Phenylethyl resorcinol proline eutectic and preparation method and application thereof

Abstract

The invention provides a phenethyl resorcinol proline eutectic crystal as well as a preparation method and application thereof, and belongs to the technical field of eutectic crystal manufacturing. The structural formula of the phenethyl resorcinol proline eutectic crystal is as shown in a formula I. The phenethyl resorcinol proline eutectic crystal has a better whitening effect and also has the advantages of lower irritation and higher stability.

Description

Technical Field

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

[0002] Phenethyl resorcinol can effectively inhibit the activity of tyrosinase (tyrosinase is the key enzyme in the formation of melanin). In addition, phenethyl resorcinol can also inhibit the formation of a large number of oxygen free radicals caused by ultraviolet radiation (oxygen free radicals can promote the formation of melanin), so phenethyl resorcinol has a good whitening and freckle removal effect. However, phenethyl resorcinol is irritating to the skin and can only be added to a few cosmetics with a maximum concentration of no more than 0.5%. In addition, phenethyl resorcinol has poor stability and is sensitive to light. Under light conditions, it will quickly change from white to beige and finally to pink, causing the phenethyl resorcinol content to drop rapidly, affecting the appearance of the cosmetics and the bioavailability of phenethyl resorcinol. Therefore, it is currently necessary 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 co-crystal and a preparation method and application thereof. The phenethylresorcinol-proline co-crystal 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: In a first aspect, an embodiment of the present application provides a phenethylresorcinol-proline co-crystal, and the structural formula of the phenethylresorcinol-proline co-crystal is shown in Formula I:

[0005] Formula I.

[0006] In the above technical solution, the double bond oxygen in proline has a lone pair of electrons, and phenethylresorcinol has functional groups such as a benzene ring and a phenolic hydroxyl group, so that phenethylresorcinol can interact with proline through intermolecular forces such as hydrogen bonds and van der Waals forces to form a crystalline material that is regularly arranged and stably exists in the same lattice. After phenethylresorcinol and proline form a eutectic, the crystal arrangement is different from that of phenethylresorcinol alone, so that the phenethylresorcinol-proline eutectic not only has the whitening effect of phenethylresorcinol, but also has the advantages of lower irritation and higher stability.

[0007] In some optional 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.

[0008] In some optional embodiments, the phenethylresorcinol-proline cocrystal is a monoclinic system 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, unit cell volume V=1154.72(7) Å 3 .

[0009] In some alternative embodiments, the proline is L-proline.

[0010] 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°.

[0011] 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: S1. In an inert atmosphere and light-proof conditions, phenethylresorcinol and proline are dissolved in an ethanol-water mixed solvent and stirred 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 phenethylresorcinol-proline eutectic; S2. The precursor solution is subjected to a cooling and crystallization treatment, and the cooling and crystallization treatment steps include: firstly, the precursor solution is subjected to a first cooling treatment, so that the temperature of the precursor solution is 20-30°C, and then the precursor solution is subjected to a second cooling treatment, 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 the temperature is maintained unchanged for crystallization treatment to obtain a solid-liquid mixture; S3. The solid-liquid mixture is subjected to a solid-liquid separation treatment and a drying treatment in sequence to obtain a phenethylresorcinol-proline eutectic.

[0012] In the above technical scheme, according to the above process, a phenethylresorcinol-proline eutectic as provided in the first aspect embodiment 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 as to obtain a phenethylresorcinol-proline eutectic in the subsequent reaction; 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 less than the cooling rate of the second cooling treatment, which can effectively reduce the probability of explosion after phenethylresorcinol and proline react to form a eutectic (the two react quickly and explosion is likely to occur, which will cause a large amount of solid to precipitate quickly, and large agglomerated particles to appear. The obtained product is usually a non-eutectic product mixed with a large amount of impurities), thereby preparing a eutectic product with high purity and yield.

[0013] In some optional embodiments, the volume proportion of water in the ethanol-water mixed solvent is 3-10%.

[0014] In the above technical solution, the volume proportion of water in the ethanol-water mixed solvent is limited to a more suitable range, which can dissolve phenylethylresorcinol and proline more quickly and thoroughly, so as to improve the preparation efficiency and yield of the eutectic product.

