Ascorbic acid-glutathione co-crystal and a preparation method thereof

Abstract

The application discloses ascorbic acid-glutathione co-crystals and a preparation method thereof, and belongs to the technical field of drug co-crystals.The ascorbic acid and glutathione are mixed according to a molar ratio of 1-5:1-5 to form ascorbic acid-glutathione co-crystals.The ascorbic acid-glutathione co-crystals prepared by the application have improved whitening performance, antioxidant performance, anti-glycation performance and transdermal rate, and the problem of poor stability of ascorbic acid in the prior art is solved.

Description

Technical Field

[0001] This invention relates to an ascorbic acid-glutathione cocrystal and its preparation method, belonging to the field of pharmaceutical cocrystal technology. Background Technology

[0002] Ascorbic acid is a natural, water-soluble vitamin that effectively improves dull skin, provides antioxidant benefits, and promotes collagen synthesis. However, its extremely low stability and poor transdermal absorption severely limit its application prospects. In practical applications, it is usually prepared as a serum or lotion with a mass concentration of 5-10%. However, such high-concentration formulations can be irritating and may cause skin stinging, redness, and other symptoms, making them unsuitable for sensitive skin. Furthermore, high-concentration formulations exhibit poor stability of ascorbic acid, making it highly susceptible to oxidation and inactivation, thus limiting its skin penetration.

[0003] Glutathione is an active peptide widely found in human cells. Its main pharmacological effects include scavenging free radicals, antioxidation, detoxification, immune regulation, and inhibition of melanin production. Studies have shown that glutathione can effectively quench reactive oxygen species, inhibit tyrosinase activity, and reduce melanin synthesis. In the cosmetics industry, glutathione is widely used in skincare products for whitening, brightening skin tone, and improving dullness due to its excellent antioxidant and skin-lightening properties. Glutathione can accelerate the excretion of metabolic waste from the skin, improve uneven skin tone and dullness, and also has the potential to soothe skin irritation and enhance the skin barrier function, gradually becoming one of the important representatives of natural active ingredients.

[0004] Currently, patent CN 120590459 A discloses an ascorbic acid-S-glutathione conjugate and its derivatives, preparation method and application; specifically, the conjugate is obtained by activating the 6-position hydroxyl group of ascorbic acid with a halogenated or sulfonic acid group, and then linking it with the thiol group of glutathione through a thioether; the stability of the conjugate is significantly improved compared with ascorbic acid, and the whitening effect is significantly better than that of the individual monomers of ascorbic acid and glutathione; however, it requires a chemical reaction and the use of organic solvents; and the operation is complicated.

[0005] A eutectic refers to a crystal structure formed by two or more molecules bound together in a specific stoichiometric ratio through weak interactions such as hydrogen bonds, electrostatic attraction, and hydrophobic interactions. Among these interactions, hydrogen bonds play a crucial role in the formation of eutectics due to their high directionality, specificity, and strong stability. Currently, no eutectic structures of ascorbic acid and glutathione have been found.

[0006] Therefore, by combining ascorbic acid and glutathione in a co-crystal, an ascorbic acid-glutathione co-crystal system can be prepared, which can improve its stability and play a synergistic role, thus having extremely high practical and economic value. Summary of the Invention

[0007] [Technical Issues] High-concentration formulations of ascorbic acid are highly irritating, have poor stability, are easily oxidized and deactivated, and have limited skin penetration.

[0008] Currently, no co-crystals of ascorbic acid and glutathione have been found.

[0009] [Technical Solution] To address the aforementioned problems, this invention provides an ascorbic acid-glutathione cocrystal and its preparation method. Specifically, this invention mixes ascorbic acid and glutathione at a molar ratio of 1-5:1-5 to form an ascorbic acid-glutathione cocrystal. The ascorbic acid-glutathione cocrystal prepared by this invention exhibits significantly improved whitening properties, antioxidant properties, anti-glycation properties, and transdermal penetration rate; and it also solves the problem of poor stability of ascorbic acid in existing technologies.

[0010] The first objective of this invention is to provide an ascorbic acid-glutathione cocrystal, the structural formula of which is as follows:

[0011] or

[0012] or

[0013] or

[0014] A second objective of this invention is to provide a method for preparing ascorbic acid-glutathione cocrystals, comprising the following steps: Method 1: Ascorbic acid and glutathione were mixed in a molar ratio of 1~5:1~5 and ground to obtain ascorbic acid-glutathione cocrystal. Method 2: Ascorbic acid and glutathione were mixed in a molar ratio of 1~5:1~5; then dissolved in water, the water was removed, and the mixture was ground to obtain ascorbic acid-glutathione cocrystal. Method 3: Ascorbic acid and glutathione were mixed in a molar ratio of 1~5:1~5; then dissolved in water, frozen, the water was removed, and the mixture was ground to obtain ascorbic acid-glutathione cocrystal. Method 4: Ascorbic acid and glutathione were mixed in a molar ratio of 1~5:1~5 to obtain ascorbic acid-glutathione cocrystal.

