A phenylethylresorcinol proline cocrystal and its preparation method and application

Abstract

This application provides a phenethylresorcinol-proline cocrystal, its preparation method, and application, belonging to the field of cocrystal manufacturing technology. The structural formula of the phenethylresorcinol-proline cocrystal is shown in Formula I. The phenethylresorcinol-proline cocrystal has a good whitening effect, and also has the advantages of low irritation and high stability.

Description

Technical Field

[0001] The present application relates to the technical field of cocrystal manufacturing, and in particular to a phenethylresorcinol-proline cocrystal and a preparation method and application thereof. Background Art

[0002] Phenethyl resorcinol effectively inhibits the activity of tyrosinase, a key enzyme in melanin formation. Furthermore, it inhibits the formation of numerous oxygen free radicals caused by UV radiation (which promote melanin formation). Therefore, phenethyl resorcinol has a strong whitening and freckle-removing effect. However, phenethyl resorcinol is irritating to the skin and is only permitted in a few cosmetics, where the maximum concentration is no more than 0.5%. Furthermore, phenethyl resorcinol is unstable and photosensitized, rapidly changing from white to beige and finally to pink under light. This causes a rapid decrease in phenethyl resorcinol content, affecting the cosmetic's appearance and bioavailability. Therefore, there is a need to develop a phenethyl resorcinol product with low irritation and high stability. Summary of the Invention

[0003] The purpose of the present application is to provide a phenethylresorcinol-proline cocrystal and its preparation method and application. The phenethylresorcinol-proline cocrystal has a good whitening effect and also has the advantages of low irritation and high stability.

[0004] The embodiment of the present application is implemented as follows:

[0005] In a first aspect, the present invention provides a phenethylresorcinol-proline cocrystal. The structural formula of the phenethylresorcinol-proline cocrystal is shown in Formula I:

[0006]

[0007] Formula I.

[0008] In the above technical solution, the double-bonded oxygen in proline has a lone pair of electrons, and phenethylresorcinol has functional groups such as a benzene ring and a phenolic hydroxyl group. This allows phenethylresorcinol to interact with proline through intermolecular forces such as hydrogen bonds and van der Waals forces, forming a crystalline material that is regularly arranged and stable within the same lattice. After phenethylresorcinol and proline form a cocrystal, the crystal arrangement differs from that of phenethylresorcinol alone. This allows the phenethylresorcinol-proline cocrystal to not only possess the whitening effects of phenethylresorcinol but also offer the advantages of lower irritation and higher stability.

[0009] In some alternative embodiments, the molecular formula of the phenethylresorcinol proline cocrystal is C 24 H 32N2O6, and in the phenethylresorcinol-proline eutectic, the molar ratio of phenethylresorcinol to proline is 1:2.

[0010] In some optional embodiments, the phenethylresorcinol proline cocrystal is monoclinic, with a space group of P21, unit cell parameters of a=8.2050(3) Å, b=9.3756(3) Å, c=15.1517(6) Å, α=90°, β=97.824(4)°, γ=90°, Z=2, and unit cell volume V=1154.72(7) Å 3 .

[0011] In some alternative embodiments, the proline is L-proline.

[0012] In some optional embodiments, the X-ray powder diffraction pattern of the phenethylresorcinol-proline cocrystal has characteristic peaks at 2θ angles of 5.94°±0.2°, 15.14°±0.2°, 17.70°±0.2°, 18.99°±0.2° and 20.12°±0.2°.

[0013] In a second aspect, the present application provides a method for preparing a phenethylresorcinol-proline cocrystal as provided in the first aspect, comprising the following steps:

[0014] S1. Under an inert atmosphere and light-proof conditions, dissolve phenylethylresorcinol and proline in an ethanol-water mixed solvent and stir the mixture at a preset temperature for reaction, wherein the volume proportion of water in the ethanol-water mixed solvent is 1-20%, and the preset temperature is 50-85°C, to obtain a precursor solution containing phenylethylresorcinol-proline cocrystal; S2. Cooling and crystallizing the precursor solution. The cooling and crystallization steps include: first cooling the precursor solution for a first time so that the temperature of the precursor solution is 20-30°C, then cooling the precursor solution for a second time so that the temperature of the precursor solution is not higher than 10°C, wherein the cooling rate of the first cooling treatment is less than the cooling rate of the second cooling treatment, and then maintaining the temperature unchanged for crystallization to obtain a solid-liquid mixture; S3. Performing solid-liquid separation and drying on the solid-liquid mixture in sequence to obtain phenylethylresorcinol-proline cocrystal.

[0015] In the above technical solution, according to the above process, a phenethylresorcinol-proline co-crystal as provided in the embodiment of the first aspect can be prepared, wherein an ethanol-water mixed system is used as a solvent and the volume proportion of water is limited to 1-20%, so that both phenethylresorcinol and proline can be fully dissolved and evenly mixed, so that the subsequent reaction can obtain the phenethylresorcinol-proline co-crystal; the stirring reaction is carried out at 50-85°C to provide a more suitable driving force so that phenethylresorcinol and proline can react and react more thoroughly; the system temperature is lowered to the crystallization treatment temperature by gradient cooling, and the cooling rate of the first cooling treatment is set to be lower than the cooling rate of the second cooling treatment, which can effectively reduce the probability of decomposition after the reaction of phenethylresorcinol and proline to form a co-crystal (the two react rapidly and decomposition is very likely to occur. Decomposition will cause a large amount of solid to precipitate rapidly, resulting in large agglomerated particles. The obtained product is usually a non-eutectic product mixed with a large amount of impurities), thereby preparing a co-crystal product with high purity and yield.

[0016] In some optional embodiments, the volume proportion of water in the ethanol-water mixed solvent is 3-10%.

[0017] In the above technical solution, the volume proportion of water in the ethanol-water mixed solvent is limited to a more appropriate range, which can dissolve phenylethylresorcinol and proline more quickly and thoroughly, thereby improving the preparation efficiency and yield of the eutectic product.

