A phenylethylresorcinol betaine eutectic and its preparation method and application

Abstract

This application provides a phenethylresorcinol betaine cocrystal, its preparation method, and application, belonging to the field of cocrystal manufacturing technology. The structural formula of the phenethylresorcinol betaine cocrystal is shown in Formula I. The phenethylresorcinol betaine cocrystal exhibits excellent whitening effects and good water solubility, resulting in ideal bioavailability and compatibility.

Description

Technical Field

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

[0002] Phenethyl resorcinol effectively inhibits tyrosinase activity (tyrosinase is a key enzyme in melanin formation). Furthermore, it inhibits the formation of numerous oxygen free radicals induced by UV radiation (oxygen free radicals promote melanin formation), resulting in its promising whitening and freckle-removing effects. However, phenethyl resorcinol is poorly water-soluble, practically insoluble in water. This extremely low solubility results in low bioavailability and poor compatibility (it can only be used with certain polyols and polar oils. The presence of these oils and polyols makes the formulated product thick and viscous, making it difficult to apply to different dosage forms). Therefore, despite its clear mechanism of action and significant efficacy, phenethyl resorcinol's poor water solubility has hampered its promotion and application. Summary of the Invention

[0003] The purpose of the present application is to provide a phenethylresorcinol betaine cocrystal and its preparation method and application. The phenethylresorcinol betaine cocrystal has good whitening effect and good water solubility, so that it has relatively ideal bioavailability and compatibility.

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

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

[0006]

[0007] Formula I.

[0008] In the above technical solution, the amino group in betaine has a strong electronegativity and forms an internal salt structure. Phenethylresorcinol has functional groups such as a benzene ring and a phenolic hydroxyl group. This allows phenethylresorcinol to interact with betaine 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 betaine form a cocrystal, the crystal arrangement is different from that of phenethylresorcinol alone. This makes the phenethylresorcinol-betaine cocrystal not only have the whitening effect of phenethylresorcinol, but also have good water solubility, making it have more ideal bioavailability and compatibility.

[0009] In some optional embodiments, the molecular formula of the phenethylresorcinol betaine cocrystal is C 19 H25 NO4, and in the phenethylresorcinol betaine eutectic, the molar ratio of phenethylresorcinol to betaine is 1:1.

[0010] In some optional embodiments, the phenethylresorcinol betaine cocrystal is monoclinic, with a space group of P21 / c, unit cell parameters of a=17.6546(9) Å, b=9.1649(5) Å, c=11.6028(6) Å, α=90°, β=104.814(5)°, γ=90°, Z=4, and unit cell volume V=1814.96(17) Å 3 .

[0011] In some optional embodiments, the X-ray powder diffraction pattern of the phenylethylresorcinol betaine cocrystal has characteristic peaks at 2θ angles of 5.29°±0.2°, 18.41°±0.2°, 20.81°±0.2°, 21.59°±0.2°, 26.08°±0.2° and 35.85°±0.2°.

[0012] In a second aspect, the present invention provides a method for preparing the phenethylresorcinol betaine cocrystal as provided in the first aspect, comprising the following steps:

[0013] S1. Under an inert atmosphere and in the dark, dissolving phenylethylresorcinol and betaine in an organic solvent to obtain a mixed solution. S2. Stirring the mixed solution at a first preset temperature, wherein the first preset temperature is 50°C to 80°C, to allow the phenylethylresorcinol and betaine to react and obtain a precursor solution containing phenylethylresorcinol betaine cocrystals. S3. Successively subjecting the precursor solution to cooling and crystallization treatments, solid-liquid separation treatments, and drying treatments to obtain phenylethylresorcinol betaine cocrystals.

[0014] According to the above process, a phenethylresorcinol-betaine co-crystal as provided in the embodiment of the first aspect can be prepared, wherein the stirring reaction is carried out at 50-80° C. to provide a more suitable driving force, so that phenethylresorcinol and betaine can react more thoroughly. Specifically, under the combined action of mechanical force and temperature, the amino groups in betaine and the hydroxyl groups in phenethylresorcinol attract each other, so that hydrogen bonds and other interaction forces are generated between the betaine and phenethylresorcinol, thereby bonding them together, thereby forming a co-crystal that is regularly arranged and stably exists in the same crystal lattice.

[0015] In some optional embodiments, the step of dissolving phenylethylresorcinol and betaine in an organic solvent under an inert atmosphere and light-proof conditions to obtain a mixed solution comprises:

[0016] In an inert atmosphere and light-proof conditions, phenylethyl resorcinol is firstly dissolved in an organic solvent to obtain a mixed solution intermediate, and then betaine is dissolved in the mixed solution intermediate to obtain a mixed solution.