[0015] In one embodiment of the present invention, the molar ratio of ascorbic acid to glutathione in the method for preparing ascorbic acid-glutathione cocrystal is 1~4:1~4, preferably 2~4:1, and specifically, 2:1, 3:1, or 4:1 can be selected.

[0016] In one embodiment of the present invention, the grinding in Method 1 is carried out at 15~35℃ (room temperature) and 80~120r / min for 1~10h; preferably, the grinding time is 1~5h; more preferably, the grinding time is 2~4h, and specifically, 2, 3, or 4h can be selected.

[0017] In one embodiment of the present invention, the ratio of the sum of the moles of ascorbic acid and glutathione to the amount of water in Method 2 is 0.01~0.1mol:500~1000g.

[0018] In one embodiment of the present invention, the water removal in Method 2 is carried out by rotary evaporation, specifically rotary evaporation at 30~70°C for 2~10 hours; preferably, the temperature is 40~60°C and the time is 2~5 hours; more preferably, the temperature is 45~55°C and the time is 2~4 hours; specifically, the temperature can be 45, 50, or 55°C and the time can be 2, 3, or 4 hours.

[0019] In one embodiment of the present invention, the grinding in Method 2 is performed at 15~35℃ (room temperature) and 80~120r / min for 5-10 minutes; specifically, the temperature can be 15, 25, or 35℃, the grinding speed can be 80, 100, or 120r / min, and the grinding time can be 5, 8, or 10 minutes.

[0020] In one embodiment of the present invention, the ratio of the sum of the moles of ascorbic acid and glutathione to the amount of water in Method 3 is 0.01~0.1mol:500~1000g.

[0021] In one embodiment of the present invention, freezing in method three is freezing at -80~-60°C for 40~60 hours.

[0022] In one embodiment of the present invention, the moisture removal in method three is achieved by freeze drying, specifically freeze drying at -70 to -40°C for 5 to 30 hours; preferably, freeze drying at -60 to -40°C for 5 to 20 hours; more preferably, freeze drying at -60 to -50°C for 10 to 20 hours; specifically, the temperature can be -60, -55, or -50°C, and the time can be 10, 15, or 20 hours.

[0023] In one embodiment of the present invention, the grinding in Method 3 is performed at 15~35℃ (room temperature) and 80~120r / min for 5~10min; specifically, the temperature can be 15, 25, or 35℃, the grinding speed can be 80, 100, or 120r / min, and the grinding time can be 5, 8, or 10min.

[0024] In one embodiment of the present invention, the mixing in Method 4 is carried out by stirring at 15~35℃ (room temperature) and 80~120r / min for 5~10min.

[0025] In one embodiment of the present invention, the particle size of the ascorbic acid-glutathione cocrystal is 4~12 μm.

[0026] The third objective of this invention is the application of the ascorbic acid-glutathione cocrystal described herein in the preparation of cosmetics or pharmaceuticals.

[0027] In one embodiment of the present invention, cosmetics include serums, lotions, and creams.

[0028] In one embodiment of the present invention, the dosage form of the medicine includes tinctures, liniments, lotions, oils, ointments, creams, gels, sprays, and film-forming agents.

[0029] The fourth objective of this invention is to provide a product that combines whitening, antioxidant, and anti-glycation properties, which utilizes the ascorbic acid-glutathione co-crystal described in this invention.

[0030] In one embodiment of the present invention, the product is an essence, lotion, face cream, shampoo, facial cleanser, etc.

[0031] The fifth objective of this invention is to provide a method for improving the stability of ascorbic acid, which employs the ascorbic acid-glutathione co-crystal described in this invention.

[0032] The sixth objective of this invention is to provide a method for simultaneously improving the whitening activity, antioxidant properties, anti-glycation properties, and transdermal penetration rate of ascorbic acid, which employs the ascorbic acid-glutathione co-crystal described in this invention.

[0033] [Beneficial Effects] (1) The ascorbic acid-glutathione cocrystal of the present invention significantly improves the stability of ascorbic acid, and at the same time, through the synergistic effect with glutathione, it significantly enhances the permeability, whitening activity, antioxidant properties, anti-glycation and other effects of ascorbic acid.

[0034] (2) The ascorbic acid-glutathione co-crystal system prepared in this invention significantly improves the physicochemical properties of ascorbic acid, including stability and transdermal performance, without changing the molecular structure of the active ingredient of the drug (ascorbic acid), thereby improving the bioavailability of the drug (ascorbic acid) and playing a synergistic role.

[0035] (3) The ascorbic acid-glutathione co-crystal system prepared by the present invention has good stability, is not easy to decompose, and is easy to store and transport.

[0036] (4) The ascorbic acid-glutathione co-crystal system prepared by the present invention is green and pollution-free, and the process is simple. It is suitable for industrial promotion, and the product can be used directly as a raw material for drugs or cosmetics.