[0015] In some optional embodiments, the preset temperature is 70-85°C.

[0016] 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.

[0017] In some optional embodiments, the cooling rate of the first cooling treatment is 0.5-3 °C / min.

[0018] In the above technical solution, the cooling rate of the first cooling treatment is limited within an appropriate range, which can more effectively reduce the probability of explosion after the reaction of phenethylresorcinol and proline, thereby obtaining a eutectic product with higher purity and yield.

[0019] In some optional embodiments, the step of obtaining a precursor solution containing phenethylresorcinol-proline cocrystal comprises: In an inert atmosphere and light-proof conditions, proline is added to an ethanol-water mixed solvent and stirred and mixed at a preset temperature so that the proline is dissolved in the ethanol-water mixed solvent to obtain a mixed solution; maintaining the temperature and stirring speed unchanged, phenethylresorcinol is added to the mixed solution so that the phenethylresorcinol is dissolved in the mixed solution and reacts with proline to obtain a precursor solution containing phenethylresorcinol-proline eutectic.

[0020] In the above technical solution, proline is dissolved at a preset temperature and under stirring conditions. On the one hand, it helps to dissolve proline quickly and thoroughly. On the other hand, it can also enable the mixed system to reach the temperature conditions required for the reaction in advance, so that phenylethyl resorcinol 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.

[0021] In some optional embodiments, the molar ratio of the amount of proline added to the amount of phenethylresorcinol added 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).

[0022] In the above technical scheme, the molar ratio of the added amount of proline and the added amount of phenylethylresorcinol is limited within the above range, so that 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.

[0023] In some optional embodiments, the temperature of the crystallization treatment is 0-10° C., and the crystallization treatment is performed under stirring conditions.

[0024] 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.

[0025] In some optional embodiments, during the crystallization process, the stirring speed is 50-600 rpm.

[0026] In the above technical solution, during the crystallization process, the adjustable range of the stirring speed is relatively wide, which is convenient for adaptive adjustment according to the volume of the mixed solution and the volume of the reaction container.

[0027] In a third aspect, an embodiment of the present application provides an application 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

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. 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 related drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a schematic diagram of the X-ray single crystal diffraction structure of the phenethylresorcinol proline co-crystal of Example 1 of the present application; Figure 2 This is the H NMR spectrum of the phenethylresorcinol proline cocrystal of Example 1 of the present application; Figure 3 This is the NMR carbon spectrum of the phenethylresorcinol proline cocrystal of Example 1 of the present application; Figure 4 The infrared spectra of the phenethylresorcinol-proline cocrystal, phenethylresorcinol and proline of Example 1 of the present application; Figure 5 A scanning electron micrograph of phenethyl resorcinol provided for this application; Figure 6 A scanning electron micrograph of proline provided for this application; Figure 7 This is a scanning electron microscope image of the phenethylresorcinol proline cocrystal of Example 1 of the present application; Figure 8 This is a scanning electron microscope image of the phenethylresorcinol proline cocrystal of Example 2 of the present application; Fig. 9 is the X-ray powder diffraction pattern of the phenethylresorcinol proline cocrystal of Example 1 of the present application; Fig.10 The inhibition curve of the phenethylresorcinol-proline co-crystal provided in the present application on the inhibition of tyrosinase (bisphenol) activity; Fig.11 The inhibition curve of the physical mixture of phenethylresorcinol and proline provided in the present application on the inhibition of tyrosinase (bisphenol) activity; Fig.12 The inhibition curve of phenethyl resorcinol on tyrosinase (bisphenol) activity provided in this application. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0031] It should be noted that “and / or” in this application, such as “Feature 1 and / or Feature 2”, refers to the three situations of “Feature 1” alone, “Feature 2” alone, or “Feature 1” plus “Feature 2”.

[0032] In addition, in the description of the present application, unless otherwise specified, the meaning of "multiple" in "one or more" refers to two or more; the range of "value a~value b" includes the two end values ​​"a" and "b", and the "unit" in "value a~value b+unit" represents the "unit" of both "value a" and "value b".

[0033] The following is a detailed description of a phenethylresorcinol proline co-crystal according to an embodiment of the present application and its preparation method and application.