[0018] In some optional embodiments, the preset temperature is 70-85°C.

[0019] In the above technical solution, the reaction temperature is further limited to the range of 70~85°C, which can provide more suitable reaction conditions, thereby obtaining a eutectic product with higher purity and yield.

[0020] In some optional embodiments, the cooling rate of the first cooling treatment is 0.5-3°C / min.

[0021] In the above technical solution, limiting the cooling rate of the first cooling treatment within an appropriate range can more effectively reduce the probability of explosive precipitation after the reaction of phenylethylresorcinol and proline, thereby obtaining a eutectic product with higher purity and yield.

[0022] In some optional embodiments, the step of obtaining a precursor solution containing phenylethylresorcinol-proline cocrystal comprises:

[0023] Under an inert atmosphere and light-shielding conditions, proline is added to an ethanol-water mixed solvent and stirred at a preset temperature to dissolve the proline in the ethanol-water mixed solvent to obtain a mixed solution; while maintaining the temperature and stirring speed, phenylethylresorcinol is added to the mixed solution to dissolve the phenylethylresorcinol in the mixed solution and react with the proline to obtain a precursor solution containing a phenylethylresorcinol-proline cocrystal.

[0024] In the above technical solution, proline is dissolved at a preset temperature and under stirring conditions. On the one hand, this helps the proline to dissolve quickly and thoroughly. On the other hand, it also allows the mixed system to reach the temperature conditions required for the reaction in advance, so that phenylethylresorcinol can be added and dissolved and then directly react with proline to obtain a eutectic product, thereby improving the preparation efficiency and purity of the eutectic product.

[0025] In some optional embodiments, the molar ratio of the added amount of proline to the added amount of phenylethylresorcinol is (1.9-2.1):1; and / or, in the mixed solution, the mass ratio of proline to the mixed solution is 1:(5-10).

[0026] In the above technical solution, by limiting the molar ratio of the added amount of proline to the added amount of phenylethylresorcinol within the above range, a eutectic product with a molar ratio closer to 2:1 can be obtained; by limiting the mass ratio of proline to the mixed solution within the above range, proline can be better dissolved and dispersed.

[0027] In some optional embodiments, the temperature of the crystallization treatment is 0-10° C., and the crystallization treatment is performed under stirring conditions.

[0028] In the above technical solution, the crystallization process is carried out under stirring conditions so that the crystal growth state is more controllable, thereby obtaining a eutectic product with better particle size uniformity.

[0029] In some optional embodiments, during the crystallization process, the stirring speed is 50-600 rpm.

[0030] In the above technical solution, during the crystallization process, the adjustable range of the stirring speed is relatively wide, which facilitates adaptive adjustment according to the volume of the mixed solution and the volume of the reaction container.

[0031] In a third aspect, an embodiment of the present application provides a use of the phenethylresorcinol proline co-crystal provided in the embodiment of the first aspect in the preparation of medicines, cosmetics or skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 Schematic diagram of the X-ray single crystal diffraction structure of the phenethylresorcinol-proline cocrystal of Example 1 of the present application;

[0034] Figure 2 This is the H NMR spectrum of the phenethylresorcinol-proline cocrystal of Example 1 of the present application;

[0035] Figure 3 This is the C NMR spectrum of the phenethylresorcinol-proline cocrystal of Example 1 of the present application;

[0036] Figure 4 The infrared spectra of the phenethylresorcinol-proline cocrystal, phenethylresorcinol, and proline of Example 1 of the present application;

[0037] Figure 5 A scanning electron micrograph of phenylethylresorcinol provided in this application;

[0038] Figure 6 A scanning electron micrograph of proline provided for this application;

[0039] Figure 7 This is a scanning electron micrograph of the phenethylresorcinol-proline cocrystal of Example 1 of the present application;

[0040] Figure 8 This is a scanning electron micrograph of the phenethylresorcinol-proline cocrystal of Example 2 of the present application;

[0041] Figure 9 This is the X-ray powder diffraction pattern of the phenethylresorcinol-proline cocrystal of Example 1 of the present application;

[0042] Figure 10 The inhibition curve of the tyrosinase (bisphenol) activity inhibition of the phenethylresorcinol-proline cocrystal provided in this application;

[0043] Figure 11 The inhibition curve of the physical mixture of phenylethylresorcinol and proline on the activity of tyrosinase (bisphenol) provided in this application;

[0044] Figure 12 The inhibition curve of phenylethyl resorcinol on tyrosinase (bisphenol) activity provided in this application. DETAILED DESCRIPTION

[0045] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0046] It should be noted that “and / or” in this application, such as “Feature 1 and / or Feature 2”, refers to three situations: “Feature 1” alone, “Feature 2” alone, or “Feature 1” plus “Feature 2”.

[0047] In addition, in the description of this application, unless otherwise specified, the "multiple" in "one or more" means two or more; the range of "value a~value b" includes the two end values "a" and "b", and the "unit of measurement" in "value a~value b+unit of measurement" represents the "unit of measurement" of both "value a" and "value b".

[0048] The following is a detailed description of a phenethylresorcinol-proline cocrystal according to an embodiment of the present application, its preparation method, and its application.

[0049] In a first aspect, the present invention provides a phenethylresorcinol-proline cocrystal. The structural formula of the phenethylresorcinol-proline cocrystal is shown in Formula I:

[0050]

[0051] Formula I.

[0052] In this application, the double-bonded oxygen in proline has lone pairs of electrons, and phenethylresorcinol has functional groups such as a benzene ring and a phenolic hydroxyl group. This allows phenethylresorcinol to interact with proline through intermolecular forces such as hydrogen bonds and van der Waals forces, forming a crystalline material that is regularly arranged and stable in the same lattice. After phenethylresorcinol and proline form a cocrystal, the crystal arrangement is different from that of phenethylresorcinol alone. This allows the phenethylresorcinol-proline cocrystal to not only have the whitening effect of phenethylresorcinol, but also have the advantages of lower irritation and higher stability.