[0017] In the above technical solution, the poorly soluble and easily deteriorating phenylethyl resorcinol is first dissolved in an organic solvent, and then the relatively stable and easily soluble betaine is dissolved. This has the advantages of a more reasonable sample addition order and easy operation.

[0018] In some optional embodiments, the step of first dissolving phenylethylresorcinol in an organic solvent is performed under ultrasonic conditions.

[0019] In the above technical solution, the vibration energy of ultrasound can reduce the interaction force between molecules in the liquid, thereby lowering the energy threshold for solute dissolution, thereby accelerating the dissolution rate and shortening the dissolution time.

[0020] In some optional embodiments, the ultrasonic frequency is 20-60 kHz, and the ultrasonic power is 700-6000 W.

[0021] In the above technical solution, by limiting the ultrasonic frequency and ultrasonic power to the above ranges, respectively, phenylethyl resorcinol can be dissolved in the organic solvent more efficiently and thoroughly.

[0022] In some optional embodiments, the step of dissolving betaine in the mixed solution intermediate is performed at a second preset temperature with stirring, wherein the second preset temperature is 50-80°C.

[0023] In the above technical solution, dissolving betaine under heating conditions can improve its dissolution efficiency. In addition, the temperature of the mixed solution can reach the reaction temperature in advance, thereby omitting the process of heating the temperature to the reaction temperature, thereby further improving the preparation efficiency of the eutectic.

[0024] In some optional embodiments, in the mixed solution, the molar ratio of phenethylresorcinol to betaine is 1:(0.9-1.2), and / or the mass ratio of phenethylresorcinol to the organic solvent is 1:(5-10).

[0025] In the above technical solution, the molar ratio of phenylethylresorcinol to betaine in the mixed solution is limited to the above range, so that a eutectic product with a molar ratio closer to 1:1 can be obtained; and the mass ratio of phenylethylresorcinol to the organic solvent in the mixed solution is limited to the above range, so that phenylethylresorcinol can be better dissolved and dispersed.

[0026] In some optional embodiments, in the step of stirring the mixed solution at a first preset temperature, the first preset temperature is 70-80° C., and the reaction time is 2-6 h.

[0027] In the above technical solution, the reaction temperature is further limited to the range of 70~80°C and the reaction time is limited to the range of 2~6 h, which can provide more suitable reaction conditions, thereby obtaining a eutectic product with higher purity and yield.

[0028] In some optional embodiments, the step of cooling and crystallizing includes: cooling the precursor solution to 0-10° C., and then maintaining the temperature unchanged for crystallization.

[0029] In the above technical solution, the crystallization treatment is carried out at 0-10°C, which has the advantage of higher crystallization efficiency.

[0030] In some optional embodiments, the crystallization process is performed under stirring conditions.

[0031] 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.

[0032] In some optional embodiments, during the crystallization process, the stirring speed is 50-600 rpm.

[0033] 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.

[0034] In a third aspect, an embodiment of the present application provides a use of the phenethylresorcinol betaine 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

[0035] 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.

[0036] Figure 1 Schematic diagram of the X-ray single crystal diffraction structure of the phenethylresorcinol betaine cocrystal of Example 1 of the present application;

[0037] Figure 2 This is the H NMR spectrum of the phenethylresorcinol betaine cocrystal of Example 1 of the present application;

[0038] Figure 3 This is the C NMR spectrum of the phenethylresorcinol betaine cocrystal of Example 1 of the present application;

[0039] Figure 4 The infrared spectra of the phenethylresorcinol betaine cocrystal, phenethylresorcinol and betaine of Example 1 of the present application;

[0040] Figure 5 A scanning electron micrograph of phenylethylresorcinol provided for this application;

[0041] Figure 6 A scanning electron microscope image of betaine provided in this application;

[0042] Figure 7 This is a scanning electron micrograph of the phenethylresorcinol betaine cocrystal of Example 1 of the present application;

[0043] Figure 8 This is a scanning electron micrograph of the phenethylresorcinol betaine cocrystal of Example 4 of the present application;

[0044] Figure 9 This is the X-ray powder diffraction pattern of the phenethylresorcinol betaine cocrystal of Example 1 of the present application;

[0045] Figure 10 The cumulative permeation results of phenylethyl resorcinol per unit area provided in this application;

[0046] Figure 11 The inhibition curve of the phenylethylresorcinol betaine cocrystal provided in this application on the inhibition of tyrosinase (bisphenol) activity;

[0047] Figure 12 The inhibition curve of tyrosinase (bisphenol) activity inhibition by phenylethylresorcinol provided in this application;

[0048] Figure 13 The inhibition graph of tyrosinase content by different types of test substances provided in this application;

[0049] Figure 14 This is a graph showing the inhibition of melanin content by different types of test substances provided in this application. DETAILED DESCRIPTION

[0050] 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.