[0034] In a first aspect, an embodiment of the present application provides a phenethylresorcinol-proline co-crystal, and the structural formula of the phenethylresorcinol-proline co-crystal is shown in Formula I:

[0035] Formula I.

[0036] In the present application, the double bond oxygen in proline has a lone pair of electrons, and phenethylresorcinol has functional groups such as a benzene ring and a phenolic hydroxyl group, so that phenethylresorcinol can interact with proline through intermolecular forces such as hydrogen bonds and van der Waals forces to form a crystalline material that is regularly arranged and stably exists in the same lattice. After phenethylresorcinol and proline form a eutectic, the crystal arrangement is different from that of phenethylresorcinol alone, so that the phenethylresorcinol-proline eutectic not only has the whitening effect of phenethylresorcinol, but also has the advantages of lower irritation and higher stability.

[0037] It should be noted that, on the one hand, phenethyl resorcinol has a strong antioxidant effect and can non-competitively inhibit the activity of tyrosinase. At the same time, phenethyl resorcinol also has an inhibitory effect on the formation of melanin, thereby reducing the production of melanin. On the other hand, proline is an amino acid component in collagen fibers and can be used as a nutrient. At the same time, proline has the effect of softening the stratum corneum, which can strengthen the function of keratin and make the skin moisturizing. In addition, proline can promote muscle growth, improve immune function, promote energy metabolism, and maintain muscle and joint vitality and improve skin sagging and wrinkling. Phenethyl resorcinol and proline form a eutectic. On the basis of retaining the efficacy of both, it can also reduce the irritation of phenethyl resorcinol and improve its stability, achieving better application effects.

[0038] As an example, the molecular formula of phenethylresorcinol proline cocrystal is C24 H 32 N2O6, and in the phenethylresorcinol-proline eutectic, the molar ratio of phenethylresorcinol to proline is 1:2.

[0039] As an example, 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 .

[0040] As an example, the proline is L-proline.

[0041] As an example, 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°.

[0042] In order to better understand the technical solution, the present application simulates and provides a schematic diagram of the molecular structure of phenethylresorcinol proline cocrystal (see Figure 1 ).

[0043] 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: S1 In an inert atmosphere and light-proof conditions, dissolve phenethylresorcinol and proline in an ethanol-water mixed solvent and react with stirring 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, the volume proportion is any one of 1%, 2%, 5%, 7%, 10%, 12%, 15%, 17% and 20% or a range between any two of them), and the preset temperature is 50-85°C (for example, but not limited to, the temperature is any one of 50°C, 60°C, 70°C, 75°C, 80°C and 85°C or a range between any two of them), to obtain a precursor solution containing phenethylresorcinol-proline eutectic.

[0044] S2 The precursor solution is subjected to a cooling and crystallization treatment, and the steps of the cooling and crystallization treatment include: firstly, the precursor solution is subjected to a first cooling treatment, so that the temperature of the precursor solution is 20-30°C (for example, but not limited to, the temperature is 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), and then the precursor solution is subjected to a second cooling treatment, 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 the temperature is maintained unchanged to perform crystallization treatment to obtain a solid-liquid mixture.

[0045] S3: performing solid-liquid separation treatment and drying treatment on the solid-liquid mixture in sequence to obtain phenethylresorcinol-proline eutectic.

[0046] In the present application, according to the above process, a phenethylresorcinol-proline eutectic as provided in the first embodiment 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 as to obtain a phenethylresorcinol-proline eutectic in the subsequent reaction; 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 temperature of the crystallization treatment by gradient cooling, and the cooling rate of the first cooling treatment is set to be less than the cooling rate of the second cooling treatment, which can effectively reduce the probability of explosion after phenethylresorcinol and proline react to form a eutectic (the two react quickly and it is easy to explode. The explosion will cause a large amount of solid to precipitate quickly, and large agglomerated particles will appear. The obtained product is usually a non-eutectic product mixed with a large amount of impurities), thereby preparing a eutectic product with high purity and yield.

[0047] 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.

