Ascorbic acid-S-glutathione conjugate and derivative as well as preparation method and application of ascorbic acid-S-glutathione conjugate

Abstract

The invention provides an ascorbic acid-S-glutathione conjugate, a derivative, a preparation method and application of the ascorbic acid-S-glutathione conjugate. The conjugate is obtained by activating ascorbic acid-6-hydroxyl by halogenation or sulfo group and then connecting the activated ascorbic acid-6-hydroxyl with sulfydryl of glutathione through thioether. The invention also discloses derivatives such as salts, esters, N-acylates, N-alkylates and the like of the compound. The stability of the series of compounds is greatly improved compared with that of vitamin C, and the whitening effect is obviously better than that of vitamin C and glutathione monomers. The invention also discloses a preparation method of the series of compounds and application of the series of compounds as antioxidants, whitening agents, nutritional supplements and the like in cosmetics, foods, feeds and medicines.

Description

Technical Field

[0001] The present invention relates to the field of chemical synthesis and application, and in particular to a new structure of an ascorbic acid and glutathione conjugate, a preparation method thereof, and application thereof in cosmetics, food, feed, and medicine. Background Art

[0002] In the field of skin whitening and anti-oxidation, vitamin C (L-ascorbic acid) has long occupied a core position as a whitening raw material due to its excellent tyrosinase inhibitory activity and reducing ability. It can effectively reduce melanin production by 62%-78% by reducing melanin intermediates and blocking the dopaquinone conversion pathway. However, this ingredient has significant application limitations: in aqueous solution, the half-life of VC is only 1-2 hours (pH=5, 25℃), and the degradation rate increases by 3-5 times when exposed to light, heat or metal ions, resulting in a retention rate of less than 30% of active substances in actual use. This characteristic makes traditional VC preparations face three major technical bottlenecks: (1) It needs to rely on complex encapsulation technology to maintain stability, but nanoliposome encapsulation can cause the transdermal rate to drop by more than 40%; (2) It is easy to cause pH fluctuations in the emulsified system, resulting in abnormal formula viscosity and phase separation risks; (3) The dehydroascorbic acid produced under light conditions does not have whitening activity and may instead induce oxidative stress reactions.

[0003] Current whitening products on the market generally suffer from efficacy shortcomings. Traditional ingredients like arbutin and kojic acid face transdermal absorption barriers (skin retention rates >85%), making them difficult to penetrate the epidermal barrier. Ingredients like niacinamide, while capable of regulating melanin transport, are ineffective in removing established spots. Even prototype VC products, touted as "fast-acting whitening," showed in human testing that after four weeks of continuous use, spot reduction was only 15%-22%, accompanied by noticeable darkening. More notably, existing products often focus on a single pathway (such as simply inhibiting tyrosinase or blocking melanin transport), failing to address the full chain of melanin production, transport, and metabolism. According to Euromonitor data, in 2022, 67% of global whitening market complaints cited "lack of lasting effects" and 49% "reappearance of dark spots after use," highlighting a market gap for long-lasting, stable whitening agents.

[0004] The essence of this technical dilemma stems from the disconnect between molecular design and delivery systems: traditional whitening ingredients suffer from (1) environmental sensitivity due to the exposure of active groups; (2) metabolic compensation effects caused by a single target; and (3) concentration cliff-like attenuation caused by a linear release curve. To achieve the synergistic effect of "high stability-strong penetration-multi-target", it is urgent to develop a new delivery system with intelligent response characteristics. Through biomimetic structural design, it can achieve gradient release and continuous replenishment of active ingredients. At the same time, it can integrate multiple whitening pathway regulatory factors to build a closed-loop whitening system from source inhibition to metabolic acceleration. Summary of the Invention

[0005] In order to solve the above-mentioned defects and deficiencies in the prior art, the present invention provides an ascorbic acid-S-glutathione conjugate and derivatives, a preparation method and applications thereof.

[0006] To solve the above technical problems, the present invention provides an ascorbic acid-S-glutathione conjugate, which is obtained by activating the 6-hydroxyl group of ascorbic acid with a halogen or sulfonic acid group and then connecting it to the sulfhydryl group of glutathione through a thioether. The structural formula is shown in formula (I): (I) In the above formula, R1 and R2 are each an H atom, or sodium, potassium, calcium, magnesium, zinc, triethanolamine salt, or a C1-C30 alkyl group; R3 and R4 are each an H atom, or a C1-C30 alkyl group; R5 and R6 are each an H atom, or sodium, potassium, calcium, magnesium, zinc, or a C1-C30 alkyl group; or a C1-C30 alkanoyl group.

[0007] Furthermore, the C1-C30 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, lauryl or benzyl; The C1-C30 alkanoyl group is acetyl, benzoyl, lauroyl, palmitoyl, BOC or Fomoc.