[0053] It should be noted that, on the one hand, phenylethylresorcinol has a strong antioxidant effect and can non-competitively inhibit the activity of tyrosinase. It also inhibits the formation of melanin, thereby reducing melanin production. On the other hand, proline is an amino acid component of collagen fibers and can be used as a nutrient. It also softens the stratum corneum, strengthening the function of the keratin and improving the skin's inherent moisturizing ability. Furthermore, proline can promote muscle growth, improve immune function, and promote energy metabolism. It also maintains muscle and joint vitality and improves skin sagging and wrinkling. The combination of phenylethylresorcinol and proline to form a cocrystal retains the efficacy of both while reducing the irritation of phenylethylresorcinol and improving its stability, achieving better application results.

[0054] As an example, the molecular formula of phenylethylresorcinol proline cocrystal is C 24 H 32 N2O6, and in the phenethylresorcinol-proline eutectic, the molar ratio of phenethylresorcinol to proline is 1:2.

[0055] As an example, the phenethylresorcinol-proline cocrystal is monoclinic with space group P21, unit cell parameters a=8.2050(3) Å, b=9.3756(3) Å, c=15.1517(6) Å, α=90°, β=97.824(4)°, γ=90°, Z=2, and unit cell volume V=1154.72(7) Å. 3 .

[0056] As an example, the proline is L-proline.

[0057] As an example, the X-ray powder diffraction pattern of the phenylethylresorcinol-proline cocrystal has characteristic peaks at 2θ angles of 5.94°±0.2°, 15.14°±0.2°, 17.70°±0.2°, 18.99°±0.2° and 20.12°±0.2°.

[0058] In order to better understand the technical solution, this application simulates and provides a schematic diagram of the molecular structure of phenylethylresorcinol proline cocrystal (see Figure 1 ).

[0059] In a second aspect, the present application provides a method for preparing a phenethylresorcinol-proline cocrystal as provided in the first aspect, comprising the following steps:

[0060] S1 Under an inert atmosphere and light-proof conditions, dissolving phenethylresorcinol and proline in an ethanol-water mixed solvent and stirring to react at a preset temperature, wherein the volume proportion of water in the ethanol-water mixed solvent is 1-20% (for example, but not limited to, any one of 1%, 2%, 5%, 7%, 10%, 12%, 15%, 17% and 20% or a range of values between any two of the volume proportions), and the preset temperature is 50-85°C (for example, but not limited to, any one of 50°C, 60°C, 70°C, 75°C, 80°C and 85°C or a range of values between any two of the volume proportions), to obtain a precursor solution containing phenethylresorcinol-proline cocrystals.

[0061] S2 performs cooling and crystallization treatment on the precursor solution, and the steps of cooling and crystallization treatment include: first performing a first cooling treatment on the precursor solution so that the temperature of the precursor solution is 20-30°C (for example, but not limited to, any one point value of 20°C, 22°C, 24°C, 26°C, 28°C and 30°C or a range value between any two of them), and then performing a second cooling treatment on the precursor solution so that the temperature of the precursor solution is not higher than 10°C, wherein the cooling rate of the first cooling treatment is less than the cooling rate of the second cooling treatment, and then maintaining the temperature unchanged to perform crystallization treatment to obtain a solid-liquid mixture.

[0062] S3: performing solid-liquid separation and drying on the solid-liquid mixture in sequence to obtain phenethylresorcinol-proline eutectic.

[0063] In the present application, according to the above process, a phenethylresorcinol-proline co-crystal as provided in the embodiment of the first aspect can be prepared, wherein an ethanol-water mixed system is used as a solvent and the volume proportion of water is limited to 1-20%, so that both phenethylresorcinol and proline can be fully dissolved and evenly mixed, so that the subsequent reaction can obtain the phenethylresorcinol-proline co-crystal; the stirring reaction is carried out at 50-85°C to provide a more suitable driving force so that phenethylresorcinol and proline can react and react more thoroughly; the system temperature is lowered to the crystallization treatment temperature by gradient cooling, and the cooling rate of the first cooling treatment is set to be lower than the cooling rate of the second cooling treatment, which can effectively reduce the probability of decomposition after the reaction of phenethylresorcinol and proline to form a co-crystal (the two react rapidly and decomposition is very likely to occur. Decomposition will cause a large amount of solid to precipitate rapidly, resulting in large agglomerated particles. The obtained product is usually a non-eutectic product mixed with a large amount of impurities), thereby preparing a co-crystal product with high purity and yield.

[0064] As an example, the volume proportion of water in the ethanol-water mixed solvent is 3~10%, for example, but not limited to, any one of 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% or a range between any two of the volume proportions.

[0065] In this embodiment, the volume ratio of water in the ethanol-water mixed solvent is limited to a more appropriate range, which can dissolve phenylethylresorcinol and proline more quickly and thoroughly, thereby improving the preparation efficiency and yield of the eutectic product.

[0066] As an example, the preset temperature is 70~85℃, such as but not limited to any point value of 70℃, 72℃, 74℃, 76℃, 78℃, 80℃, 82℃, 84℃ and 85℃, or a range value between any two of them.

[0067] In this embodiment, the reaction temperature is further limited to the range of 70-85° C., which can provide more suitable reaction conditions, thereby obtaining a eutectic product with higher purity and yield.

[0068] As an example, the cooling rate of the first cooling treatment is 0.5~3 ℃ / min, for example, but not limited to, the cooling rate is any one of 0.5 ℃ / min, 0.6 ℃ / min, 0.7 ℃ / min, 0.8 ℃ / min, 0.9 ℃ / min, 1 ℃ / min, 1.5 ℃ / min, 2 ℃ / min, 2.5 ℃ / min and 3 ℃ / min, or a range between any two of them.

[0069] In this embodiment, limiting the cooling rate of the first cooling treatment to an appropriate range can more effectively reduce the probability of explosion after the reaction of phenylethylresorcinol and proline, thereby obtaining a eutectic product with higher purity and yield.