[0051] 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”.

[0052] 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".

[0053] The following is a detailed description of a phenethylresorcinol betaine cocrystal according to an embodiment of the present application, its preparation method, and its application.

[0054] In a first aspect, the present invention provides a phenethylresorcinol betaine cocrystal. The structural formula of the phenethylresorcinol betaine cocrystal is shown in Formula I:

[0055]

[0056] Formula I.

[0057] In this application, the amino group in betaine has a strong electronegativity and forms an internal salt structure. Phenethylresorcinol has functional groups such as a benzene ring and a phenolic hydroxyl group. This allows phenethylresorcinol to interact with betaine 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 betaine form a cocrystal, the crystal arrangement is different from that of phenethylresorcinol alone. This allows the phenethylresorcinol-betaine cocrystal to not only have the whitening effect of phenethylresorcinol, but also have good water solubility, resulting in more ideal bioavailability and compatibility.

[0058] It should be noted that betaine is an alkaloid, chemically known as N,N,N-trimethylglycine, with a chemical structure similar to amino acids. Betaine is widely present in plants and animals and plays a crucial role in nutrient metabolism. As a byproduct of metabolism, it is a crucial osmotic regulator, promoting fat metabolism and intracellular protein formation, revitalizing cells, keeping the skin youthful and vibrant, and maintaining skin elasticity. Furthermore, betaine has excellent biocompatibility and is highly soluble in water. As a carrier of water molecules, betaine releases these molecules into the surrounding environment when physical conditions change, where they are then absorbed and utilized by cells, achieving a hydrating effect.

[0059] As an example, the molecular formula of phenylethylresorcinol betaine cocrystal is C 19 H 25 NO4, and in the phenethylresorcinol-betaine eutectic, the molar ratio of phenethylresorcinol to betaine is 1:1.

[0060] As an example, the phenethylresorcinol betaine cocrystal is monoclinic with space group P21 / c, unit cell parameters a=17.6546(9) Å, b=9.1649(5) Å, c=11.6028(6) Å, α=90°, β=104.814(5)°, γ=90°, Z=4, and unit cell volume V=1814.96(17) Å. 3 .

[0061] As an example, the X-ray powder diffraction pattern of the phenylethylresorcinol betaine cocrystal has characteristic peaks at 2θ angles of 5.29°±0.2°, 18.41°±0.2°, 20.81°±0.2°, 21.59°±0.2°, 26.08°±0.2° and 35.85°±0.2°.

[0062] In order to better understand the technical solution, this application simulates and provides a schematic diagram of the molecular structure of phenylethylresorcinol betaine cocrystal (see Figure 1 ).

[0063] In a second aspect, the present application provides a method for preparing the phenethylresorcinol betaine cocrystal as provided in the first aspect, comprising the following steps:

[0064] S1. Under an inert atmosphere and in the dark, dissolving phenylethylresorcinol and betaine in an organic solvent to obtain a mixed solution. S2. Stirring the mixed solution for reaction at a first preset temperature, wherein the first preset temperature is 50°C to 80°C (for example, but not limited to, any one of 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C, or a range between any two thereof), to allow the phenylethylresorcinol and betaine to react to obtain a precursor solution containing phenylethylresorcinol betaine cocrystals. S3. The precursor solution is sequentially subjected to cooling and crystallization treatments, solid-liquid separation treatments, and drying treatments to obtain phenylethylresorcinol betaine cocrystals.

[0065] In the present application, according to the above process, a phenethylresorcinol-betaine co-crystal as provided in the embodiment of the first aspect can be prepared, wherein the stirring reaction is carried out at 50-80° C. to provide a more suitable driving force so that phenethylresorcinol and betaine can react and react more thoroughly; specifically, under the combined action of mechanical force and temperature, the amino group in betaine and the hydroxyl group of phenethylresorcinol attract each other, so that hydrogen bonds and other interaction forces are generated between betaine and phenethylresorcinol, thereby combining them together, thereby forming a co-crystal that is regularly arranged and stably exists in the same crystal lattice.

[0066] It should be noted that the type of organic solvent is not limited and can be selected according to conventional methods in the art. For example, the organic solvent can be at least one of isopropanol, ethanol, acetone and methanol.