[0008] The present invention also provides a derivative of an ascorbic acid-S-glutathione conjugate, wherein the derivative is a salt, an ester, an N-acylated product or an N-alkylated product of the ascorbic acid-S-glutathione conjugate.

[0009] The present invention also provides a method for preparing an ascorbic acid-S-glutathione conjugate, comprising the following steps: The 6-hydroxyl group of ascorbic acid is activated by halogenation or sulfonic acid, and the activated ascorbic acid is reacted with glutathione to form a thioether bond between the activated group at the 6-position of ascorbic acid and the sulfhydryl group of glutathione, thereby obtaining an ascorbic acid-S-glutathione conjugate.

[0010] Furthermore, the activated ascorbic acid reacts with glutathione at a reaction temperature of 0-80° C., a reaction time of 1-24 hours, and an organic solvent.

[0011] Furthermore, the N-acylated compound is prepared by subjecting an ascorbic acid-S-glutathione conjugate to an N-acylation reaction.

[0012] Furthermore, the N-alkylated compound is prepared by subjecting an ascorbic acid-S-glutathione conjugate to an N-alkylation reaction.

[0013] The present invention also provides the use of the ascorbic acid-S-glutathione conjugate or a derivative of the ascorbic acid-S-glutathione conjugate in the preparation of cosmetics.

[0014] The present invention also provides the use of the ascorbic acid-S-glutathione conjugate or a derivative of the ascorbic acid-S-glutathione conjugate in the preparation of food, feed or medicine.

[0015] The beneficial technical effects achieved by the present invention are as follows: The present invention provides an ascorbic acid-S-glutathione conjugate, which converts the 6-hydroxyl group of ascorbic acid (vitamin C) into a highly reactive group through specific chemical modification, and forms a stable thioether bond with the sulfhydryl group of glutathione. The conjugate comprises the following core structural features: (1) the ascorbic acid nucleus remains intact, and its 2,3-ene diol structure is not destroyed; (2) the 6-hydroxyl group is activated by halogenation (-Cl, -Br) or sulfonation (-SO3⁻M⁺, M=Na / K); (3) a directional connection with the cysteine ​​residue in the glutathione molecule is achieved through a thioether bond (-S-CH2-). The compound also includes its pharmaceutically acceptable salt form (such as sodium salt, magnesium salt), esterified derivatives (C1-C6 fatty acid esters), N-acylated products (acetyl, succinyl modification) and N-alkylated derivatives (methyl, benzyl substitution) and other derivatives.

[0016] In terms of the synthesis process, the present invention adopts a two-step method to accurately construct the target molecule: first, the 6-hydroxyl group of ascorbic acid is activated by selective halogenation or sulfonation, preferably in a mixed solvent of acetic acid and pyridine / ethyl acetate, using hydrogen bromide / acetic acid, hydrogen chloride / acetic acid, phosphorus tribromide / acetic acid, N-chlorosuccinimide (NCS), or N-bromosuccinimide (NBS) as halogenation reagents, and controlling the temperature at 0-35°C to achieve regioselective substitution; then, in a weakly alkaline buffer system (pH 7.5-8.5), a nucleophilic substitution reaction is carried out using the activated ascorbic acid and the sulfhydryl group of glutathione, and the target product with a purity of >98% is obtained after purification by silica gel column chromatography.

[0017] This series of compounds exhibits significant performance advantages: significant improvements in stability, anti-free radical, whitening effects, etc.

[0018] The compound of the present invention has multi-dimensional application value: in the cosmetics field, it can be used as a multifunctional additive in essences, emulsions and other products, with antioxidant, whitening and skin barrier repair functions; in the food industry, it can be used as a natural color preservative to effectively maintain the color stability of fruit and vegetable juices; in the pharmaceutical field, it can be developed into a transdermal preparation for the treatment of chloasma. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0020] The present invention is further described below with reference to the embodiments.

[0021] Example 1: Synthesis of Ascorbic Acid-S-Glutathione (GSH-VC)

[0022] Under nitrogen, add 10g of ascorbic acid (purity ≥99%) and 100mL of glacial acetic acid to the flask in sequence. Stirring is initiated, and the temperature is maintained below 10°C in an ice bath. Slowly add 50g of a 40% hydrogen bromide / acetic acid solution dropwise over approximately 1 hour. The temperature is then slowly raised to 25°C, and the reaction is continued for 4.5 hours (TLC monitoring indicates the disappearance of ascorbic acid spots). Concentrate under reduced pressure, add 50mL of ethyl acetate, and stir for 2 hours to precipitate an off-white solid. Filter, wash with ethyl acetate, and vacuum dry to obtain an off-white crystalline solid for later use. The reaction process is as follows:

[0023] The above bromide and 15 g of glutathione were dissolved in 20 g of water. 25 mL of methanol and 11.5 g of sodium bicarbonate were added, and the mixture was reacted at 20°C for 12 hours. After the reaction, the mixture was separated and purified by column chromatography to obtain the ascorbic acid-S-glutathione (GSH-VC) conjugate with a yield of 75.2%. The reaction process is shown below:

[0024] Example 2: Synthesis of Ascorbic Acid-S-Glutathione (GSH-VC) Under nitrogen, add 10g of ascorbic acid and 200mL of glacial acetic acid to the flask in sequence. Stirring is initiated, and the temperature is maintained below 10°C in an ice bath. Slowly add 12g of phosphorus tribromide (PBr3) dropwise over approximately 1 hour. The temperature is raised to 25°C, and the reaction is stirred for 6 hours (TLC monitoring indicates the disappearance of ascorbic acid spots). The solution is concentrated, and 50mL of ethyl acetate is added. Stirring precipitates an off-white solid, which is filtered, washed with ethyl acetate, and vacuum-dried to obtain an off-white crystalline solid for later use. The reaction process is as follows:

[0025] The activated solution was concentrated, and 16.5 g of glutathione dissolved in 25 g of water was added. 50 mL of ethanol and 45 g of triethylamine were then added. The reaction was allowed to react at room temperature for 8 hours. After TLC monitoring, column chromatography was used to separate the ascorbic acid-S-glutathione (GSH-VC) conjugate with a yield of 83.5%. The reaction process is shown below:

[0026] Example 3: Synthesis of Ascorbic Acid-S-Glutathione (GSH-VC) Dissolve 10g of ascorbic acid in 50mL of glacial acetic acid, add 25g of NBS, and stir at 25°C for 7 hours to complete the bromination activation of the 6-hydroxyl group of ascorbic acid. The reaction process is as follows:

[0027] The activated solution was concentrated and then reacted with 15g of glutathione dissolved in 20g of water and an appropriate amount of sodium carbonate in methanol at 40°C for 12 hours. After the reaction, the product was separated and purified by column chromatography to obtain the ascorbic acid-S-glutathione (GSH-VC) conjugate with a yield of 75.9%. The reaction process is shown below:

[0028] Example 4: Synthesis of Ascorbic Acid-S-Glutathione (GSH-VC) Dissolve 10g of ascorbic acid in 100mL of 40% hydrogen chloride / glacial acetic acid solution and stir at 25°C for 7 hours to complete the chlorination activation of the 6-hydroxyl group of ascorbic acid. The reaction process is as follows:

[0029] The activated solution was concentrated, and 15 g of glutathione was dissolved in 25 g of water. 5.5 g of sodium carbonate was added and stirred to dissolve. 25 mL of methanol was then added and allowed to react at 40°C for 12 hours. After the reaction, the product was separated and purified by column chromatography to obtain the ascorbic acid-S-glutathione (GSH-VC) conjugate with a yield of 69.2%. The reaction process is shown below:

[0030] Example 5: Synthesis of Ascorbic Acid-S-Glutathione (GSH-VC) Sodium Salt

[0031] 5g of the ascorbic acid-S-glutathione (GSH-VC) conjugate prepared in Example 1 was added to a three-necked flask. 100mL of anhydrous methanol was added. With stirring, sodium methoxide solution (pre-prepared as a 20% methanol solution) was slowly added at a rate of 1 drop / second. The reaction temperature was maintained at 10±2°C, and the pH was controlled between 7 and 8. Stirring was continued for 2 hours. The mixture was filtered and vacuum dried at 50°C for 8 hours to obtain 5.1g of a light yellow powdery solid with a yield of 82%. The reaction process is as follows:

[0032] Example 6: Synthesis of N-acetylascorbic acid-S-glutathione (Ac-GSH-VC) 5.0 g of ascorbic acid-S-glutathione (GSH-VC) prepared in Example 1 was added to a three-necked flask and dissolved in 100 mL of dichloromethane. Cool in an ice-water bath. 6.0 mL of acetic anhydride was slowly added dropwise, along with 2.0 mL of N-methylmorpholine. The temperature was maintained at 0-5°C and stirred for 1 hour. 0.1 g of DMAP was added, the mixture was warmed to room temperature, and stirring was continued for 4 hours. After TLC (dichloromethane / methanol = 10:1) revealed the disappearance of the GSH-VC starting material spot, the solvent was removed by rotary evaporation to obtain the crude product. The crude product was dissolved in ethyl acetate and washed sequentially with 5% sodium bicarbonate solution and saturated sodium chloride solution. After separation, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. Purification by column chromatography (dichloromethane / methanol = 20:1) yielded Ac-GSH-VC as a light yellow solid in a 92.1% yield. The reaction process is shown below:

[0033] Example 7: Synthesis of Tetraacetyl Ascorbic Acid-S-Glutathione (tAc-GSH-VC)

