioneine, butylene glycol, AVC, and water, while Example 20 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glycyrrhizin, hydroxypropyl β-cyclodextrin, and water. The difference between the two is that Example 20 contained 0.06 wt% of a combination of glycyrrhizin and hydroxypropyl β-cyclodextrin. With the contents of the other components being the same, under light irradiation testing, the composition sample prepared in Example 20 performed significantly better than the composition sample prepared in Example 28. This further clarifies that the combination of glycyrrhizin and hydroxypropyl β-cyclodextrin can help inhibit the photodegradation of ergothioneine in the composition.

[0191] Under alkaline conditions (pH=8), the composition samples prepared in Examples 29-36 all showed significant changes in taste. This was mainly due to the decomposition of ergothioneine in the samples caused by simulated sunlight exposure. This indicates that even if an appropriate amount of glycyrrhizin or a combination of glycyrrhizin and hydroxypropyl β-cyclodextrin is added to the composition, it is still not possible to effectively inhibit the photodegradation of ergothioneine.

[0192] At the same pH value (pH=8), when all or part of the composition of glycyrrhizin or the composition of glycyrrhizin and hydroxypropyl β-cyclodextrin was replaced with 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 light irradiation test of the composition sample still showed a significant change in taste and could not effectively inhibit the photodegradation of ergothionein.

[0193] In conclusion, under acidic conditions, glycyrrhizin or a combination of glycyrrhizin and hydroxypropyl β-cyclodextrin can significantly help inhibit the photodegradation of ergothionein.

[0194] Test Example 2: High Temperature Test Experiment

[0195] Test samples: Composition samples prepared in Examples 1-42

[0196] 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.

[0197] The samples from Examples 1-42 were poured into glass bottles, the caps were tightened, and the bottles were placed in an Incucell incubator at 48°C for 14 days to observe the changes in the products.

[0198] 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.

[0199] Classification Odor and strength standards Level 1 No discernible change in taste Level 2 Slight change in taste Level 3 Significant taste change

[0200] Table 2 High-temperature tests of the composition samples prepared in Examples 1-42

[0201]

[0202] After 14 days of high-temperature testing at 48°C, under acidic conditions (pH=4), the composition samples containing ergothioneine, butylene glycol, AVC, glycyrrhizin, and water prepared in Examples 1-4 and 6-8 showed no perceptible change in taste; the composition sample prepared in Example 5 showed only a slight change in taste.

[0203] At the same pH value (pH=4), when all or part of the glycyrrhizin in the composition was replaced with 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.

[0204] Specifically as follows:

[0205] Example 9 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, glycyrrhizin, and water. The former contained 0.2 wt% less glycyrrhizin than the latter, and with the contents of the remaining components being the same, the composition sample prepared in Example 2 showed significantly better performance than the composition sample prepared in Example 9 under high-temperature stability testing. This indicates that glycyrrhizin has a positive stabilizing effect on ergothioneine in the composition.

[0206] Example 10 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Example 6 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glycyrrhizin, hydroxypropyl β-cyclodextrin, and water. The former contained 0.1 wt% hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), while the latter contained 0.06 wt% glycyrrhizin and hydroxypropyl β-cyclodextrin. With the remaining components present in the same amounts, the composition sample prepared in Example 6 showed significantly better performance than the composition sample prepared in Example 10 under high-temperature stability testing. This indicates that glycyrrhizin and hydroxypropyl β-cyclodextrin have a positive stabilizing effect on ergothioneine in the composition. Furthermore, it also demonstrates that hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) cannot inhibit the photodegradation of ergothioneine.

[0207] Example 11 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Example 6 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glycyrrhizin, hydroxypropyl β-cyclodextrin, and water. The former contained 0.1 wt% sodium hyaluronate (molecular weight 10K-100KDa), while the latter contained 0.06 wt% glycyrrhizin and hydroxypropyl β-cyclodextrin. With the remaining component contents being identical in both examples, the composition sample prepared in Example 6 showed significantly better performance than the composition sample prepared in Example 11 under high-temperature stability testing. This indicates that glycyrrhizin and hydroxypropyl β-cyclodextrin 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.

[0208] Example 12 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 3K-10KDa), glycyrrhizin, and water. Under high-temperature stability testing, a slight change in taste was observed. This indicates that the combination of glycyrrhizin and hyaluronic acid (molecular weight 3K-10KDa) can also inhibit the photodegradation of ergothioneine.