[0067] It should be noted that the form in which phenylethylresorcinol and betaine are dissolved in the organic solvent is not limited. For example, they can be dissolved in the organic solvent simultaneously or in a stepwise manner.

[0068] As an example, under an inert atmosphere and light-proof conditions, the steps of dissolving phenylethylresorcinol and betaine in an organic solvent to obtain a mixed solution include:

[0069] In an inert atmosphere and light-proof conditions, phenylethyl resorcinol is firstly dissolved in an organic solvent to obtain a mixed solution intermediate, and then betaine is dissolved in the mixed solution intermediate to obtain a mixed solution.

[0070] In this embodiment, the poorly soluble and easily deteriorating phenylethyl resorcinol is first dissolved in the organic solvent, and then the relatively stable and easily soluble betaine is dissolved. This has the advantages of a more reasonable sample addition order and easy operation.

[0071] As an example, the step of dissolving phenylethylresorcinol in an organic solvent is performed under ultrasonic conditions.

[0072] In this embodiment, the vibration energy of the ultrasonic wave can reduce the interaction force between molecules in the liquid, thereby lowering the energy threshold for solute dissolution, thereby accelerating the dissolution rate and shortening the dissolution time.

[0073] As an example, the ultrasonic frequency is 20~60 kHz, for example but not limited to the ultrasonic frequency of any one point value of 20 kHz, 30 kHz, 40 kHz, 50 kHz, and 60 kHz, or a range value between any two of them; the ultrasonic power is 700~6000 W, for example but not limited to the ultrasonic power of any one point value of 700 W, 1000 W, 2000 W, 3000 W, 4000 W, 5000 W and 6000 W, or a range value between any two of them.

[0074] In this embodiment, by limiting the ultrasonic frequency and the ultrasonic power to the above ranges, phenylethylresorcinol can be dissolved in the organic solvent more efficiently and thoroughly.

[0075] As an example, the step of dissolving betaine in the mixed solution intermediate is performed at a second preset temperature and under stirring conditions, wherein the second preset temperature is 50-80°C, for example, but not limited to, any one of 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C, or a range between any two of the temperatures.

[0076] In this embodiment, dissolving betaine under heating conditions can improve its dissolution efficiency. In addition, the temperature of the mixed solution can reach the reaction temperature in advance, thereby omitting the process of heating the temperature to the reaction temperature, thereby further improving the preparation efficiency of the eutectic.

[0077] As an example, in the mixed solution, the molar ratio of phenethylresorcinol to betaine is 1:(0.9~1.2), for example, but not limited to, a molar ratio of 1:0.9, 1:0.95, 1:1, 1:1.15 and 1:1.2, or a range of values between any two of them; and / or, the mass ratio of phenethylresorcinol to the organic solvent is 1:(5~10), for example, but not limited to, a mass ratio of 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10, or a range of values between any two of them.

[0078] In this embodiment, the molar ratio of phenethylresorcinol to betaine in the mixed solution is limited to the above range, so that a eutectic product with a molar ratio closer to 1:1 can be obtained; and the mass ratio of phenethylresorcinol to the organic solvent in the mixed solution is limited to the above range, so that phenethylresorcinol can be better dissolved and dispersed.

[0079] As an example, in the step of stirring the mixed solution at a first preset temperature, the first preset temperature is 70~80℃, for example, but not limited to, any one of 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃ and 80℃, or a range between any two of them; the reaction time is 2~6 h, for example, but not limited to, any one of 2 h, 3 h, 4 h, 5 h and 6 h, or a range between any two of them.

[0080] In this embodiment, the reaction temperature is further limited to the range of 70-80° C. and the reaction time is limited to the range of 2-6 h, which can provide more suitable reaction conditions, thereby obtaining a eutectic product with higher purity and yield.

[0081] It should be noted that the temperature of the crystallization treatment is not limited, as long as the phenylethylresorcinol betaine eutectic in the precursor solution can be precipitated.

[0082] As an example, the steps of cooling and crystallizing treatment include: cooling the precursor solution to 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 then maintaining the temperature unchanged for crystallization treatment.

[0083] In this embodiment, the crystallization treatment is performed at 0-10° C., which has the advantage of higher crystallization efficiency.

[0084] As an example, the crystallization process is performed under stirring conditions.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] In other possible implementations, during the crystallization process, a static crystallization method may be adopted.

[0089] 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.

[0090] In this embodiment, vacuum drying is adopted, which has the advantages of lower drying temperature and less prone to product denaturation.

[0091] It should be noted that any process or step not specifically described or limited in the preparation of phenylethylresorcinol betaine cocrystals may be arranged according to conventional selections in the art.