[0034] 6.0 g of ascorbic acid-S-glutathione (GSH-VC) prepared in Example 1 was added to a three-necked flask and dissolved in 150 mL of dichloromethane. Cool in an ice-water bath. 10.0 mL of acetic anhydride was slowly added dropwise, along with 3.0 mL of triethylamine. The temperature was maintained at 0-5°C and stirred for 1.5 hours. 0.15 g of DMAP was added, the mixture was warmed to room temperature, and stirring was continued for 5 hours. After TLC (dichloromethane / methanol = 15:1) revealed the disappearance of the GSH-VC starting material spot, the solvent was removed by rotary evaporation to obtain the crude product. The crude product was dissolved in ethyl acetate and washed sequentially with 5% sodium bicarbonate solution and saturated sodium chloride solution. After separation, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. Purification by column chromatography (dichloromethane / methanol = 30:1) yielded tAc-GSH-VC as a light yellow solid in a 97.2% yield. The reaction process is as follows:

[0035] Example 8: N-palmitoyl ascorbyl-S-glutathione (C 16 Synthesis of β-GSH-VC 5.5 g of ascorbic acid-S-glutathione (GSH-VC) prepared in Example 1 was added to a three-necked flask and dissolved in 120 mL of dichloromethane. Cool in an ice-water bath. 8.0 mL of palmitoyl chloride was slowly added dropwise, along with 3.5 mL of triethylamine. The temperature was maintained at 0-5°C and stirred for 2 hours. 0.12 g of DMAP was added, the mixture was warmed to room temperature, and stirring was continued for 6 hours. After TLC (dichloromethane / methanol = 20:1) revealed the disappearance of the GSH-VC starting material spot, the solvent was removed by rotary evaporation to obtain the crude product. The crude product was dissolved in ethyl acetate and washed sequentially with 5% sodium bicarbonate solution and saturated sodium chloride solution. After separation, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. Purification by column chromatography (dichloromethane / methanol = 40:1) yielded C16-GSH-VC as an off-white solid in an 86.3% yield. The reaction process is as follows:

[0036] Example 9: Synthesis of N-octanoyl ascorbic acid-S-glutathione (C8-GSH-VC) 5.0 g of ascorbic acid-S-glutathione (GSH-VC) prepared in Example 1 was added to a three-necked flask and dissolved in 100 mL of dichloromethane. Cool in an ice-water bath. 5.0 mL of octanoyl chloride was slowly added dropwise, along with 3.0 mL of triethylamine. The temperature was maintained at 0-5°C and stirred for 1.5 hours. 0.1 g of DMAP was added, the mixture was warmed to room temperature, and stirring was continued for 5 hours. After TLC (dichloromethane / methanol = 15:1) revealed the disappearance of the GSH-VC starting material spot, the solvent was removed by rotary evaporation to obtain the crude product. The crude product was dissolved in ethyl acetate and washed sequentially with 5% sodium bicarbonate solution and saturated sodium chloride solution. After separation, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. Purification by column chromatography (dichloromethane / methanol = 30:1) yielded C8-GSH-VC as a light yellow solid in a 95.1% yield. The reaction process is shown below:

[0037] Example 10: Synthesis of Ascorbic Acid-S-Glutathione Methyl Ester (GSH-VC-Me)

[0038] Add 5.0 g of ascorbic acid-S-glutathione (GSH-VC) prepared in Example 1 to a three-necked flask, dissolve in 100 mL of dichloromethane, and cool in an ice-water bath. Slowly add a pre-prepared 20% sodium methoxide methanol solution dropwise at a rate of 1 drop / second. Keep the reaction temperature at 10 ± 2°C and the pH between 7 and 8, and stir continuously for 2 hours. Filter and vacuum dry at 50°C for 8 hours to obtain a light yellow powdery solid with a yield of 82.0%. The reaction process is as follows:

[0039] Example 11: Synthesis of Ascorbic Acid-S-Glutathione Ethyl Ester (GSH-VC-Et) 5g of ascorbic acid-S-glutathione (GSH-VC) prepared in Example 1 was added to a three-necked flask, followed by 50mL of anhydrous ethanol and 1g of concentrated sulfuric acid. The mixture was heated under reflux for 6 hours. After the reaction, the mixture was concentrated, extracted with ethyl acetate, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated to obtain ascorbic acid-S-glutathione ethyl ester in a yield of 92.3%. The reaction process is as follows:

[0040] Example 12: Synthesis of N-benzoyl ascorbic acid-S-glutathione (Bz-GSH-VC)

[0041] The ascorbic acid-S-glutathione (GSH-VC) conjugate prepared in Example 1 was dissolved in dimethylformamide, and benzoyl chloride and triethylamine were added. The mixture was reacted at room temperature for 8 hours to carry out the N-acylation reaction. After completion of the reaction, the mixture was filtered, washed, and dried to obtain the ascorbic acid-S-glutathione (GSH-VC) conjugate N-benzoylated with a yield of 68.5%. The reaction process is as follows:

[0042] Example 13: Synthesis of N-butyl ascorbic acid-S-glutathione (Bu-GSH-VC)

[0043] 5 g of the ascorbic acid-S-glutathione (GSH-VC) conjugate prepared in Example 1, 10 mL of deionized water, and 20 mL of ethanol were added sequentially to a flask and stirred until dissolved. N-butyl ascorbic acid-S-glutathione (GSH-VC) conjugate was then added, and the mixture was heated under reflux for 5 hours. The mixture was cooled to 0-5°C, and 6.5 g of sodium borohydride was added in portions. The reaction was allowed to proceed for 1 hour. After a further 3.5 hours at room temperature, sulfuric acid was added to adjust the pH to 2-3. The mixture was then distilled under reduced pressure and separated by column chromatography to obtain N-butyl ascorbic acid-S-glutathione (Bu-GSH-VC) with a yield of 42.1%. The reaction process is shown below:

[0044] Example 14: Synthesis of N-Boc-ascorbic acid-S-glutathione (Boc-GSH-VC)

[0045] 5 g of the ascorbic acid-S-glutathione (GSH-VC) conjugate prepared in Example 1, 30 mL of deionized water, and 20 mL of acetone were added to a flask in sequence, stirred until dissolved, and cooled to 0-5°C. Di-tert-butyl dicarbonate was added portionwise and stirred for 3 hours. After the reaction, the acetone was recovered by vacuum distillation, filtered, washed, and separated by column chromatography to obtain N-Boc-ascorbic acid-S-glutathione (Boc-GSH-VC) with a yield of 57.3%. The reaction process is shown below:

[0046] Example 15: Stability Study The ascorbic acid-S-glutathione (GSH-VC) conjugate prepared in Example 1 of the present invention, standard vitamin C, and glutathione monomer were each precisely prepared with purified water to a 1% (w / v) aqueous solution. Three replicates of each solution were prepared to ensure the reliability of the experimental data. Each sample was placed in a constant temperature and humidity incubator, strictly controlled at 40°C and 75% relative humidity. The active ingredient content of each sample was measured using high-performance liquid chromatography (HPLC) every 7 days, with the experiment lasting up to 42 days. The test results are shown in Table 1. Under the same environmental conditions, the active ingredient content of the vitamin C solution dropped to 40% after 21 days, and the active ingredient content of the glutathione solution dropped to 50% after 28 days. However, the active ingredient content of the GSH-VC conjugate solution of the present invention remained above 80% after 42 days. As shown in Table 1, the GSH-VC conjugate of the present invention exhibits enhanced stability due to the interaction of vitamin C and glutathione.

[0047] Table 1: Changes of active ingredient content over time Time (days) GSH-VC VC GSH 0 100% 100% 100% 7 98% 80% 85% 14 96% 60% 75% 21 94% 40% 60% 28 92% - 50% 35 90% - - 42 ≥80% - -

[0048] Example 16: Melanin Inhibitory Effect Thirty fresh human skin tissue samples from healthy donors were selected, each approximately 0.5 mm thick and 1 cm x 1 cm in size. The samples were randomly divided into three groups of 10 each and placed in culture dishes containing a special culture medium to maintain viability. Each of the three groups of skin samples was then smeared with a cream containing 1% vitamin C, 1% glutathione, and 1% ascorbic acid-S-glutathione (GSH-VC) conjugate prepared in Example 1, once daily for 28 days. During the incubation period, the culture dish environment was strictly controlled at a temperature of 37°C and a humidity of 95%, with a continuous flow of 5% CO2. Using a high-precision skin colorimeter, melanin content in skin samples was measured regularly at the start of the experiment, and on the 7th, 14th, 21st, and 28th days. The results showed that melanin content in skin samples treated with vitamin C decreased by 15%, and that in skin samples treated with glutathione decreased by 20%. Furthermore, melanin content in skin samples treated with the GSH-VC conjugate of the present invention decreased significantly by 40%, demonstrating that the conjugate of the present invention exhibits significant whitening efficacy, significantly outperforming existing products.

[0049] Example 17: Scavenging effect of DPPH

[0050] The classic DPPH method was used to evaluate the free radical scavenging ability of the ascorbic acid-S-glutathione (GSH-VC) conjugate prepared in Example 2 of the present invention. A 0.1 mM solution of the GSH-VC conjugate was precisely prepared, along with a vitamin C solution of the same concentration as a control. The conjugate solution and the vitamin C solution were each mixed with a DPPH ethanol solution in a specific proportion. After reacting at room temperature in the dark for 30 minutes, the absorbance was measured at 517 nm using a UV-visible spectrophotometer. The scavenging rate was calculated according to the formula, as shown in Table 2. The DPPH scavenging rate of the 0.1 mM GSH-VC conjugate was as high as 92.5%, while the vitamin C control had a scavenging rate of 65.3%. This demonstrates that the conjugate of the present invention possesses enhanced free radical scavenging ability and significantly improved antioxidant efficacy.