[0037] (5) The ascorbic acid-glutathione cocrystal prepared by this invention still maintains an effective component content of over 80% after 49 days, and the DPPH and ABTS scavenging rates are both higher than 90%; the cumulative permeation within 24 hours is 4.86 times that of pure ascorbic acid, reaching 12.54 mg / cm³. 2 The inhibition rate of AGEs reached over 20%. Attached Figure Description

[0038] Figure 1 The image shows the Gaussian plot of the ascorbic acid-glutathione cocrystal of Example 1; where (a) and (b) are the electrostatic potential analysis plot and the intermolecular interaction force analysis plot, respectively.

[0039] Figure 2 The infrared spectrum of the ascorbic acid-glutathione cocrystal of Example 1 is shown.

[0040] Figure 3 Differential scanning calorimetry (DSC) of the ascorbic acid-glutathione cocrystal of Example 1.

[0041] Figure 4 The image shows the powder X-ray diffraction pattern of the ascorbic acid-glutathione cocrystal of Example 1.

[0042] Figure 5 This is a permeability test diagram of the ascorbic acid-glutathione cocrystal of Example 1.

[0043] Figure 6 The image shows the Gaussian plot of the ascorbic acid-glutathione cocrystal of Example 2; where (a) and (b) are the electrostatic potential analysis plot and the intermolecular interaction force analysis plot, respectively.

[0044] Figure 7 The image shows the Gaussian plot of the ascorbic acid-glutathione cocrystal of Example 3; where (a) and (b) are the electrostatic potential analysis plot and the intermolecular interaction force analysis plot, respectively. Detailed Implementation

[0045] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0046] Test method: 1. Gaussian calculation: Density functional theory (DFT) calculations were performed at the B3LYP / 6-311G(d,p) level using Gaussian 16 software. Electrostatic potentials (ESPs) were calculated, and the molecular configurations of ascorbic acid and glutathione were constructed. Independent gradient models based on Hirshfeld partitioning (IGMH) and atoms-in-molecular (AIM) methods were used. Intermolecular interactions in Examples 1-3 were analyzed using Multiwfn and VMD software. VMD 1.9.4 was used to process and graphically display the results.

[0047] 2. Stability Test: A 1% (w / v, g / 100 mL) aqueous solution was precisely prepared using deionized water, with three replicates for each solution to ensure the reliability of the experimental data. All samples were placed in a constant temperature and humidity incubator, with environmental conditions strictly controlled at 45℃ and 75% relative humidity. High-performance liquid chromatography (HPLC) was used to detect the content of the active ingredient in each sample every 7 days, with the experimental period lasting up to 49 days.

[0048] 3. Fourier transform infrared spectroscopy test: The parameters are as follows: Machine specifications: Nicolet iS50; Machine manufacturer: Thermo Fisher Scientific, USA; Experimental parameters: Scanning range: 500~4000cm. -1 The resolution is 4cm. -1 .

[0049] 4. Differential scanning calorimetry: The parameters are as follows: Machine specifications: TGA / DSC1 / 1100SF, Machine manufacturer: Mettler Toledo AG, Switzerland, Experimental parameters: Nitrogen atmosphere, flow rate 50 mL / min, temperature range 50~240℃, heating rate 1℃ / min.

[0050] 5. Powder X-ray diffraction test: The parameters are as follows: Machine specifications: D8 model, Manufacturer: Bruker AXS GmbH, Germany, Experimental parameters: Degree range 5~40°, Scanning speed 10° / min.

[0051] 6. Tyrosinase inhibitory activity assay: 30 μL of solutions containing different concentrations (1–100 μg / mL, diluted with deionized water) of the examples, comparative examples, and ascorbic acid, 10 μL of PBS, and 10 μL of tyrosinase solution were mixed as the sample group; 40 μL of PBS and 10 μL of tyrosinase solution were mixed as the control group; and 50 μL of PBS solution was used as the blank group. The mixture was incubated at 37 °C for 10 min, followed by the addition of 10 μL of 1 mg / mL L-DOPA solution, and then incubated again at 37 °C for 10 min.

[0052] The absorbance of the reaction system was measured at 475 nm using a microplate reader. Kojic acid (KA) was used as a positive inhibition control for comparison. The percentage of tyrosinase inhibition was calculated using the following formula, IC50. 50 The values ​​were obtained by fitting using Origin software:

[0053] Among them, A sample A represents the absorbance of the sample solution. blank A represents the absorbance of the blank solution; control The absorbance is the value of the control group solution.

[0054] 7. Determination of free radical scavenging rate: The strength of its antioxidant properties was verified by testing the scavenging efficiency of the sample against DPPH and ABTS free radicals.

[0055] Ascorbic acid and the examples and comparative examples were diluted with methanol to 20 μg / mL to prepare the test sample solution. The scavenging rate of the test sample solution against DPPH and ABTS free radicals was tested according to the method of GB / T39100-2020.