[0070] As an example, the steps of obtaining a precursor solution containing phenylethylresorcinol-proline cocrystal include:

[0071] Under an inert atmosphere and light-shielding conditions, proline is added to an ethanol-water mixed solvent and stirred at a preset temperature to dissolve the proline in the ethanol-water mixed solvent to obtain a mixed solution; while maintaining the temperature and stirring speed, phenylethylresorcinol is added to the mixed solution to dissolve the phenylethylresorcinol in the mixed solution and react with the proline to obtain a precursor solution containing a phenylethylresorcinol-proline cocrystal.

[0072] In this embodiment, dissolving proline at a preset temperature and under stirring conditions, on the one hand, helps the proline to dissolve quickly and thoroughly, and on the other hand, allows the mixed system to reach the temperature conditions required for the reaction in advance, so that phenylethylresorcinol can be added and dissolved and then directly react with proline to obtain a eutectic product, thereby improving the preparation efficiency and purity of the eutectic product.

[0073] As an example, the molar ratio of the added amount of proline to the added amount of phenylethylresorcinol is (1.9~2.1):1, for example, but not limited to, the molar ratio is any one of 1.9:1, 1.95:1, 2:1, 2.05:1 and 2.1:1, or a range between any two of them; and / or, in the mixed solution, the mass ratio of proline to the mixed solution is 1:(5~10), for example, but not limited to, the mass ratio is any one of 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10, or a range between any two of them.

[0074] In this embodiment, the molar ratio of the added amount of proline and the added amount of phenylethylresorcinol is limited within the above range, and a eutectic product with a molar ratio closer to 2:1 can be obtained; and the mass ratio of proline to the mixed solution is limited within the above range, so that proline can be better dissolved and dispersed.

[0075] It should be noted that the temperature of the crystallization treatment is not limited as long as the phenethylresorcinol-proline eutectic in the precursor solution can be precipitated.

[0076] As an example, the temperature of the crystallization treatment is 0~10℃, for example, but not limited to, the temperature is any one of 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃ and 10℃, or a range between any two of them; and the crystallization treatment is carried out under stirring conditions.

[0077] In this embodiment, the crystallization process is carried out under stirring conditions so that the crystal growth state is more controllable, thereby obtaining a eutectic product with better particle size uniformity.

[0078] As an example, during the crystallization process, the stirring speed is 50~600 rpm, for example, but not limited to, any one of 50 rpm, 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm and 600 rpm, or a range between any two of them.

[0079] In this embodiment, during the crystallization process, the adjustable range of the stirring speed is relatively wide, which facilitates adaptive adjustment according to the volume of the mixed solution and the volume of the reaction container.

[0080] In other possible implementations, during the crystallization process, a static crystallization method may be adopted.

[0081] As an example, the drying method is vacuum drying, wherein the drying temperature is 50~55°C, for example, but not limited to, the temperature is any one of 50°C, 51°C, 52°C, 53°C, 54°C and 55°C, or a range between any two of them; the drying time is 12~48 h, for example, but not limited to, the drying time is any one of 12 h, 15 h, 30 h, 40 h and 48 h, or a range between any two of them.

[0082] In this embodiment, vacuum drying is adopted, which has the advantages of lower drying temperature and less prone to product denaturation.

[0083] It should be noted that any process or step not specifically described or limited in the preparation of the phenylethylresorcinol-proline cocrystal may be arranged according to conventional selection in the art.

[0084] In a third aspect, an embodiment of the present application provides a use of the phenethylresorcinol proline co-crystal provided in the embodiment of the first aspect in the preparation of medicines, cosmetics or skin care products.

[0085] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0086] Example 1

[0087] The present invention provides a method for preparing a phenethylresorcinol-proline cocrystal, comprising the following steps:

[0088] S1. Under an inert atmosphere (nitrogen) and light-shielding conditions, 51.8 g of proline was added to 300 g of an ethanol-water mixed solvent (the volume ratio of ethanol to water was 95:5) and stirred at a preset temperature, wherein the preset temperature was 85°C and the rotation speed was 400 rpm, so that the proline was dissolved in the ethanol-water mixed solvent to obtain a mixed solution; maintaining the temperature and stirring speed constant, 48.2 g of phenylethylresorcinol was added to the mixed solution to dissolve the phenylethylresorcinol in the mixed solution and react with the proline to obtain a precursor solution containing a phenylethylresorcinol-proline eutectic.

[0089] S2: The precursor solution is first cooled for the first time so that the temperature of the precursor solution is 25°C, and then the precursor solution is cooled for the second time so that the temperature of the precursor solution is 0°C. The cooling rate of the first cooling treatment is 0.5°C / min, and the cooling rate of the second cooling treatment is 4°C / min. The temperature is then maintained constant and a stirring crystallization treatment is performed for 3 h. The stirring speed is 200 rpm to obtain a solid-liquid mixture.

[0090] S3: The solid-liquid mixture was filtered, and the obtained solid was dried in a vacuum drying oven at a drying temperature of 50° C. for 24 h to obtain phenethylresorcinol-proline cocrystals in a yield of 89.6%.

[0091] Example 2

[0092] This embodiment of the present application provides a method for preparing phenethylresorcinol-proline cocrystals, which differs from Example 1 only in that the volume ratio of ethanol to water is 97:3, and the crystallization method is static crystallization, with a yield of 87.4%.

[0093] Example 3

[0094] This embodiment of the present application provides a method for preparing phenethylresorcinol-proline cocrystal, which differs from Example 1 only in that the volume ratio of ethanol to water is 90:10, and the yield is 87.9%.

[0095] Comparative Example 1

[0096] The comparative example of the present application provides a method for preparing phenethylresorcinol-proline cocrystal, which differs from Example 1 only in that: in step S2, the temperature is directly lowered to the crystallization temperature (0°C) at a cooling rate of 4°C / min.

[0097] Comparative Example 2

[0098] The comparative example of the present application provides a method for preparing phenethylresorcinol-proline cocrystal, which differs from Example 1 only in that the volume ratio of ethanol to water is 50:50.