[0051] Table 2: Free radical scavenging rate (DPPH method) data table

[0052] Example 18: Inhibition of tyrosinase In order to verify the inhibitory activity of the ascorbic acid-S-glutathione (GSH-VC) conjugate prepared in Example 3 of the present invention on tyrosinase, a colorimetric method was used for quantitative analysis.

[0053] Reagents and instruments: Sample: GSH-VC conjugate (prepared according to the method of Example 3, purity ≥98%); Reference substance: arbutin (positive control, purity ≥99%); Enzyme source: mushroom tyrosinase (EC 1.14.18.1, Sigma, activity ≥2000 U / mg); Substrate: L-dopa (Sigma, purity ≥99%); Instrument: UV-visible spectrophotometer (model: UV-2600, wavelength accuracy ±1 nm).

[0054] Experimental process: 1) Dissolve GSH-VC conjugate and arbutin in phosphate buffered saline (PBS, pH 6.8, 0.1 M) to prepare solutions with final concentrations of 5, 10, 20, 40, and 80 μM.

[0055] 2) Take 2.0 mL of sample solution of different concentrations, add 50 μL of tyrosinase solution (100 U / mL), mix well, and preincubate in a 37°C constant temperature water bath for 10 min. Then, add 50 μL of L-DOPA solution (2 mM), mix well, and start the reaction.

[0056] 3) Use PBS instead of enzyme solution to eliminate interference from substrate autooxidation; use PBS instead of sample solution to determine the baseline of enzyme activity.

[0057] Detection method: Absorbance measurement: The absorbance of the reaction system was continuously monitored at 475 nm, and the linear growth slope of the OD value within 3 min (ΔOD / min) was recorded.

[0058] Calculation of inhibition rate:

[0059] IC50 value determination: Dose-inhibition rate curves were fitted using GraphPad Prism software, and the half-maximal inhibitory concentration (IC50) was calculated.

[0060] Experimental results: GSH-VC conjugate: IC50 = 18.7 ± 1.2 μM (n = 3), concentration-dependent inhibition; Arbutin control: IC50=42.3±2.1μM (n=3); Blank control: absorbance change in the PBS group ≤ 5% (excluding interference from non-enzymatic reactions).

[0061] Conclusion: The inhibitory activity of GSH-VC conjugate against tyrosinase was significantly stronger than that of arbutin (p < 0.01), and its IC50 value was only 44% of that of the latter. This indicates that the conjugate has a highly effective tyrosinase inhibitory ability and can be used as a potential whitening agent in the cosmetic field.

[0062] Example 19: Preparation and efficacy verification of whitening essence The ascorbic acid-S-glutathione (GSH-VC) conjugate prepared in Example 7 was used for formula optimization and efficacy verification of a whitening essence. The formula of the whitening essence is shown in Table 3 below.

[0063] Table 3

[0064] Preparation of aqueous phase: Heat deionized water to 70°C, add glycerin, butylene glycol, and sodium hyaluronate, stir until completely dissolved, and keep warm for later use.

[0065] Dispersion of active ingredients: Slowly add the GSH-VC conjugate (sieved to 80 mesh) into the aqueous phase and stir homogenously (1000 rpm, 10 min) until uniformly dispersed.

[0066] Preservation and filling: After cooling to 40℃, add phenoxyethanol and ethylhexylglycerin, stir evenly, and aseptically fill into brown light-proof bottles.

[0067] Efficacy verification: Volunteer screening: Inclusion criteria: 50 healthy women, aged 20-45 years, Fitzpatrick skin type III-IV, dark skin color on face and neck (melanin index ≥180), no history of skin disease or allergy.

[0068] Experimental Design: Test area: Symmetrical areas on both cheeks (approximately 4 cm² in area).

[0069] Grouping: GSH-VC essence group (experimental group), basic formula group (without GSH-VC) (control group).

[0070] Usage: Apply after cleansing every morning and evening, massage gently until absorbed, for 28 consecutive days.

[0071] Detection indicators: Melanin index: L (lightness), a (red-green), and b (yellow-blue) values ​​were measured using a CM-2600d spectrophotometer (Minolta), and the melanin content was calculated (Melanin Index = 100 × [(L control group - L experimental group) + (b control group - b* experimental group)] / 100).

[0072] Subjective evaluation: Volunteers rated skin tone improvement, translucency, and satisfaction on a 10-point scale.

[0073] Results and statistics: Melanin index: The experimental group decreased by an average of 35.2% (p < 0.01), and the control group decreased by 8.7% (p > 0.05).