[0048] In this embodiment, the volume ratio of water in the ethanol-water mixed solvent is limited to a more suitable range, which can dissolve phenethylresorcinol and proline more quickly and thoroughly, thereby improving the preparation efficiency and yield of the eutectic product.

[0049] As an example, the preset temperature is 70~85℃, for example 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.

[0050] 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.

[0051] 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.

[0052] In this embodiment, the cooling rate of the first cooling treatment is limited within an appropriate range, which can more effectively reduce the probability of explosion after the reaction of phenethylresorcinol and proline, thereby obtaining a eutectic product with higher purity and yield.

[0053] As an example, the steps of obtaining a precursor solution containing phenethylresorcinol-proline cocrystal include: In an inert atmosphere and light-proof conditions, proline is added to an ethanol-water mixed solvent and stirred and mixed at a preset temperature so that the proline is dissolved in the ethanol-water mixed solvent to obtain a mixed solution; maintaining the temperature and stirring speed unchanged, phenethylresorcinol is added to the mixed solution so that the phenethylresorcinol is dissolved in the mixed solution and reacts with proline to obtain a precursor solution containing phenethylresorcinol-proline eutectic.

[0054] 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 phenylethyl resorcinol 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.

[0055] As an example, the molar ratio of the amount of proline added to the amount of phenethylresorcinol added is (1.9~2.1):1, for example but not limited to the molar ratio of 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 of any one of 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 or a range between any two of them.

[0056] In this embodiment, the molar ratio of the added amount of proline and the added amount of phenylethylresorcinol is limited within the above range, so that 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.

[0057] 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.

[0058] As an example, the temperature of the crystallization treatment is 0~10°C, for example but not limited to the temperature of any one point value among 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C and 10°C, or a range value between any two of them; and the crystallization treatment is carried out under stirring conditions.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] In other possible implementations, during the crystallization process, a static crystallization method may be adopted.

[0063] 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 being 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 being any one of 12 h, 15 h, 30 h, 40 h and 48 h or a range between any two of them.

[0064] In this embodiment, vacuum drying is adopted, which has the advantages of lower drying temperature and less prone to product denaturation.

[0065] It should be noted that any process or step not specifically described or limited in the preparation process of phenethylresorcinol-proline cocrystal may be arranged according to conventional selection in the art.

[0066] In a third aspect, an embodiment of the present application provides an application of the phenethylresorcinol proline co-crystal provided in the embodiment of the first aspect in the preparation of medicines, cosmetics or skin care products.

[0067] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0068] Example 1 The present application provides a method for preparing a phenethylresorcinol-proline cocrystal, comprising the following steps: S1 In an inert atmosphere (nitrogen) and light-proof conditions, 51.8 g of proline is added to 300 g of an ethanol-water mixed solvent (the volume ratio of ethanol to water is 95:5) and stirred and mixed at a preset temperature, wherein the preset temperature is 85°C and the rotation speed is 400 rpm, so that the proline is dissolved in the ethanol-water mixed solvent to obtain a mixed solution; maintaining the temperature and the stirring speed unchanged, 48.2 g of phenethylresorcinol is added to the mixed solution, so that the phenethylresorcinol is dissolved in the mixed solution and reacts with proline, to obtain a precursor solution containing phenethylresorcinol-proline eutectic.

[0069] S2: First, the precursor solution is subjected to the first cooling treatment so that the temperature of the precursor solution is 25°C, and then the precursor solution is subjected to the second cooling treatment so that the temperature of the precursor solution is 0°C, wherein 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, and then the temperature is maintained unchanged to carry out stirring crystallization treatment for 3 h, wherein the stirring speed is 200 rpm, to obtain a solid-liquid mixture.

[0070] S3: filtering the solid-liquid mixture, and drying the obtained solid in a vacuum drying oven, wherein the drying time is 24 h and the drying temperature is 50° C., to obtain phenethylresorcinol-proline cocrystals with a yield of 89.6%.

[0071] Example 2 The present application example provides a method for preparing phenethylresorcinol-proline cocrystal, 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%.

[0072] Example 3 The present application example 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%.

[0073] Comparative Example 1 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.

[0074] Comparative Example 2 The comparative example of the present application provides a method for preparing phenethylresorcinol-proline cocrystal, which is different from Example 1 only in that the volume ratio of ethanol to water is 50:50.