[0099] Test Example 1

[0100] Phenethylresorcinol-proline cocrystals were prepared according to the preparation methods of Examples 1-3 and Comparative Examples 1-2, and then each sample was numbered. Then, the element content of each sample was tested using an element analyzer, and the results were statistically summarized in Table 1.

[0101] Table 1

[0102]

[0103] It should be noted that the theoretical value is calculated based on a standard molar ratio of phenylethylresorcinol to proline of 1:2.

[0104] Referring to Table 1, the test results of Examples 1 to 3 are basically consistent with the theoretical results, indicating that Examples 1 to 3 all successfully prepared phenethylresorcinol-proline cocrystals; the test results of Comparative Example 1 differed significantly from the theoretical values, indicating that the cooling rate was too fast, resulting in decomposition during the preparation process, which in turn made it difficult to prepare the cocrystal product; in Comparative Example 2, due to the unreasonable volume ratio of the ethanol-water mixed solvent, phenethylresorcinol was difficult to disperse and dissolve in the ethanol-water mixed solvent, which made it difficult to continue the reaction, and therefore no cocrystal product was obtained.

[0105] Test Example 2

[0106] Qualitative Analysis of Phenethylresorcinol-Proline Cocrystal

[0107] Test method:

[0108] The phenethylresorcinol-proline cocrystal obtained in Example 1 was qualitatively analyzed.

[0109] Table 2 Single crystal data of phenylethylresorcinol-proline cocrystal

[0110]

[0111] Table 3 Atomic coordinates and isotropic atomic displacement parameters of phenylethylresorcinol-proline cocrystal

[0112]

[0113] It should be noted that U(eq) is defined as one third of the trace of the orthogonal Uij tensor.

[0114] Table 4 Anisotropic atomic displacement parameters of phenylethylresorcinol-proline cocrystal

[0115]

[0116] It should be noted that the anisotropic atomic displacement factor power is: -2π 2 [h 2 a* 2 U 11 +2hka*b*U 12 +…].

[0117] Table 5 Bond lengths of phenylethylresorcinol-proline cocrystals

[0118]

[0119] Table 6 Chemical bond angles of phenylethylresorcinol-proline cocrystal

[0120]

[0121] Table 7 Hydrogen bonding parameters of phenylethylresorcinol-proline cocrystal

[0122]

[0123] 1 -1+X,+Y,+Z; 2 2-X,1 / 2+Y,1-Z; 3 1-X,-1 / 2+Y,1-Z.

[0124] Table 8 Torsion angle parameters of phenylethylresorcinol-proline cocrystal

[0125]

[0126] Table 9 Hydrogen atom coordinates of phenylethylresorcinol-proline cocrystal (Å×10 4 ) and the isotropic atomic displacement parameters (Å 2 ×10 3 )

[0127]

[0128] Test Example 3

[0129] H NMR spectroscopy ( 1 H-NMR) characterization

[0130] Test method:

[0131] The phenylethylresorcinol proline cocrystal obtained in Example 1 was subjected to nuclear magnetic resonance spectroscopy ( 1 H-NMR) characterization, and MeOD solvent was selected as the test solvent.

[0132] See Figure 2 From the H NMR spectrum, we can clearly find 12 hydrogen atoms of phenethylresorcinol and 14 hydrogen atoms on proline. The rest are a small amount of residual water peaks and deuterated reagent peaks. No obvious impurity peaks are seen. This shows that in the phenethylresorcinol-proline cocrystal, phenethylresorcinol and proline exist in a molar ratio of 1:2.

[0133] Test Example 4

[0134] C NMR spectroscopy ( 13 C-NMR) characterization

[0135] Test method:

[0136] The phenylethylresorcinol proline cocrystal obtained in Example 1 was subjected to C NMR spectroscopy ( 13 C-NMR) characterization, and MeOD solvent was selected as the test solvent.

[0137] See Figure 3 The test results show that phenethylresorcinol and proline in the phenethylresorcinol-proline cocrystal exist in a molar ratio of 1:2.

[0138] Test Example 5

[0139] Infrared spectroscopy characterization

[0140] Test method:

[0141] The phenethylresorcinol-proline cocrystal obtained in Example 1 was characterized by infrared spectroscopy, wherein the test parameter was transmittance, and the test wave number was 400 cm -1 ~4000cm -1 , the test mode is ATR.

[0142] See Figure 4 The infrared spectrum of the phenethylresorcinol-proline cocrystal has different absorption peaks from proline and phenethylresorcinol, and is not a simple superposition of the characteristic peaks of the two precursors, indicating that phenethylresorcinol and proline form a cocrystal, which is a new structure.

[0143] Test Example 6

[0144] Morphological characterization

[0145] Test method:

[0146] Phenethylresorcinol, proline, and the phenethylresorcinol-proline cocrystals obtained in Example 1 and Example 2 were characterized by scanning electron microscopy.

[0147] See Figure 5 、 Figure 6 and Figure 7 The morphology of ethylresorcinol-proline cocrystal is prismatic block with smooth surface and clear boundary, which is completely different from the morphology of phenylethylresorcinol and proline.

[0148] See Figure 7 and Figure 8 Compared with stirred crystallization, the particles of phenylethylresorcinol proline cocrystals obtained by static crystallization are larger, with a rougher surface and debris, indicating that the stirred crystallization method is better.

[0149] Test Example 7

[0150] X-ray powder diffraction (XRD) test

[0151] Test method:

[0152] The phenethylresorcinol-proline cocrystal obtained in Example 1 was subjected to an X-ray powder diffraction test, wherein the test conditions were 5° / min and the test range was 3°~50°.

[0153] See Figure 9 ,The results showed that the phenethylresorcinol-proline cocrystal had characteristic peaks at 2θ angles of approximately 5.94°±0.2°, 15.14°±0.2°, 17.70°±0.2°, 18.99°±0.2°, and 20.12°±0.2°.