[0056] 8. In vitro skin permeability measurement: In vitro skin permeation tests were performed using a Franz diffusion cell. Undamaged, clean pigskin was placed on the cell with the stratum corneum facing upwards. Ascorbic acid was diluted with deionized water to a concentration of 30 mg / mL as the test sample. The receiving chamber was filled with physiological saline, and 2 mL of each test sample was added to the diffusion cell. The mixture was continuously stirred at a constant temperature (37±0.1℃), and 0.5 mL of the receiving solution was collected at 1, 2, 4, 6, 12, and 24 hours, with an equal volume of fresh receiving solution added. The collected samples were filtered through a 0.45 μm microporous filter and then subjected to HPLC analysis. The cumulative drug permeation was calculated using the following formula:

[0057] Among them, Q S Cumulative drug penetration per unit area (mg / cm²)2 ); C sn V represents the drug concentration (mg / mL) in the receptor fluid measured within the sampling interval. S For the volume of the receptor pool, It is the cumulative drug concentration in the recipient fluid, S is the sampling volume, and A is the cumulative drug concentration in the recipient fluid. S It is the effective diffusion area.

[0058] 9. Determination of the inhibitory effect on human skin melanin: Fresh human skin tissue samples from healthy donors were selected, each approximately 0.5 mm thick and 1 cm × 1 cm in area. Ten samples were placed in each group in petri dishes containing a special culture medium to maintain viability. The skin samples were then coated with either 1% ascorbic acid or 1% ascorbic acid from the examples and comparative studies. The glutathione cocrystal cream was applied once daily for 28 days. During the incubation period, the temperature of the culture dish was strictly controlled at 37 ℃ and the humidity at 95%, with a continuous purging of 5% CO2 gas.

[0059] The cream formula is shown in Table 1 below.

[0060] A high-precision skin colorimeter was used to measure the melanin content in skin samples.

[0061] Table 1

[0062] The specific preparation method of the cream is as follows: (1) Add ingredients 1-3 and 5-8 from the formula to the main pot, heat to 85°C, and homogenize and disperse evenly; (2) Add ingredients 9 to 14 in the formula to the oil pot, heat to 85°C, and stir until completely dissolved; (3) Pump all the substances (i.e., the oil phase) from the oil pot into the main pot, stir and homogenize for 5 minutes, and keep warm at 85°C for 25 minutes; (4) Start cooling down to 55°C and add ingredient No. 4 into the main pot; (5) Continue to cool down to 45°C, add ingredients No. 15-17 into the main pot, and stir to mix evenly; (6) Cool down to below 38°C and discharge to obtain cream.

[0063] 10. Determination of the rate of reduction in skin melanin content after serum use: The examples, comparative examples, and ascorbic acid were applied to the formulation optimization and efficacy verification of the whitening serum. The whitening serum formulation is shown in Table 2 below.

[0064] Table 2

[0065] The specific preparation method for the serum is as follows: (1) Add ingredients 1-3 and 5-8 from the formula to the main pot, heat to 70°C, homogenize and disperse evenly, and keep warm for 30 minutes; (2) Start cooling down to 50°C and add ingredient No. 4 into the main pot; (3) Continue to cool down to 40℃, add raw materials No. 9-10, and stir to mix evenly; (4) Cool down to below 38°C and discharge to obtain the essence.

[0066] Efficacy verification of serum: Volunteer selection: 50 healthy women aged 20-40, Fitzpatrick skin type IIIIV, with dark skin on the face and neck (melanin index ≥180), and no history of skin diseases or allergies.

[0067] Experimental Design: Test area: Symmetrical areas of both cheeks (approximately 4 cm in area) 2 ).

[0068] Grouping: The group using the serum from the example (experimental group) and the group using the basic formula (control group).

[0069] Usage: Apply after cleansing in the morning and evening, and gently massage until absorbed for 28 consecutive days.

[0070] Testing indicators: Using CM The 2600d spectrophotometer (Minolta) measures the L (lightness), a (red-green), and b (yellow-blue) values, and calculates the melanin content using the following formula: Melanin content = 100% × [(L control group)] L experimental group) + (b control group) b experimental group) / 100 11. Anti-glycation assay: Using pH 7.4 phosphate buffer as the matrix, a solution containing 5 g / L bovine serum albumin and 0.1 mol / L glucose (a high-glucose protein solution) was prepared. This solution was used to prepare sample solutions (100 μg / mL) for the examples, comparative examples, and ascorbic acid, respectively. Positive control groups and blank groups were also prepared. The positive control group consisted of aminoguanidine (4 g / L), and the blank group consisted of a high-glucose protein solution without the analyte. All liquids were sterilized by filtration through a 0.2 μm filter and aliquoted into 1.5 mL conical centrifuge tubes. The samples were incubated continuously at 37°C under aerobic and light-protected conditions for 28 days. All samples were then frozen at -20°C.

[0071] Protein glycation products were measured using a fluorescence spectrophotometer with an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The assay was repeated three times, and the inhibition rate was determined according to the following formula: Inhibition rate = (Fluorescence intensity of blank group - Fluorescence intensity of sample group) / Fluorescence intensity of blank group × 100% The content of AGEs (advanced glycation end products) is positively correlated with fluorescence intensity; the higher the inhibition rate, the better the product inhibits AGEs.