[0075] Test Example 1 Phenethylresorcinol proline cocrystals were prepared according to the preparation methods of Examples 1 to 3 and Comparative Examples 1 to 2, respectively. Then, each sample was numbered, and then, the element content of each sample was tested using an element analyzer, and the results were statistically summarized in Table 1.

[0076] Table 1

[0077] It should be noted that the theoretical value is calculated based on the standard that the molar ratio of phenethylresorcinol to proline is 1:2.

[0078] 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 co-crystals; the test result of Comparative Example 1 is quite different from the theoretical value, indicating that the cooling rate is too fast, resulting in detonation during the preparation process, which makes it difficult to prepare the co-crystal product; in Comparative Example 2, due to the unreasonable setting of the volume ratio of the ethanol-water mixed solvent, phenethylresorcinol is difficult to disperse and dissolve in the ethanol-water mixed solvent, which makes it difficult to continue the reaction, so no co-crystal product is obtained.

[0079] Test Example 2 Qualitative Analysis of Phenethyl Resorcinol-Proline Cocrystal Test method: The phenethylresorcinol-proline co-crystal obtained in Example 1 was qualitatively analyzed.

[0080] Table 2 Single crystal data of phenethylresorcinol-proline cocrystal

[0081] Table 3 Atomic coordinates and isotropic atomic displacement parameters of phenethylresorcinol-proline cocrystal

[0082] It should be noted that U(eq) is defined as one third of the trace of the orthogonal Uij tensor.

[0083] Table 4 Anisotropic atomic displacement parameters of phenethylresorcinol-proline cocrystal

[0084] It should be noted that the anisotropic atomic displacement factor power is expressed as: -2π 2 [h 2 a* 2 U 11 +2hka*b*U 12 +…].

[0085] Table 5 The chemical bond lengths of phenethylresorcinol-proline cocrystals

[0086] Table 6 Bond angles of phenethylresorcinol-proline cocrystal

[0087] Table 7 Hydrogen bonding parameters of phenethylresorcinol-proline cocrystal

[0088] 1 -1+X,+Y,+Z; 2 2-X,1 / 2+Y,1-Z; 3 1-X,-1 / 2+Y,1-Z.

[0089] Table 8 Torsion angle parameters of phenethylresorcinol-proline cocrystal

[0090] Table 9 Hydrogen atom coordinates of phenethylresorcinol-proline cocrystal (Å×10 4 ) and the isotropic atomic displacement parameters (Å 2 ×10 3 )

[0091] Test Example 3 H NMR spectroscopy ( 1 H-NMR) characterization Test method: The phenethylresorcinol proline cocrystal obtained in Example 1 was subjected to hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR) characterization, and MeOD solvent was selected as the test solvent.

[0092] See also Figure 2From the H NMR spectrum, we can clearly find 12 hydrogen atoms of phenethylresorcinol and 14 hydrogen atoms on proline, and the rest are a small amount of residual water peaks and deuterated reagent peaks. No obvious impurity peaks are seen, which shows that in the phenethylresorcinol-proline cocrystal, phenethylresorcinol and proline exist in a molar ratio of 1:2.

[0093] Test Example 4 C NMR spectroscopy ( 13 C-NMR) characterization Test method: The phenethylresorcinol proline cocrystal obtained in Example 1 was subjected to carbon nuclear magnetic resonance spectroscopy ( 13 C-NMR) characterization, and MeOD solvent was selected as the test solvent.

[0094] See also Figure 3 The test results show that phenethylresorcinol and proline in the phenethylresorcinol-proline eutectic are present in a molar ratio of 1:2.

[0095] Test Example 5 Infrared spectroscopy characterization Test method: 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.

[0096] See also Figure 4 The infrared spectrum of 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.

[0097] Test Example 6 Morphological characterization Test method: The phenethylresorcinol, proline, and the phenethylresorcinol-proline co-crystals obtained in Example 1 and Example 2 were characterized by scanning electron microscopy.

[0098] See also Figure 5 , Figure 6 and Figure 7 The morphology of ethylresorcinol-proline eutectic is prismatic block with smooth surface and distinct boundary, which is completely different from the morphology of phenylethylresorcinol and proline.