[0154] Test Example 8

[0155] Stability testing

[0156] Test method:

[0157] The samples prepared in Examples 1 to 3 and phenylethylresorcinol monomer were used as test samples. Each sample group was divided into four equal parts, one group was placed at room temperature, one group was heated, one group was refrigerated, and one group was exposed to light (sunlight). The stability of the products under different conditions was measured, and the results are statistically summarized in Table 10.

[0158] Table 10

[0159]

[0160] Referring to Table 10, the phenethylresorcinol-proline cocrystals obtained in Examples 1 to 3 have better stability at room temperature, high temperature, low temperature, and sunlight than the phenethylresorcinol monomer, indicating that the phenethylresorcinol and proline cocrystals have excellent stability.

[0161] Test Example 9

[0162] In vitro cytotoxicity assay

[0163] (1) Detection method: human melanocyte cytotoxicity MTT assay.

[0164] (2) Experimental materials: phenethylresorcinol-proline cocrystal and phenethylresorcinol obtained in Example 1.

[0165] (3) Experimental method: Prepare cell suspension and adjust the cell concentration to 3.0×10 4Cell suspensions were plated in 96-well cell culture plates at a concentration of 100 μL per well and cultured for 24 hours. The original culture medium was discarded and 100 μL of test sample at various concentrations, as well as a control group (Conrol control: DMEM medium with 10% FBS, Blank control: DMEM medium) were added to each well. The cells were incubated in an incubator for 72 ± 0.5 hours. Cell morphology and characteristics were observed under a phase-contrast inverted microscope. 20 μL of MTT solution was added to each well and incubated in an incubator for 3 ± 0.5 hours. The liquid was removed and 100 μL of DMSO was added to each well. The cells were shaken on a shaker for 10-15 minutes, and the absorbance was measured at 570 nm using a microplate reader. Data are presented as mean ± standard deviation. Relative cell viability (viability) was calculated, with the cell viability of the control group set as 100%. The results are summarized in Tables 11 and 12, respectively.

[0166]

[0167] Among them, OD TA Refers to the absorbance value of the sample group, OD Blank Refers to the absorbance value of the Blank control group, OD Control Refers to the absorption value of the Conrrol control group.

[0168] Table 11 Experimental results of phenethylresorcinol-proline eutectic (Mean±SD)

[0169]

[0170] Table 12 Experimental results of phenylethylresorcinol (Mean±SD)

[0171]

[0172] Referring to Tables 11 and 12 (test data were fitted using GraphPad Prism5), the concentration of phenethylresorcinol-proline cocrystals at which the cell viability was 90% was 0.001016 mg / mL (CI95%: 0.0004482-0.002103 mg / mL); the concentration of phenethylresorcinol at which the cell viability was 90% was 0.0006012 mg / mL (CI95%: 0.0004017-0.0008847 mg / mL), indicating that the safe concentration of phenethylresorcinol-proline cocrystals was higher than that of phenethylresorcinol.

[0173] Test Example 10

[0174] Tyrosinase activity inhibition assay

[0175] (1) Experimental principle: In the biosynthesis of skin melanin, tyrosinase is the key enzyme. It acts on dopa to form dopaquinone, which then spontaneously undergoes a series of reactions to finally form melanin. In a Na2HPO4-citric acid buffer solution with a pH of 6.8, tyrosinase can catalyze the conversion of dopa into dopaquinone, and the absorbance value can be measured at 475 nm using a spectrophotometer. Raw materials with an inhibitory effect on tyrosinase activity can reduce the conversion of dopa into dopaquinone, thereby reducing the absorbance value. Based on the change in the absorbance value, the inhibitory effect of the raw materials on tyrosinase activity is evaluated.

[0176] (2) Experimental materials: The phenylethyl resorcinol-proline co-crystal obtained in Example 1, a physical mixture of phenylethyl resorcinol and proline with a molar ratio of 1:2, individual phenylethyl resorcinol, and kojic acid (positive control group).

[0177] (3) Testing method: T / SHRH 015-2018 "Cosmetics - Test Method for Tyrosinase Activity Inhibition". Then, the test results are respectively statistically analyzed in Tables 13, 14, and 15. At the same time, graphs are drawn based on the statistical results.

[0178] Table 13

[0179]

[0180] * The data in the table are mean ± relative deviation.

[0181] * Statistical method: Analysis is carried out using the t-test method, with a significance level of α = 0.05; P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference; P < 0.001 indicates an extremely significant difference.

[0182] Table 14

[0183]

[0184] * The data in the table are mean ± relative deviation. <00004​​​​​​​​​​​​​*Statistical method: The t-test method was used for analysis, and the significance level α = 0.05; P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference; P < 0.001 indicates an extremely significant difference.

[0190] Refer to Table 13, Table 14, and Table 15 and Figure 10 、 Figure 11 and Figure 12 It can be seen that the half-inhibitory concentration IC 50 = 0.043 mg / mL (95% confidence interval IC 50 : 0.039 mg / mL ~ 0.047 mg / mL) of the eutectic of phenethyl resorcinol and proline on the activity inhibition of tyrosinase (bisphenol) has a whitening effect. The half-inhibitory concentration IC 50 = 0.085 mg / mL (95% confidence interval IC 50 : 0.076 mg / mL ~ 0.094 mg / mL) of the physical mixture of phenethyl resorcinol and proline on the activity inhibition of tyrosinase (bisphenol) has a whitening effect. The half-inhibitory concentration IC 50 = 0.037 mg / mL (95% confidence interval IC 50 : 0.034 mg / mL ~ 0.040 mg / mL) of phenethyl resorcinol on the activity inhibition of tyrosinase (bisphenol) has a whitening effect. It shows that the ability of the eutectic of phenethyl resorcinol and proline to inhibit the activity of tyrosinase (bisphenol) is better than that of the physical mixture of phenethyl resorcinol and proline and is equivalent to that of phenethyl resorcinol. However, under the same test concentration, since the content of phenethyl resorcinol in the eutectic of phenethyl resorcinol and proline is less, it shows that the eutectic of phenethyl resorcinol and proline can also exert a good whitening effect with a lower addition amount and less irritation.