[0072] Unless otherwise specified, the solvent used in the test method is water, and unless otherwise specified, the percentage refers to mass percentage.

[0073] Raw materials used in the examples: Ascorbic acid, with a purity of 99.7%, was purchased from Shanghai Haohong Biomedical Technology Co., Ltd. Glutathione, 99% pure, reduced form, purchased from Shanxi Fushengda Biotechnology Co., Ltd.

[0074] Example 1 An ascorbic acid-glutathione cocrystal, the structural formula of which is as follows:

[0075] The preparation method is as follows: 0.03 mol ascorbic acid and 0.01 mol glutathione were mixed evenly and ground at 25℃ (room temperature) and 80 r / min for 2 h to obtain a white solid product, namely: ascorbic acid-glutathione cocrystal (particle size 4~12 μm).

[0076] The obtained ascorbic acid-glutathione cocrystal was subjected to performance testing, and the test results are as follows: (1) Characterization of eutectic structure: Figure 1 The image shows the Gaussian plot of the ascorbic acid-glutathione cocrystal from Example 1; where (a) and (b) are the electrostatic potential analysis plot and the intermolecular interaction force analysis plot, respectively. Figure 1 It can be seen that there are obvious hydrogen bonds, van der Waals interactions and other weak interactions between ascorbic acid and glutathione components. All these interactions jointly promote the formation of ascorbic acid-glutathione cocrystal.

[0077] Figure 2 The image shows the infrared spectrum of the ascorbic acid-glutathione cocrystal from Example 1. Figure 2 It can be seen that the ascorbic acid-glutathione cocrystal in Example 1 is at a temperature of 2800~3600. -1There is only one broader absorption peak within the range, due to the strong hydrogen bond formed between ascorbic acid and glutathione, which covers the triplet peak of the primary alcohol. Meanwhile, the ascorbic acid-glutathione cocrystal shows a peak at 1639°C. -1 The presence of only a characteristic absorption peak from the -C=O group indicates that the formation of hydrogen bonds in the system disrupts intramolecular vibrational coupling, causing the vibrational modes of the two carbonyl groups to converge. This demonstrates that a hydrogen bond is formed between the carboxyl group of ascorbic acid and the nitrogen atom of glutathione, and the resulting intermolecular interactions in the system lead to the shift in the infrared absorption peak.

[0078] Figure 3 Differential scanning calorimetry (DSC) of the ascorbic acid-glutathione cocrystal from Example 1. Figure 3 It can be seen that ascorbic acid and glutathione exhibit single melting peaks at 196℃ and 201℃, respectively, while the melting point of the ascorbic acid-glutathione eutectic system in Example 1 is 153℃. The appearance of a single melting point that is different from that of the raw material components indicates the formation of the eutectic system.

[0079] Figure 4 The image shows the powder X-ray diffraction pattern of the ascorbic acid-glutathione cocrystal from Example 1. Figure 4 It can be seen that the ascorbic acid-glutathione cocrystal in Example 1 exhibits a novel characteristic diffraction peak at 35.19°, while the intensity of some characteristic diffraction peaks of ascorbic acid and glutathione is significantly reduced or disappears. This indicates that the cocrystal system is not a physical mixture, but rather forms a new crystal structure.

[0080] Figure 5 This is a permeability test diagram of the ascorbic acid-glutathione cocrystal from Example 1. From... Figure 5 It can be seen that the cumulative permeation of the ascorbic acid-glutathione cocrystal in Example 1 within 24 hours was 4.86 times that of ascorbic acid, reaching 12.54 mg / cm³. 2 Notably, at 4 hours after transdermal contact, the cumulative permeation of the ascorbic acid-glutathione cocrystal was 15.8 times that of the ascorbic acid group, reaching 3.04 mg / cm³. 2 The transdermal delivery of ascorbic acid gradually reaches saturation over time. The enhanced skin delivery of ascorbic acid via the cocrystal system can be attributed to the strong intermolecular interactions present in the system, which influence the structural integrity of the skin lipid network, thereby promoting the transdermal delivery of ascorbic acid.

[0081] Example 2 An ascorbic acid-glutathione cocrystal, the structural formula of which is as follows:

[0082] The preparation method is as follows: 0.02 mol ascorbic acid and 0.01 mol glutathione were mixed and placed in a round-bottom flask. After dissolving in 1000 g of deionized water, the water was removed by rotary evaporation at 45 °C for 3 h. The mixture was then ground at 25 °C (room temperature) at 80 r / min for 5 min to obtain a white powder product, namely: ascorbic acid-glutathione cocrystal (particle size 4~12 μm).

[0083] Example 3 An ascorbic acid-glutathione cocrystal, the structural formula of which is as follows:

[0084] The preparation method is as follows: 0.04 mol ascorbic acid and 0.01 mol glutathione were mixed and placed in a round-bottom flask. After dissolving in 1000 g of deionized water, the water was removed by rotary evaporation at 55 °C for 4 h. The mixture was then ground at 35 °C (room temperature) at 100 r / min for 8 min to obtain a white powder product, namely: ascorbic acid-glutathione cocrystal (particle size 4~12 μm).