[0099] See also Figure 7 and Figure 8Compared with stirred crystallization, the particles of phenethylresorcinol proline eutectic obtained by static crystallization are larger, the surface is rougher and there are debris, indicating that the stirred crystallization method is better.

[0100] Test Example 7 X-ray powder diffraction (XRD) test Test method: 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°.

[0101] See also Fig. 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°.

[0102] Test Example 8 Stability test Test method: The groups of samples prepared in Examples 1 to 3 and phenethyl resorcinol monomer were used as test samples. Each group of samples was divided into 4 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 were statistically summarized in Table 10.

[0103] Table 10

[0104] 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 phenethylresorcinol and proline have excellent stability after forming a cocrystal.

[0105] Test Example 9 In vitro cytotoxicity assay (1) Detection method: human melanocyte cytotoxicity MTT assay.

[0106] (2) Experimental materials: phenethylresorcinol-proline cocrystal and phenethylresorcinol obtained in Example 1.

[0107] (3) Experimental method: Prepare cell suspension and adjust the cell concentration to 3.0×10 4 / mL, the cell suspension was inoculated into a 96-well cell culture plate, 100 μL per well, and cultured for 24 h. The original culture medium in the well was discarded, and 100 μL of test samples of different concentrations and a control group (Conrrol control: DMEM medium containing 10% FBS, Blank control: DMEM medium) were added to each well. Incubate in the incubator for 72±0.5h. 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 the incubator for 3±0.5 h. The liquid in the well was removed, 100 μL of DMSO was added to each well, and after oscillating on an oscillator for 10-15 min, the absorbance was measured at a wavelength of 570 nm on an ELISA reader. The data of each group were expressed as mean ± standard deviation, and the cell activity of the control group was 100%, and the relative cell activity (Viability) of each group was calculated, and the test results were statistically reported in Tables 11 and 12.

[0108]

[0109] 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.

[0110] Table 11 Experimental results of phenethylresorcinol-proline eutectic (Mean±SD)

[0111] Table 12 Experimental results of phenethyl resorcinol (Mean±SD)

[0112] Referring to Tables 11 and 12 (the test data were fitted by GraphPad Prism5), the concentration of phenethylresorcinol-proline cocrystal 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 cocrystal was higher than that of phenethylresorcinol.

[0113] Test Example 10 Tyrosinase activity inhibition test (1)Experimental principle: In the biosynthesis of skin melanin, tyrosinase is the key enzyme that 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 inhibitory effects 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 can be evaluated.

[0114] (2)Experimental materials: The phenethyl resorcinol-proline cocrystal obtained in Example 1, a physical mixture of phenethyl resorcinol and proline with a molar ratio of 1:2, individual phenethyl resorcinol, and kojic acid (positive control group).

[0115] (3)Testing method: T / SHRH 015-2018 "Cosmetics - Test Method for Tyrosinase Activity Inhibition", and then the test results were respectively tabulated in Tables 13, 14, and 15. Meanwhile, graphs were plotted based on the statistical results.

[0116] Table 13

[0117] *Data in the table are mean ± relative deviation.

[0118] *Statistical method: Analyzed 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.

[0119] Table 14

[0120] *Data in the table are mean ± relative deviation.

[0121] *Statistical method: Analyzed 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.

[0122] Table 15

[0123] *Data in the table are mean ± relative deviation.

[0124] *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 significant difference; P < 0.01 indicates very significant difference; P < 0.001 indicates extremely significant difference.

[0125] Refer to Table 13, Table 14 and Table 15 and Fig.10 、 Fig.11 and Fig.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 phenethyl resorcinol proline eutectic on the inhibition of tyrosinase (bisphenol) activity 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 inhibition of tyrosinase (bisphenol) activity 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 inhibition of tyrosinase (bisphenol) activity has a whitening effect. It shows that the ability of phenethyl resorcinol proline eutectic to inhibit tyrosinase (bisphenol) activity is superior to 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 phenethyl resorcinol proline eutectic is less, it shows that phenethyl resorcinol proline eutectic can also exert good whitening effect with lower addition amount and less irritation.