[0191] Test Example 11

[0192] Detection of the ability to inhibit the activity of human melanocyte tyrosinase

[0193] (1) Experimental principle: The inhibition of melanin production is mainly achieved through two aspects: one is to inhibit the activity of tyrosinase, and the other is to inhibit the signal pathway of melanin production. Some whitening agents, such as kojic acid and its derivatives and arbutin, inhibit melanin production by inhibiting the activity of tyrosinase. There are also inhibitors that act on the melanin production signal pathway, such as inhibitors of the α-melanocyte stimulating hormone (α-MSH) receptor MCIR protein. In addition, products with antioxidant effects can reduce dopaquinone to levodopa, which can be used to a certain extent in the development of whitening products. This method uses α-MSH to induce melanocytes and uses colorimetry to detect the effect of the sample on tyrosinase inhibition to evaluate its efficacy.

[0194] (2) Experimental materials: phenethylresorcinol-proline eutectic obtained in Example 1, a physical mixture of phenethylresorcinol and proline at a molar ratio of 1:1, phenethylresorcinol and kojic acid (positive control).

[0195] (3) Test method: Cells were seeded into 96-well plates and cultured for 18-24 h. The original culture medium was removed and culture medium containing different concentrations of the test substance and α-MSH was added. The culture was continued for 3 days. After the exposure, the culture medium was removed and the cells were washed twice with PBS. 90 μL of cell lysis solution was added and the cells were lysed by repeated freeze-thaw treatment. The well plate was pre-warmed at 37°C for 5 min and 10 μL of L-DOPA solution was quickly added. After shaking, the absorbance was measured at a wavelength of 405 nm. After reacting at 37°C for 30 min, the absorbance was measured again. The data were analyzed using SPSS and expressed as mean ± standard deviation. If p < 0.05, the difference was considered statistically significant. The test results were then statistically summarized in Tables 16 and 17.

[0196]

[0197] Table 16

[0198]

[0199] Note: # indicates that the difference is statistically significant compared with the blank control group (NC) (p < 0.05);

[0200] *Indicates that the difference is statistically significant compared with the model control group (M) (p<0.05).

[0201] Table 17

[0202]

[0203] As shown in Tables 16 and 17, the tyrosinase activity in the model control group was significantly increased compared with the blank control group (p < 0.05). The relative tyrosinase activity in the positive control group was significantly decreased compared with the model control group (p < 0.05), indicating successful modeling. Compared with the model control group, the relative tyrosinase activity of the phenylethylresorcinol-proline cocrystal at a test concentration of 0.000235 mg / mL was significantly decreased (p < 0.05), by 7.77%, indicating an inhibitory effect on tyrosinase activity. The relative tyrosinase activity of the phenylethylresorcinol-proline cocrystal physical mixture at a test concentration of 0.000235 mg / mL was significantly decreased (p < 0.05), by 4.52%, indicating an inhibitory effect on tyrosinase activity. The relative tyrosinase activity of phenylethylresorcinol at a test concentration of 0.000113 mg / mL was significantly decreased (p < 0.05), by 5.79%, indicating an inhibitory effect on tyrosinase activity. The significant differences between the test substance groups with the same concentration showed that the phenethylresorcinol-proline cocrystal at a concentration of 0.000235 mg / mL had significant differences from the physical mixture of phenethylresorcinol-proline cocrystal and phenethylresorcinol, indicating that the phenethylresorcinol-proline cocrystal had the best inhibitory effect on tyrosinase activity.

[0204] Test Example 12

[0205] Detection of melanin production inhibition ability in human melanocytes

[0206] (1) Experimental principle: The inhibition of melanin production is mainly achieved in two ways: one is to inhibit the activity of tyrosinase, and the other is to inhibit the signal pathway of melanin production. Some whitening agents, such as kojic acid and its derivatives and arbutin, inhibit melanin production by inhibiting the activity of tyrosinase. There are also inhibitors that act on the melanin production signal pathway, such as inhibitors of the α-melanocyte stimulating hormone (α-MSH) receptor MCIR protein. In addition, products with antioxidant effects can reduce dopaquinone to levodopa, which can be used to a certain extent in the development of whitening products. This method uses α-MSH to induce melanocytes and uses colorimetry to detect the effect of the sample on the inhibition of melanin production to evaluate its efficacy.

[0207] (2) Experimental materials: phenethylresorcinol-proline eutectic obtained in Example 1, a physical mixture of phenethylresorcinol-proline at a molar ratio of 1:1, and kojic acid (positive control).

[0208] (3) Test method:

[0209] Cells were seeded into 6-well plates and cultured for 18–24 hours. The original culture medium was removed and replaced with culture medium containing varying concentrations of the test substance and α-MSH. Cultures were continued for 5 days, with the medium changed once. At the end of exposure, the culture medium was removed, the cells were washed once with PBS, and each well was harvested using a cell scraper. Cells were lysed with 1 mol / L NaOH solution (containing 10% DMSO) to obtain cell fluid. The cell fluid was heated at 80°C for 30 minutes to lyse melanosomes. The absorbance of the cell fluid was measured at 490 nm. Data were analyzed using SPSS and presented as mean ± SD. Differences were considered statistically significant if p < 0.05. The results are summarized in Tables 18 and 19.

[0210]

[0211] Table 18

[0212]

[0213] Note: # indicates that the difference is statistically significant compared with the blank control group (NC) (p < 0.05);

[0214] *Indicates that the difference is statistically significant compared with the model control group (M) (p<0.05).