[0085] Example 4 An ascorbic acid-glutathione cocrystal, the structural formula of which is as follows:

[0086] The preparation method is as follows: 0.03 mol ascorbic acid and 0.01 mol glutathione were mixed in a round-bottom flask, dissolved in 700 g of deionized water, and the water was removed by rotary evaporation at 50 °C for 2 h. The mixture was then ground at 25 °C (room temperature) at 120 r / min for 10 min to obtain a white powder product, namely: ascorbic acid-glutathione cocrystal (particle size 4~12 μm).

[0087] Example 5 An ascorbic acid-glutathione cocrystal, the structural formula of which is as follows:

[0088] The preparation method is as follows: 0.03 mol ascorbic acid and 0.01 mol glutathione were mixed evenly and placed in a material tray. They were dissolved in 700 g of deionized water and pre-frozen (frozen at -80℃ for 40 h). Then, the mixture was treated with a freeze dryer at -60℃ for 10 h to remove moisture. Finally, it was ground at 25℃ (room temperature) at 80 r / min for 5 min to obtain a white powder product, namely: ascorbic acid-glutathione cocrystal (particle size 4~12 μm).

[0089] Example 6 An ascorbic acid-glutathione cocrystal, the structural formula of which is as follows:

[0090] The preparation method is as follows: Mix 0.02 mol ascorbic acid and 0.01 mol glutathione evenly, place them in a material tray, dissolve them with 1000 g of deionized water and pre-freeze (freeze at -70℃ for 50 h), then treat with a freeze dryer at -55℃ for 15 h to remove moisture, and grind at 25℃ (room temperature) at 80 r / min for 5 min to obtain a white powder product, namely: ascorbic acid-glutathione cocrystal (particle size 4~12 μm).

[0091] Example 7 An ascorbic acid-glutathione cocrystal, the structural formula of which is as follows:

[0092] The preparation method is as follows: Mix 0.04 mol ascorbic acid and 0.01 mol glutathione evenly, place them in a material tray, dissolve them with 1000 g of deionized water and pre-freeze (freeze at -60℃ for 60 h), then treat with a freeze dryer at -50℃ for 20 h to remove moisture, and grind at 30℃ (room temperature) at 100 r / min for 10 min to obtain a white powder product, namely: ascorbic acid-glutathione cocrystal (particle size 4~12 μm).

[0093] Example 8 An ascorbic acid-glutathione cocrystal, the structural formula of which is as follows:

[0094] The preparation method is as follows: Mix 0.03 mol ascorbic acid and 0.01 mol glutathione, stir at 25℃ (room temperature) and 80 r / min for 5 min to obtain a white powder product, namely: ascorbic acid-glutathione cocrystal (particle size 4~12 μm).

[0095] Example 9 An ascorbic acid-glutathione cocrystal, the structural formula of which is as follows:

[0096] The preparation method is as follows: 0.01 mol ascorbic acid and 0.01 mol glutathione were mixed evenly and ground at 25℃ (room temperature) and 100 r / min for 4 h to obtain a white solid product, namely: ascorbic acid-glutathione cocrystal (particle size 4~12 μm).

[0097] Comparative Example 1 Based on Example 1, the amount of ascorbic acid was changed to 0.06 mol, and the remaining steps were the same as in Example 1.

[0098] The results showed that after increasing the amount of ascorbic acid, the two components did not come into complete contact during the entire grinding process. Subsequent tests revealed that a high-purity eutectic system could not be obtained, and there was an excess of ascorbic acid.

[0099] Comparative Example 2 Based on Example 1, the amount of ascorbic acid was changed to 0.01 mol, the amount of glutathione was changed to 0.06 mol, and the remaining steps were the same as in Example 1.

[0100] The results showed that after increasing the amount of glutathione, the two components did not come into complete contact during the entire grinding process. Subsequent tests revealed that a high-purity co-crystal system could not be obtained, and there was an excess of glutathione.

[0101] Comparative Example 3 Based on Example 1, the grinding time was changed to 0.5 hours, and the remaining steps were the same as in Example 1.

[0102] The results showed that after shortening the grinding time, the two components did not come into complete contact during the entire grinding process. Subsequent tests revealed that a high-purity eutectic system could not be obtained, and the system's self-assembly was incomplete.

[0103] Comparative Example 4 Based on Example 1, the grinding time was changed to 11 hours, and the remaining steps were the same as in Example 1.

[0104] The results show that extending the grinding time will cause the "building effect" of mechanical force to turn into a "destructive effect", ultimately leading to a deterioration of the eutectic effect and the inability to obtain a high-purity eutectic system.

[0105] Comparative Example 5 Based on Example 2, the temperature of the rotary evaporator was changed to 20°C, and the remaining steps were the same as in Example 2.