[0126] Test Example 11 Detection of the inhibitory ability of human melanocyte tyrosinase (1) Experimental principle: The inhibition of melanogenesis is mainly achieved through two aspects. One is to inhibit the activity of tyrosinase, and the other is to inhibit the signal pathway of melanogenesis. Some whitening agents such as kojic acid and its derivatives and arbutin, etc., inhibit the production of melanin by inhibiting the activity of tyrosinase. There are also those that act on the melanogenesis signal pathway, such as inhibitors targeting the α-melanocyte stimulating hormone (α-MSH) receptor MC1R protein. In addition, products with antioxidant effects can reduce dopaquinone to L-dopa, which can be used for the development of whitening products to a certain extent. In this method, α-MSH is used to induce melanocytes, and the colorimetric method is used to detect the effect of the sample on the inhibition of tyrosinase to evaluate its efficacy.

[0127] (2) Experimental materials: the phenethylresorcinol-proline eutectic obtained in Example 1, a physical mixture of phenethylresorcinol-proline at a molar ratio of 1:1, and phenethylresorcinol and kojic acid (positive control).

[0128] (3) Test method: Inoculate cells into 96-well plates and culture for 18-24 h; remove the original culture medium, add culture medium containing different concentrations of the test substance and α-MSH, and culture continuously for 3 days; after exposure, remove the culture medium, wash twice with PBS, add 90 μL of cell lysis solution, and treat by repeated freezing and thawing to lyse the cells; preheat the plate at 37°C for 5 min, quickly add 10 μL L-DOPA solution, shake and measure the absorbance at 405 nm. After reacting at 37°C for 30 min, measure the absorbance again. Data were analyzed using SPSS and expressed as mean ± standard deviation. If p < 0.05, the difference was considered statistically significant, and the test results were statistically reported in Tables 16 and 17.

[0129]

[0130] Table 16

[0131] Note: # indicates that the difference is statistically significant compared with the blank control group (NC) (p<0.05); *Indicates that the difference was statistically significant compared with the model control group (M) (p<0.05).

[0132] Table 17

[0133] As shown in Table 16 and Table 17, compared with the blank control group, the tyrosinase activity of the model control group was significantly increased (p<0.05); compared with the model control group, the relative content of tyrosinase activity of the positive control group was significantly decreased (p<0.05), indicating that the model was successfully established. Compared with the model control group, the relative content of tyrosinase activity of phenethylresorcinol-proline cocrystal at the test concentration of 0.000235 mg / mL was significantly reduced (p<0.05), decreased by 7.77%, and had the effect of inhibiting tyrosinase activity; the relative content of tyrosinase activity of phenethylresorcinol-proline cocrystal physical mixture at the test concentration of 0.000235 mg / mL was significantly reduced (p<0.05), decreased by 4.52%, and had the effect of inhibiting tyrosinase activity; the relative content of tyrosinase activity of phenethylresorcinol at the test concentration of 0.000113 mg / mL was significantly reduced (p<0.05), decreased by 5.79%, and had the effect of inhibiting 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.

[0134] Test Example 12 Detection of melanin production inhibition ability in human melanocytes (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 the production of melanin 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 in the development of whitening products to a certain extent. 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.

[0135] (2) Experimental materials: the 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).

[0136] (3) Test method: The cells were inoculated into 6-well plates and cultured for 18-24 h; the original culture medium was removed, and the culture medium containing different concentrations of the test substance and α-MSH was added, and the culture was continued for 5 days, with the medium changed once in the middle; at the end of exposure, the culture medium was removed, washed once with PBS, and the cells in each well were collected by cell scraping; 1 mol / L NaOH solution (containing 10% DMSO) was added to dissolve the cells and obtain cell fluid; the cell fluid was heated at 80°C for 30 min to lyse the melanosomes; the cell fluid was taken to measure the absorbance value at a wavelength of 490 nm. The data were analyzed using SPSS and expressed as mean ± standard deviation. If p < 0.05, the difference was considered statistically significant, and then the test results were statistically reported in Tables 18 and 19.

[0137]

[0138] Table 18

[0139] Note: # indicates that the difference is statistically significant compared with the blank control group (NC) (p<0.05); *Indicates that the difference was statistically significant compared with the model control group (M) (p<0.05).