[0215] Table 19

[0216]

[0217] As shown in Tables 18 and 19, the relative melanin content in the model control group was significantly increased compared with the blank control group (p < 0.05); the relative melanin content in the positive control group was significantly decreased compared with the model control group (p < 0.05), indicating successful modeling. Compared with the model control group, the relative melanin content of the phenylethylresorcinol-proline cocrystal at a test concentration of 0.000235 mg / mL was significantly reduced (p < 0.05), by 20.49%, indicating an inhibitory effect on melanin production. The relative melanin content of the phenylethylresorcinol-proline physical mixture at a test concentration of 0.000235 mg / mL was significantly reduced (p < 0.05), by 14.42%, indicating an inhibitory effect on melanin production. The relative melanin content of phenylethylresorcinol at a test concentration of 0.000113 mg / mL was significantly reduced (p < 0.05), by 15.98%, indicating an inhibitory effect on melanin production. The significant differences between the test substance groups with the same concentration showed that the phenethylresorcinol-proline cocrystal at a concentration of 0.000235 mg / mL had significant differences from the physical mixture of phenethylresorcinol-proline cocrystal and phenethylresorcinol, indicating that the phenethylresorcinol-proline cocrystal had the best effect in inhibiting melanin production.

[0218] Test Example 13

[0219] Irritation test

[0220] (1) Experimental materials: 1% emulsion of phenylethylresorcinol-proline eutectic; negative control: blank + filter disc; number of participants: 30.

[0221] (2) Test method: Select qualified patch test equipment and use the closed patch test method. Place 0.020 g to 0.025 g of the test substance in the patch test equipment and apply it to the flexed side of the subject's forearm with a low-allergenic tape. Remove the test substance after 24 hours. Observe the skin reaction 0.5, 24, and 48 hours after removal. Evaluate the stimulation intensity according to Table 20 and summarize the test results in Table 21.

[0222] Table 20

[0223]

[0224] Table 21

[0225]

[0226] As shown in Table 21, due to the use of the same-body self-comparison method, no irritation was observed in the negative control group, which consisted of 30 regions. This demonstrates the validity of the experiment and the reliability of the results. The phenylethylresorcinol-proline cocrystal group showed no irritation at 0.5, 24, or 48 hours, demonstrating the mild and low irritation properties of the phenylethylresorcinol-proline cocrystal.

[0227] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

Claims

1. A phenethylresorcinol-proline eutectic, characterized in that: The structural formula of the phenethylresorcinol proline co-crystal is shown in Formula I: Formula I; The X-ray powder diffraction pattern of the phenethylresorcinol proline cocrystal has characteristic peaks at 2θ angles of 5.94°±0.2°, 15.14°±0.2°, 17.70°±0.2°, 18.99°±0.2° and 20.12°±0.2°.

2. The phenethylresorcinol-proline cocrystal according to claim 1, characterized in that The molecular formula of the phenethylresorcinol proline eutectic is C 24 H 32 N2O6, and in the phenethylresorcinol-proline eutectic, the molar ratio of the phenethylresorcinol to the proline is 1:

2.

3. The phenethylresorcinol-proline cocrystal according to claim 1, wherein The phenethylresorcinol-proline cocrystal is a monoclinic system with a space group of P21, a unit cell parameter of a=8.2050(3) Å, b=9.3756(3) Å, c=15.1517(6) Å, α=90°, β=97.824(4)°, γ=90°, Z=2, and a unit cell volume of V=1154.72(7) Å. 3 .

4. The phenethylresorcinol proline cocrystal according to any one of claims 1 to 3, characterized in that The proline is L-proline.

5. A method for preparing a phenethylresorcinol-proline cocrystal according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Under an inert atmosphere and in the dark, dissolving phenethylresorcinol and proline in an ethanol-water mixed solvent and stirring the mixture at a preset temperature, wherein the volume proportion of water in the ethanol-water mixed solvent is 1-20%, and the preset temperature is 50-85° C., to obtain a precursor solution containing the phenethylresorcinol-proline cocrystal; S2: performing a cooling and crystallization treatment on the precursor solution, wherein the cooling and crystallization treatment step comprises: first performing a first cooling treatment on the precursor solution so that the temperature of the precursor solution is 20-30° C., then performing a second cooling treatment on the precursor solution so that the temperature of the precursor solution is not higher than 10° C., wherein the cooling rate of the first cooling treatment is lower than the cooling rate of the second cooling treatment, and then maintaining the temperature unchanged to perform a crystallization treatment to obtain a solid-liquid mixture; S3: performing solid-liquid separation and drying on the solid-liquid mixture in sequence to obtain the phenethylresorcinol-proline eutectic.

6. The preparation method according to claim 5, characterized in that The volume proportion of water in the ethanol-water mixed solvent is 3-10%.

7. The preparation method according to claim 5, characterized in that The preset temperature is 70~85℃.

8. The preparation method according to claim 5, wherein The cooling rate of the first cooling treatment is 0.5-3°C / min.

9. The preparation method according to any one of claims 5 to 8, characterized in that The step of obtaining the precursor solution containing the phenethylresorcinol proline cocrystal comprises: Under an inert atmosphere and light-shielding conditions, adding proline to the ethanol-water mixed solvent and stirring and mixing at the preset temperature to dissolve the proline in the ethanol-water mixed solvent to obtain a mixed solution; The temperature and stirring speed are maintained unchanged, and the phenethylresorcinol is added to the mixed solution to dissolve the phenethylresorcinol in the mixed solution and react with the proline to obtain a precursor solution containing the phenethylresorcinol-proline cocrystal.

10. The preparation method according to claim 9, characterized in that The molar ratio of the amount of proline added to the amount of phenylethylresorcinol added is (1.9-2.1):1; And / or, in the mixed solution, the mass ratio of the proline to the mixed solution is 1:(5-10).

11. The preparation method according to any one of claims 5 to 8, characterized in that The crystallization treatment is performed at a temperature of 0-10° C., and the crystallization treatment is performed under stirring conditions.

12. The preparation method according to claim 11, characterized in that During the crystallization process, the stirring speed is 50-600 rpm.

13. Use of the phenethylresorcinol proline co-crystal according to any one of claims 1 to 4 in the preparation of cosmetics or skin care products.

Images