[0209] Example 13 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Under high-temperature stability testing, a significant change in taste was observed. Hyaluronic acid (molecular weight 100K-1000KDa) did not help inhibit the photodegradation of ergothioneine.

[0210] After 14 days of high-temperature stability testing, under acidic conditions (pH=6), the composition samples containing ergothioneine, butylene glycol, AVC, glycyrrhizin, and water prepared in Examples 18 and 20-22 showed no perceptible change in taste; the composition samples prepared in Examples 15-17 and 19 showed only slight changes in taste.

[0211] At the same pH value (pH=6), when all or part of the glycyrrhizin in the composition was replaced with 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.

[0212] Specifically as follows:

[0213] Example 23 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydroxypropyl β-cyclodextrin, and water. Example 16 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glycyrrhizin, hydroxypropyl β-cyclodextrin, and water. The difference between the former and the latter is that the latter has an additional 0.2 wt% glycyrrhizin. With the contents of the remaining components being the same, the composition sample prepared in Example 16 performed better than the composition sample prepared in Example 23 in the high-temperature stability test. This indicates that glycyrrhizin can help inhibit the photodegradation of ergothioneine in the composition. However, hydroxypropyl β-cyclodextrin cannot help inhibit the photodegradation of ergothioneine and its effect is less than that of glycyrrhizin.

[0214] Example 24 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hydrolyzed hyaluronic acid (molecular weight 3K-10KDa), and water. Under high-temperature stability testing, the composition sample prepared in Example 24 showed a significant change in taste. This indicates that hydrolyzed hyaluronic acid (molecular weight 3K-10KDa) does not help inhibit the photodegradation of ergothioneine.

[0215] Example 25 prepared a composition sample containing ergothioneine, butylene glycol, AVC, sodium hyaluronate (molecular weight 10K-100KDa), and water. Under high-temperature stability testing, the composition sample prepared in Example 25 showed a significant change in taste. This indicates that sodium hyaluronate (molecular weight 10K-100KDa) cannot help inhibit the photodegradation of ergothioneine.

[0216] Example 26 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glycyrrhizin, hyaluronic acid (molecular weight 3K-10KDa), and water. Under high-temperature stability testing, the composition sample prepared in Example 26 showed a significant change in taste. This indicates that the combination of hyaluronic acid (molecular weight 3K-10KDa) and glycyrrhizin does not help inhibit the photodegradation of ergothioneine.

[0217] Example 27 prepared a composition sample containing ergothioneine, butylene glycol, AVC, hyaluronic acid (molecular weight 100K-1000KDa), and water. Under high-temperature stability testing, the composition sample prepared in Example 27 showed a significant change in taste. This indicates that hyaluronic acid (molecular weight 100K-1000KDa) does not help inhibit the photodegradation of ergothioneine.

[0218] Example 28 prepared a composition sample containing ergothioneine, butylene glycol, AVC, and water, while Example 20 prepared a composition sample containing ergothioneine, butylene glycol, AVC, glycyrrhizin, hydroxypropyl β-cyclodextrin, and water. The difference between the two is that Example 20 contained 0.06 wt% of a combination of glycyrrhizin and hydroxypropyl β-cyclodextrin. With the contents of the other components being the same, the composition sample prepared in Example 20 showed significantly better performance than the composition sample prepared in Example 28 under high-temperature stability testing. This further clarifies that the combination of glycyrrhizin and hydroxypropyl β-cyclodextrin can help inhibit the photodegradation of ergothioneine in the composition.

[0219] Under alkaline conditions (pH=8), the composition samples prepared in Examples 29-36 all showed significant changes in taste. This was mainly due to the decomposition of ergothioneine in the samples under high-temperature stability testing. This indicates that even if an appropriate amount of glycyrrhizin or a combination of glycyrrhizin and hydroxypropyl β-cyclodextrin is added to the composition, it is still not possible to effectively inhibit the photodegradation of ergothioneine.

[0220] At the same pH value (pH=8), when all or part of the composition of glycyrrhizin or the composition of glycyrrhizin and hydroxypropyl β-cyclodextrin was replaced with 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 high-temperature stability test of the composition sample still showed a significant change in taste and could not effectively inhibit the photodegradation of ergothionein.

[0221] In conclusion, under acidic conditions, glycyrrhizin or a combination of glycyrrhizin and hydroxypropyl β-cyclodextrin can significantly help inhibit the photodegradation of ergothionein.