[0074] Subjective rating: 90% of the volunteers believed that the skin tone on the experimental side was more even and the translucency was improved, and 85% expressed their willingness to use it long-term.

[0075] Conclusion: This whitening essence, with GSH-VC conjugate as its core active ingredient, is optimized through formulation and processing to ensure stability and skin feel. Human trials have demonstrated that it significantly reduces melanin production (p < 0.01), improves skin brightness and radiance, and demonstrates a good safety profile.

[0076] Example 20: Beverage Preparation The ascorbic acid-S-glutathione (GSH-VC) conjugate prepared in Example 5 was applied to orange juice beverage.

[0077] Formula and process: Addition amount: GSH-VC conjugate is added at 0.05% (w / v) of the total mass of the beverage (i.e. 10 mg / 200 mL).

[0078] Preparation process: Orange juice (soluble solids ≥11°Bx) was mixed with GSH-VC solution (pre-dissolved in a small amount of warm water), homogenized (20 MPa, 2 times), and pasteurized (65°C, 30 min) after filling.

[0079] Stability test: Accelerated conditions: The samples were placed in a thermostat at 37°C and 75% relative humidity, protected from light for 12 weeks.

[0080] Detection indicators: Color: L, a, and b* values ​​were measured every 2 weeks using a colorimeter (CR-400, Konica Minolta) and changes in ΔE were observed.

[0081] Component analysis: HPLC (C18 column, mobile phase 0.1% phosphoric acid-methanol gradient elution) was used to detect the retention rates of ascorbic acid and glutathione.

[0082] Antioxidant activity: evaluated by DPPH free radical scavenging assay (λ=517 nm) and FRAP assay (Fe³⁺ reducing ability).

[0083] result: Color stability: ΔE≤1.5 (unnoticeable change to the human eye), control group ΔE=3.2.

[0084] Ingredient retention rate: ascorbic acid retention rate 95.2%, glutathione retention rate 91.8%.

[0085] Antioxidant activity: The DPPH scavenging rate increased by 28.6% compared with the control group (p<0.01), and the FRAP value increased by 1.5 times.

[0086] Conclusion: GSH-VC conjugate significantly improves the antioxidant activity and stability of beverages and is suitable for heat-sensitive beverage systems.

[0087] Example 21: Feed Additive The GSH-VC conjugate prepared in Example 4 was added to tilapia feed.

[0088] Feed preparation: basic feed, crude protein 32%, crude fat 6%, cellulose 5% (in line with the nutritional standards of tilapia); experimental group, added 0.1% (w / w) GSH-VC conjugate (sieved to 80 mesh to ensure uniform mixing); control group: an equal amount of maltodextrin replaced the conjugate.

[0089] Animal experiment: Sixty healthy tilapia (initial body weight ≈50 g) were randomly divided into two groups (n=30) and cultured in a recirculating water system (water temperature 28±1°C, dissolved oxygen ≥5 mg / L). They were fed twice daily (8:00 and 16:00) at a feed intake of 3% of their body weight for 30 days.

[0090] Antioxidant enzyme activity was assessed by homogenizing liver tissue and centrifuging it at 4°C, 3000 rpm, for 10 min. SOD (WST-1 assay) and GSH-Px (DTNB assay) activities were measured using kits. Meat quality analysis involved measuring L, a, and b values ​​of back muscles after slaughter using a colorimeter. The redness value (a / L*) was calculated. Data were analyzed using SPSS t-tests, with a significance level of p < 0.05.

[0091] Results: The SOD activity in the experimental group (125.3±8.7 U / mg prot) vs the control group (96.2±6.5 U / mg prot) was 30.3% (p=0.002).

[0092] GSH-Px activity: experimental group (45.1±3.2 U / mg prot) vs control group (35.9±2.8 U / mg prot), 25.6% (p=0.004).

[0093] Fish flesh color: a / L value of the experimental group was 0.28±0.03, and that of the control group was 0.24±0.02, with redness increased by 16.7% (p=0.011).

[0094] Conclusion: GSH-VC conjugate can effectively enhance the antioxidant capacity and improve the meat quality of tilapia, and is recommended as an aquatic feed additive.

[0095] Example 22: Tablet Preparation The GSH-VC prepared in Example 1 was developed into oral tablets for the treatment of chloasma. The specific process is as follows: Preparation formula: GSH-VC 0.5 g / tablet (purity ≥98%); microcrystalline cellulose (filler, 30 mg), sodium starch glycolate (disintegrant, 5 mg), magnesium stearate (lubricant, 1 mg).

[0096] Preparation process: wet granulation (80% ethanol to make soft material), tableting pressure 150 N, tablet weight controlled at 0.6±0.02 g.