[0106] The results showed that after lowering the temperature of the rotary evaporator, the two components did not come into complete contact during the entire process. Subsequent tests revealed that a high-purity eutectic system could not be obtained, and the system's self-assembly was incomplete.

[0107] Comparative Example 6 Based on Example 2, the temperature of the rotary evaporator was changed to 80°C, and the remaining steps were the same as in Example 2.

[0108] The results showed that increasing the temperature of the rotary evaporator resulted in excessively high temperatures throughout the process, which damaged the stability of ascorbic acid and glutathione, leading to a decrease in the activity of the final product.

[0109] Comparative Example 7 Based on Example 2, the operating time of the rotary evaporator was changed to 11 hours, and the remaining steps were the same as in Example 2.

[0110] The results showed that extending the operating time of the rotary evaporator disrupted the stability of ascorbic acid and glutathione during prolonged rotary evaporation, leading to a decrease in the activity of the final product.

[0111] Comparative Example 8 Based on Example 2, the operating time of the rotary evaporator was changed to 1 hour, and the remaining steps were the same as in Example 2.

[0112] The results showed that after shortening the operating time of the rotary evaporator, ascorbic acid and glutathione did not get sufficient contact, and a stable eutectic system was not formed, resulting in incomplete self-assembly of the system.

[0113] Comparative Example 9 Based on Example 5, the temperature of the freeze dryer was changed to -80°C, and the remaining steps were the same as in Example 5.

[0114] The results showed that lowering the temperature of the freeze dryer inhibited the diffusion of eutectic molecules, affected the formation of the eutectic system, and impacted the activity of the product.

[0115] Comparative Example 10 Based on Example 5, the temperature of the freeze dryer was changed to -30°C, and the remaining steps were the same as in Example 5.

[0116] The results showed that when the temperature of the freeze dryer was increased, the adsorbed water on the eutectic surface melted rapidly throughout the process, which destroyed the eutectic system and affected the activity of the product.

[0117] Comparative Example 11 Based on Example 5, the freeze dryer usage time was changed to 4 hours, and the remaining steps were the same as in Example 5.

[0118] The results showed that after shortening the usage time of the freeze dryer, the two components did not come into complete contact during the entire process. Subsequent tests revealed that a high-purity eutectic system could not be obtained, and the system's self-assembly was incomplete.

[0119] Comparative Example 12 Based on Example 5, the freeze dryer usage time was changed to 40 hours, and the remaining steps were the same as in Example 5.

[0120] The results showed that prolonged use of the freeze dryer and long-term vacuum extraction damaged the stability of ascorbic acid and glutathione, affecting the activity of the final product.

[0121] Comparative Example 13 Based on Example 1, nicotinamide was used instead of glutathione, and the remaining steps were the same as in Example 1.

[0122] Comparative Example 14 Based on Example 2, nicotinic acid was used to replace glutathione, and the remaining steps were the same as in Example 1.

[0123] Comparative Example 15 Based on Example 5, glucose was used instead of glutathione, and the remaining steps were the same as in Example 1.

[0124] Comparative Example 16 A method for preparing ascorbic acid S The method for producing glutathione conjugates includes the following steps: Under nitrogen protection, 10 g of ascorbic acid (purity ≥99%) and 100 mL of glacial acetic acid (≥99.5%) were added sequentially to a flask. Stirring was started (300 rpm), and the temperature was maintained at 8°C in an ice bath. 50 g of a hydrogen bromide / acetic acid solution (40% concentration, water as solvent) was slowly added dropwise over approximately 1 hour. The temperature was then slowly raised to 25°C, and the reaction continued for 4.5 hours (TLC monitoring showed the disappearance of ascorbic acid spots). The mixture was concentrated under reduced pressure, and 50 mL of ethyl acetate was added. The mixture was stirred at 300 rpm for 2 hours, resulting in the precipitation of a white solid. This solid was filtered, washed with ethyl acetate, and dried under vacuum to obtain a white crystalline solid. The brominated product (white crystalline solid) and 15 g of glutathione were dissolved in 20 g of water, and 25 mL of methanol and 11.5 g of sodium bicarbonate were added. The mixture was reacted at 20°C for 12 hours. After the reaction, the ascorbic acid was purified by column chromatography. S Glutathione conjugates.

[0125] The obtained product was subjected to performance testing, and the test results are as follows: Tables 3-5 show the stability monitoring (active ingredient content test results) of the examples, comparative examples, ascorbic acid, and glutathione. As can be seen from Tables 3-5, under the same environmental conditions, the active ingredient content of the ascorbic acid solution decreased to 37% after 21 days, and the active ingredient content of the glutathione solution decreased to 41% after 28 days. However, the active ingredient content of the ascorbic acid-glutathione co-crystal solution of the examples remained at 80% after 49 days, far exceeding that of the comparative example. This indicates that the ascorbic acid-glutathione of the present invention has higher stability than ascorbic acid, glutathione, and the comparative example, and the formation of the co-crystal system can improve the stability of the active ingredient.