[0222] Test Example 3: Detection of Ergothionein by Ultra-High Performance Liquid Chromatography

[0223] Test samples: Composition samples prepared in Examples 1-42 after the light irradiation test in Test Example 1; Composition samples prepared in Examples 1-42 after the high temperature stability test in Test Example 2.

[0224] Test standard: NY / T 3872-2021 (UPLC)

[0225] 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.

[0226] Testing steps:

[0227] 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).

[0228] 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.

[0229] 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.

[0230] Results Calculation: Quantitative analysis using the external standard method; the formula for calculating the ergothionein content in the sample is as follows:

[0231]

[0232] In the formula:

[0233] X — Mass fraction of the analyte in the sample, %

[0234] C—The concentration of ergothioneine in the sample solution obtained from the standard curve, in micrograms per milliliter (μg·mL). -1 );

[0235] V—The total volume of the sample after dilution, in milliliters (mL);

[0236] m — Sample mass, in grams (g);

[0237] The ergothioneine content in the composition samples after light exposure or high temperature stability testing is shown in the table below:

[0238] Table 3. Ergothionein content in the composition samples after light or high temperature stability tests.

[0239]

[0240] 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.

[0241] Firstly, under alkaline conditions, ergothioneine is more easily decomposed. Due to its unique structure, containing hydroxyl and carbonyl groups, ergothioneine reacts chemically with hydroxide ions under alkaline conditions, resulting in changes in its structure, properties, and odor. Regardless of concentration or under light and high temperature conditions, the structural integrity and existence of ergothioneine are significantly challenged, leading to a noticeable change in taste, specifically a foul odor. However, even under these stringent conditions, we unexpectedly discovered that the combined effect of ergothioneine and glycyrrhizin, as described in Example 31, exhibited a relatively good ergothioneine retention rate. Specifically, at a 1:2 ratio of glycyrrhizin to ergothioneine, the abundant hydroxyl groups on the surface effectively protected the ergothioneine through hydrogen bonding. This condition and ratio provide a very good solution and technology for the application of ergothioneine in alkaline products.

[0242] Acidic pH is the recommended direction for ergothioneine application. A comparison of pH=4 and pH=6 shows that a lower pH under acidic conditions is more conducive to the stability of ergothioneine. Unexpectedly, by adjusting the proportions of different ergothioneine compositions at various concentrations, several different combinations and concentrations were found to have excellent protective functions. Examples 14, 28, and 42 demonstrate that under different pH conditions, the ergothioneine content degraded by more than 30-50% within two weeks.

[0243] In contrast to existing technologies, such as the Chinese patent application CN110327242B which discloses a method for inhibiting the photodegradation of ergothioneine using hyaluronic acid salts, the composition samples prepared in Examples 10-11, 13, 24-25, 27, 38-39, and 41 of this application were used to simultaneously investigate the inhibitory effect of hyaluronic acid and its derivatives on ergothioneine in the prior art. Compared with ergothioneine under the same concentration and pH conditions, no significant improvement in photodegradation and high-temperature degradation stability was observed. On the contrary, the content of ergothioneine under these conditions decreased to some extent. It is speculated that the possible reason is that different types of hyaluronic acid, including hyaluronic acid with a relatively small molecular weight but still having a molecular weight of several thousand Daltons, did not form a complete and tight encapsulation structure, resulting in ergothioneine concentration differences and escape.

[0244] Under pH=4 conditions, the composition samples prepared in Examples 1-3 and Examples 6-8 unexpectedly obtained excellent protective effects against ergothioneine. After 14 days of light exposure or 14 days of high temperature stability test, the content in the sample was retained at more than 82.1%.

[0245] The composition samples prepared in Examples 4-5 also showed an inhibitory effect on ergothioneine. After 14 days of light exposure or 14 days of high temperature stability testing, the content in the samples remained at over 79%.

[0246] Under pH 6 conditions, the compositions prepared in Examples 18 and 20-22 also unexpectedly exhibited excellent protective effects against ergothioneine. After 14 days of light exposure or 14 days of high-temperature stability testing, the ergothioneine content in the samples remained above 73%.

[0247] In summary, when the pH is acidic, the glabridin molecule in the composition sample has abundant hydroxyl groups, resulting in numerous binding sites within the cyclodextrin inclusions. This binding is a spontaneous reaction primarily driven by hydrophobic interactions. Reports on molecular docking suggest that the addition of glabridin may have altered the internal water transport environment of ergothioneine, leading to a more stable binding. This also essentially ensured that the ergothioneine content remained above 70%.