[0097] Participants: 50 patients with melasma (Fitzpatrick type III-IV, MI ≥ 150), aged 25-55 years, were randomly divided into the experimental group (n = 25) and the placebo group (n = 25) in a double-blind manner.

[0098] Dosage regimen: 1 tablet twice a day for 12 weeks.

[0099] Evaluation indicators: Pigmentation index: The IT / EI ratio was measured using Mexameter® MX18 (CK Company).

[0100] Results: The MI decreased by 42.3%±5.7% in the experimental group (p<0.01), and by 7.8%±3.2% in the placebo group (p>0.05); 70% of patients achieved ≥30% pigment improvement (VS 12% in the placebo group).

[0101] Example 23: Patch Preparation The C16-GSH-VC hydrogenated gel prepared in Example 8 was developed into a melasma patch, and the specific research was as follows: Patch preparation: 2% C16-GSH-VC gel (Carbomer 940 as base, glycerin as humectant, pH=6.5) was applied to non-woven fabric (drug loading 4.2 mg / cm²), covered with a release layer, and wrapped in aluminum foil.

[0102] Clinical trial: 30 female patients with melasma (area ≥ 2 cm², MI ≥ 180), aged 30-50 years, with bilateral facial self-control (one side with patch and one side with blank patch). Five times a week, before bedtime, for 6 hours after cleansing, for 8 weeks.

[0103] Detection method: The stratum corneum was collected by tape stripping method, and quantified by HPLC after methanol extraction; pigment improvement: VISIA-CR imaging system was used to analyze the MI and the proportion of erythema area; tolerability: stinging and erythema scores (grade 0-3).

[0104] Results: Retention amount, 4.2±0.3 μg / cm² (consistent with in vitro release); efficacy, MI on the treated side decreased by 68.2% (p<0.001), and the erythema area decreased by 52.7%; safety: 93% of the subjects had no irritation (VS 100% in the blank patch group).

[0105] Conclusion: C16-GSH-VC can improve melasma pigmentation in a targeted manner.

[0106] It should be noted that the experimental methods used to verify the effects of Examples 15 to 23 are general methods, and other methods can also be used for verification.

[0107] The present invention has been disclosed above with preferred embodiments, which are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. An ascorbic acid-S-glutathione conjugate, characterized in that: The conjugate is obtained by activating the 6-hydroxyl group of ascorbic acid with a halogen or sulfonic acid group, and then connecting it to the sulfhydryl group of glutathione through a thioether. The structural formula is shown in formula (I): （I） In the above formula, R1 and R2 are each an H atom, or sodium, potassium, calcium, magnesium, zinc, triethanolamine salt, or a C1-C30 alkyl group; R3 and R4 are each an H atom, a C1-C30 alkanoyl group, or a C1-C30 alkyl group; R5 and R6 are each an H atom, or sodium, potassium, calcium, magnesium, zinc, a C1-C30 alkanoyl group, or a C1-C30 alkyl group.

2. The ascorbic acid-S-glutathione conjugate according to claim 1, wherein: The C1-C30 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, lauryl or benzyl; The C1-C30 alkanoyl group is acetyl, benzoyl, lauroyl, palmitoyl, BOC or Fomoc.

3. A derivative of an ascorbic acid-S-glutathione conjugate, characterized in that: The derivative is a salt, an ester, an N-acylated compound or an N-alkylated compound of the ascorbic acid-S-glutathione conjugate.

4. A method for preparing an ascorbic acid-S-glutathione conjugate, characterized in that The following steps are involved: The 6-hydroxyl group of ascorbic acid is activated by halogenation or sulfonic acid, and the activated ascorbic acid is reacted with glutathione to form a thioether bond between the activated group at the 6-position of ascorbic acid and the sulfhydryl group of glutathione, thereby obtaining an ascorbic acid-S-glutathione conjugate.

5. The method for preparing the ascorbic acid-S-glutathione conjugate according to claim 4, wherein: The activated ascorbic acid reacts with glutathione at a reaction temperature of 0-80° C., a reaction time of 1-24 hours, and an organic solvent.

6. The derivative of the ascorbic acid-S-glutathione conjugate according to claim 3, characterized in that: The N-acylated compound is prepared by subjecting an ascorbic acid-S-glutathione conjugate to an N-acylation reaction.

7. The derivative of the ascorbic acid-S-glutathione conjugate according to claim 3, characterized in that: The N-alkylated compound is prepared by subjecting ascorbic acid-S-glutathione conjugate to N-alkylation reaction.

8. Use of the ascorbic acid-S-glutathione conjugate according to any one of claims 1 to 2, or a derivative of the ascorbic acid-S-glutathione conjugate according to claim 3, in the preparation of cosmetics.

9. Use of the ascorbic acid-S-glutathione conjugate according to any one of claims 1 to 2, or a derivative of the ascorbic acid-S-glutathione conjugate according to claim 3, in the preparation of food, feed or medicine.

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