[0126] Table 3. Stability monitoring of the examples, ascorbic acid, and glutathione (results of active ingredient content test).

[0127] Note: "-" indicates that the concentration of the active ingredient is below 50%, and further stability monitoring is not required.

[0128] Table 4. Stability monitoring results of comparative examples 1-8 (results of active ingredient content test).

[0129] Note: "-" indicates that the concentration of the active ingredient is below 50%, and further stability monitoring is not required.

[0130] Table 5. Stability monitoring results of comparative examples 9-16 (results of active ingredient content test).

[0131] Note: "-" indicates that the concentration of the active ingredient is below 50%, and further stability monitoring is not required.

[0132] Table 6 shows the whitening, antioxidant, and penetration performance tests.

[0133] As can be seen from Table 6: (1) IC of the embodiment 50 The value was significantly lower than that of ascorbic acid and the comparative IC50. 50 The value fully demonstrates that the co-crystal of the present invention has a stronger ability to inhibit tyrosinase activity, and the whitening effect is significantly improved.

[0134] (2) The DPPH and ABTS scavenging rates of the 20 μg / mL ascorbic acid-glutathione cocrystal were both higher than 90%, which was significantly higher than the free radical scavenging rates of ascorbic acid and the comparative ratio. This fully demonstrates that the cocrystal of the present invention has a stronger free radical scavenging ability and significantly improved antioxidant effect.

[0135] (3) The cumulative permeation of the ascorbic acid-glutathione cocrystal was much greater than that of the comparative example; the cumulative permeation of Example 1 within 24 hours was 4.86 times that of pure ascorbic acid, reaching 12.54 mg / cm³. 2 .

[0136] Table 6 Whitening, Antioxidant, and Penetration Performance Tests

[0137] Table 7 shows other performance tests. As can be seen from Table 7: (1) Effect of inhibiting melanin in human skin: The melanin content of the skin samples in the application example was significantly reduced, which was much higher than that in the comparative example and the ascorbic acid group; (2) Characterization of the whitening essence's efficacy: The melanin content in the skin of volunteers who applied the essence of the example significantly decreased, far exceeding that of the comparative example and the ascorbic acid group, indicating that the cocrystal of the present invention has a significant advantage in whitening efficacy. Therefore, the whitening essence uses ascorbic acid-glutathione cocrystal as the core active ingredient, and its stability and skin feel are ensured through optimized formulation and process. Human trials have confirmed that it can significantly reduce melanin production (p<0.01), improve skin brightness and radiance, and has good safety.

[0138] (3) Anti-glycation characterization: The results of the examples were higher than those of the comparative group and the ascorbic acid group, and close to the positive control group (24.53%), indicating that the cocrystals of the examples have a good AGEs inhibition function and have a certain anti-glycation effect.

[0139] Table 7 Other performance tests

[0140] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person 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.

Claims

1. An ascorbic acid-glutathione co-crystal, characterized in that, The structure is as follows: or or or 。 2. A method for preparing the ascorbic acid-glutathione co-crystal of claim 1, characterized in that, Includes the following steps: Method 1: Ascorbic acid and glutathione were mixed in a molar ratio of 1~5:1~5 and ground to obtain ascorbic acid-glutathione cocrystal. Method 2: Ascorbic acid and glutathione were mixed in a molar ratio of 1~5:1~5; then dissolved in water, the water was removed, and the mixture was ground to obtain ascorbic acid-glutathione cocrystal. Method 3: Ascorbic acid and glutathione were mixed in a molar ratio of 1~5:1~5; then dissolved in water, frozen, the water was removed, and the mixture was ground to obtain ascorbic acid-glutathione cocrystal. Method 4: Ascorbic acid and glutathione were mixed in a molar ratio of 1~5:1~5 to obtain ascorbic acid-glutathione cocrystal.

3. The method according to claim 2, characterized in that, In Method 1, grinding is carried out at 15~35℃ (room temperature) and 80~120r / min for 1~10h.

4. The method according to claim 2, characterized in that, In Method 2, moisture is removed by rotary evaporation, specifically at 30-70°C for 2-10 hours.

5. The method according to claim 2, characterized in that, In method three, freezing involves freezing at -80 to -60°C for 40 to 60 hours.

6. The method according to claim 2, characterized in that, In method three, moisture is removed by freeze drying, specifically freeze drying at -70 to -40°C for 5 to 30 hours.

7. The use of the ascorbic acid-glutathione cocrystal according to claim 1 in the preparation of cosmetics or pharmaceuticals.

8. A product that combines whitening, antioxidant, and anti-glycation properties, characterized in that, The ascorbic acid-glutathione co-crystal described in claim 1 was used.

9. A method for improving the stability of ascorbic acid, characterized in that, The ascorbic acid-glutathione co-crystal described in claim 1 was used.

10. A method for simultaneously improving the whitening activity, antioxidant properties, anti-glycation properties, and transdermal penetration rate of ascorbic acid, characterized in that, The ascorbic acid-glutathione co-crystal described in claim 1 was used.

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