[0248] For example, a 2:1 mass ratio of ergothioneine to glycyrrhizin is considered an optimal concentration. However, as shown in Example 4, a higher concentration of ergothioneine resulted in accelerated degradation. Even when the concentration of glycyrrhizin was further reduced to a 5:1 or 4:1 mass ratio, a relatively good effect was maintained. However, a concentration of 10:1 did not provide adequate protection. Therefore, given the relatively high cost of glycyrrhizin, its ability to achieve good protective effects at lower concentrations is an unexpected achievement.

[0249] Compared to Example 9, simply using cyclodextrin to encapsulate ergothioneine has proven to be an ineffective way to address its photodegradation and high-temperature stability.

[0250] In terms of different concentrations, the ratio of ergothioneine to glycyrrhizin is best when the ratio is between 2:1 and 5:1. In addition, Example 12 illustrates the use of glycyrrhizin and hyaluronic acid in combination, but no synergistic effect or usefulness of hyaluronic acid was observed.

[0251] Test Example 4: Evaluation of Antioxidant Performance

[0252] Test samples: Composition samples prepared in Examples 1-3, 7-8, 14, 21-22, 28 and 42.

[0253] Experimental methods:

[0254] 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).

[0255] 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.

[0256] 3) UVB irradiation: According to the test groups, the groups exposed to UVB irradiation were subjected to 300mJ / cm² irradiation. 2 UVB irradiation.

[0257] 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.

[0258] 5) Statistical Analysis of Results: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0259] Table 4. Evaluation results of antioxidant performance of some examples

[0260]

[0261] Note: Mean fluorescence intensity (MFI) reflects the ROS content. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0262] 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.

[0263] Comparing the antioxidant properties of ergothioneine under different pH conditions, it was found that commonly used vitamin E (as a control group) exhibited superior antioxidant performance. Vitamin E is a substance with strong antioxidant activity and is a crucial antioxidant in the human body, blocking free radical chain reactions. It plays an indispensable role in stabilizing and protecting cell membranes and in normal human metabolism. Vitamin E plays an important role in inhibiting inflammatory responses, fighting free radicals, anti-aging, and anti-cancer processes by influencing inflammatory factors and regulating cell signal transduction. Vitamin E has been widely used to treat various skin diseases, including photoaging, inflammatory skin diseases, connective tissue diseases, viral skin diseases, and herpes. Regarding ergothioneine itself, the compositions prepared in Examples 14, 28, and 42 also significantly improved antioxidant properties compared to vitamin E.

[0264] Comparing the combinations of ergothioneine and glycyrrhizin under different pH conditions, a significant increase in antioxidant capacity was unexpectedly found in all cases, with most examples showing an increase of over 100%. In particular, Example 2 showed a 91.02% improvement in the antioxidant capacity of the in vivo sample, representing a 106.2% increase compared to the 44.15% antioxidant capacity of ergothioneine. Figure 1 As shown.

[0265] Test Example 5: Comparison of Anti-Glycation Efficacy (Detection Index AGEs)

[0266] Test examples: Composition samples prepared in Examples 1-3, 7-8, and 21-22;

[0267] Experimental methods:

[0268] Obtain ExVivo tissue (subcutaneous adipose tissue removed), thaw in PBS solution containing penicillin and antibiotics, and take tissue blocks with a diameter of 4-6 mm. Repeat the freeze-thaw cycle 5 times in liquid nitrogen and a 37°C water bath. Place the excised skin tissue blocks in a 24-well plate, add 1 ml of PBS containing penicillin and antibiotics, and incubate with CO2 for 18±2 h. Perform the glycation reaction as follows:

[0269] 1) Take a new 24-well plate and add 0.5 mM methylglyoxal, 1 ml / well;

[0270] 2) According to 10J / cm 2 Irradiation with UVA radiation dose;

[0271] 3) After irradiation, the orifice plate was transferred to a 45°C oven for saccharification reaction for 72 hours;

[0272] 4) After 72 hours, aspirate and discard the saccharification reaction working solution, and add freshly prepared saccharification reaction working solution, i.e., 3mM aminoguanidine sulfate as a positive control. Repeat steps 2) to 4) with 3% of the example sample and control. The above experiments use isolated skin tissue soaked in PBS as a blank control, and isolated skin tissue without any sample added under normal induction conditions as a negative control.