[0140] Table 19

[0141] Referring to Table 18 and Table 19, compared with the blank control group, the relative content of melanin in the model control group was significantly increased (p<0.05); compared with the model control group, the relative content of melanin in the positive control group was significantly decreased (p<0.05), indicating that the model was successfully established. Compared with the model control group, the relative content of melanin in the phenethylresorcinol-proline cocrystal was significantly reduced at a test concentration of 0.000235 mg / mL (p<0.05), a decrease of 20.49%, which had the effect of inhibiting melanin production; the relative content of melanin in the phenethylresorcinol-proline physical mixture was significantly reduced at a test concentration of 0.000235 mg / mL (p<0.05), a decrease of 14.42%, which had the effect of inhibiting melanin production; the relative content of melanin in the phenethylresorcinol was significantly reduced at a test concentration of 0.000113 mg / mL (p<0.05), a decrease of 15.98%, which had the effect of inhibiting 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.

[0142] Test Example 13 Irritation test (1) Experimental materials: 1% emulsion of phenethylresorcinol-proline eutectic; negative control: blank + filter disc; number of subjects: 30.

[0143] (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 curved side of the subject's forearm with a low-allergenic tape. Remove the test substance after 24 hours and observe the skin reaction at 0.5, 24, and 48 hours after removal. Evaluate the stimulation intensity according to Table 20 and summarize the test results in Table 21.

[0144] Table 20

[0145] Table 21

[0146] As shown in Table 21, due to the use of the same body self-comparison method, the negative control group had a total of 30 regions, and no irritation occurred in the negative control group, proving that the experiment was effective and the results were credible. The phenethylresorcinol proline eutectic group had no irritation at 0.5 h, 24 h, and 48 h, proving that the phenethylresorcinol proline eutectic was mild and low in irritation.

[0147] 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 the 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 work are within the scope of protection of the present 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.

2. The phenethylresorcinol-proline eutectic 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, characterized in that The phenethylresorcinol-proline eutectic is a monoclinic crystal 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 .

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

5. The phenethylresorcinol proline co-crystal according to any one of claims 1 to 3, characterized in that 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°.

6. A method for preparing the phenethylresorcinol proline cocrystal according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. In an inert atmosphere and light-proof conditions, dissolving phenethylresorcinol and proline in an ethanol-water mixed solvent and stirring and reacting 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 eutectic; S2: performing a cooling and crystallization treatment on the precursor solution, wherein the cooling and crystallization treatment step comprises: firstly performing a first cooling treatment on the precursor solution so that the temperature of the precursor solution is 20-30°C, 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 a crystallization treatment to obtain a solid-liquid mixture; S3: performing solid-liquid separation and drying treatments on the solid-liquid mixture in sequence to obtain the phenethylresorcinol-proline eutectic.

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

8. The preparation method according to claim 6, characterized in that: The preset temperature is 70~85℃.

9. The preparation method according to claim 6, characterized in that The cooling rate of the first cooling treatment is 0.5-3°C / min.

10. The preparation method according to any one of claims 6 to 9, characterized in that The step of obtaining the precursor solution containing the phenethylresorcinol proline eutectic comprises: Under an inert atmosphere and light-proof conditions, adding proline to the ethanol-water mixed solvent and stirring and mixing at the preset temperature so that the proline is dissolved in the ethanol-water mixed solvent to obtain a mixed solution; The temperature and stirring speed are maintained unchanged, and the phenethyl resorcinol is added to the mixed solution, so that the phenethyl resorcinol is dissolved in the mixed solution and reacts with the proline to obtain a precursor solution containing the phenethyl resorcinol-proline eutectic.

11. The preparation method according to claim 10, characterized in that: The molar ratio of the amount of proline added to the amount of phenethyl resorcinol 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).

12. The preparation method according to any one of claims 6 to 9, characterized in that The crystallization treatment is carried out at a temperature of 0-10° C., and the crystallization treatment is carried out under stirring conditions.

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

14. Use of the phenethylresorcinol proline co-crystal according to any one of claims 1 to 5 in the preparation of medicines, cosmetics or skin care products.

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