[0273] 5) UVA irradiation was performed 3 times. After the 4th irradiation, the detached skin was cleaned and transferred to a new glycosylation reaction working solution. Steps 3) and 4) were repeated for a total of 21 days.

[0274] 6) After the glycosylation reaction is completed, the isolated skin tissue is transferred to a 1.5 mL EP tube, fixed with 4% paraformaldehyde, embedded, sectioned, and stained with AGEs immunohistochemistry.

[0275] GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups.

[0276] Relative improvement rate (%) = (Negative control - Relative average of examples) / Negative control x 100%

[0277] Table 5. Results of anti-glycation efficacy (detection index AGEs) of each embodiment

[0278]

[0279] The anti-glycation results of the compositions prepared in Examples 1-3, 7-8, and 21-22 unexpectedly revealed that ergothioneine has a very good synergistic effect with glycyrrhizin in anti-glycation. Specifically:

[0280] From the perspective of improvement rate, the sample with better ergothioneine protection in Example 2 showed the best synergistic effect at 28.82%, which was 6% higher than the positive control. No anti-glycation effect was observed in the ergothioneine sample itself. However, while glycyrrhizin itself had a good anti-glycation effect, its anti-glycation effect was unexpectedly more significant when combined with ergothioneine.

[0281] 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.

[0282] The composition samples prepared in Examples 1-8, 12, and 15-22 were used for the preparation of topical skin agents. The topical skin agents are preferably cosmetic compositions, such as lotions, serums, and creams. The weight percentage of the composition samples prepared in Examples 1-8, 12, and 15-22 in the topical skin agent is 0.0001 wt%-90 wt%. A preferred weight percentage is 0.001 wt%-10 wt%. A more preferred weight percentage is 0.001 wt%-5 wt%.

[0283] The following are examples of the specific applications of the compositions prepared in Examples 1-8, 12, and 15-22 in topical skin preparations, along with the formulations and preparation methods of these dosage forms. In the following tables, "-" indicates no additives.

[0284] Application Example 1: Preparation of Face Cream

[0285]

[0286] Application Example 2: Emulsion Preparation

[0287]

[0288] Application Example 3: Preparation of Gel

[0289]

[0290] Application Example 4: Preparation of Toner

[0291]

[0292] Application Example 5: Preparation of Serum

[0293]

[0294] Application Example 6: Preparation of Facial Masks

[0295]

[0296] Application Example 7: Preparation of Eye Cream

[0297]

[0298] Application Example 8: Preparation of Spray

[0299]

[0300] Application Example 9: Preparation of Shower Gel

[0301]

[0302] Application Example 10: Preparation of Facial Cleanser

[0303]

[0304] Application Example 11: Preparation of Essence Water

[0305]

[0306] In the examples of specific applications of all the above topical skin agents, the composition samples of Examples 1-8, Examples 15-22, Examples 31 and 36 can be used directly in the formulation, either partially or completely, to replace deionized water, or can be used directly as a formulation.

Claims

1. A composition for inhibiting the photodegradation of ergothioneine based on glycyrrhizin, comprising glycyrrhizin, ergothioneine, butanediol, acrylamide dimethyl taurate ammonium / VP copolymer, hydroxypropyl β-cyclodextrin, and water, wherein, The content of glycyrrhizin is 0.05-0.4 wt%. The weight ratio of glycyrrhizin to ergothioneine is 0.5-4:1-8. The content of the acrylamide dimethyl taurate ammonium / VP copolymer is 0.8 wt%. The content of the hydroxypropyl β-cyclodextrin is 0.05-2.4 wt%. The ergothioneine content is 0.1-0.8 wt%. The pH value of the composition is 4.

2. The application of a composition based on glycyrrhizin in inhibiting the photodegradation of ergothioneine, said composition comprising glycyrrhizin, ergothioneine, butanediol, acrylamide dimethyl taurate ammonium / VP copolymer, hydroxypropyl β-cyclodextrin and water, wherein, The content of glycyrrhizin is 0.05-0.4 wt%. The weight ratio of glycyrrhizin to ergothioneine is 0.5-4:1-8. The content of the acrylamide dimethyl taurate ammonium / VP copolymer is 0.8 wt%. The content of the hydroxypropyl β-cyclodextrin is 0.05-2.4 wt%. The ergothioneine content is 0.1-0.8 wt%. The pH value of the composition is 4.

3. Use of the composition of claim 1 for non-therapeutic purposes of anti-oxidation or anti-glycation.

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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