[0092] In a third aspect, an embodiment of the present application provides a use of the phenethylresorcinol betaine co-crystal provided in the embodiment of the first aspect in the preparation of medicines, cosmetics or skin care products.

[0093] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0094] Example 1

[0095] The present invention provides a method for preparing a phenethylresorcinol betaine cocrystal, comprising the following steps:

[0096] S1 Under an inert atmosphere and light-proof conditions, 50 g of phenylethylresorcinol and 300 g of isopropanol were added to a reaction vessel, and then dissolved under ultrasonic conditions, wherein the ultrasonic frequency was 40 kHz and the ultrasonic power was 1500 W, until the phenylethylresorcinol was completely dissolved to obtain a mixed solution intermediate; then, 28.5 g of betaine was added to the mixed solution intermediate so that the molar ratio of phenylethylresorcinol to betaine was 1:1, and then dissolved by stirring at 80°C, wherein the stirring speed was 600 rpm, until the betaine was completely dissolved to obtain a mixed solution.

[0097] S2: maintaining the temperature of the mixed solution constant (ie, 80° C.) and stirring at 600 rpm for 3 h to allow phenylethylresorcinol and betaine to react, thereby obtaining a precursor solution containing phenylethylresorcinol-betaine cocrystals.

[0098] S3: The temperature of the precursor solution was cooled to 5°C and stirred at 400 rpm for crystallization for 3 hours. Solid-liquid separation was then performed, and the resulting solid was dried in a vacuum drying oven for 20 hours at 50°C to obtain phenylethylresorcinol betaine cocrystals in a yield of 88.9%.

[0099] Example 2

[0100] This embodiment of the present application provides a method for preparing a phenethylresorcinol-betaine cocrystal, which differs from Example 1 only in that the molar ratio of phenethylresorcinol to betaine is 1:0.9, and the yield is 87.5%.

[0101] Example 3

[0102] This embodiment of the present application provides a method for preparing a phenethylresorcinol-betaine cocrystal, which differs from Example 1 only in that the molar ratio of phenethylresorcinol to betaine is 1:1.2, and the yield is 87.1%.

[0103] Example 4

[0104] This embodiment of the present application provides a method for preparing phenethylresorcinol betaine cocrystal, which differs from Example 1 only in that the stirring crystallization method is adjusted to a static crystallization method, and the yield is 81.1%.

[0105] Comparative Example 1

[0106] This embodiment of the present application provides a method for preparing phenethylresorcinol betaine cocrystal, which differs from Example 1 only in that all isopropyl alcohol is replaced with water.

[0107] Comparative Example 2

[0108] This embodiment of the present application provides a method for preparing phenethylresorcinol betaine cocrystal, which differs from Example 1 only in that the reaction temperature in step S2 is adjusted to 40°C.

[0109] Test Example 1

[0110] Phenethylresorcinol betaine cocrystals were prepared according to the preparation methods of Examples 1 to 4 and Comparative Examples 1 to 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 reported in Table 1.

[0111] Table 1

[0112]

[0113] It should be noted that the theoretical value is calculated based on a standard molar ratio of phenylethylresorcinol to betaine of 1:1.

[0114] Referring to Table 1, the test results of Examples 1 to 4 are basically consistent with the theoretical results, indicating that Examples 1 to 4 all successfully prepared phenethylresorcinol betaine cocrystals.

[0115] According to the test results of Example 1 and Comparative Example 1, if water is used as the solvent, phenethylresorcinol betaine cocrystal cannot be prepared because phenethylresorcinol is difficult to dissolve in water.

[0116] The test results of Example 1 and Comparative Example 2 show that the test results of Comparative Example 2 are significantly different from the theoretical results, indicating that the prepared substance is not a cocrystal. In other words, the reaction temperature is too low, and the driving force provided is insufficient to successfully prepare the phenylethylresorcinol betaine cocrystal.

[0117] Test Example 2

[0118] Qualitative Analysis of Phenethyl Resorcinol Betaine Cocrystal

[0119] Test method:

[0120] The phenethylresorcinol betaine cocrystal obtained in Example 1 was qualitatively analyzed.

[0121] Table 2 Single crystal data of phenylethylresorcinol betaine eutectic

[0122]

[0123] Table 3 Atomic coordinates and isotropic atomic displacement parameters of phenylethylresorcinol betaine cocrystal

[0124]

[0125] It should be noted that U(eq) is defined as one third of the trace of the orthogonal Uij tensor.

[0126] Table 4 Anisotropic atomic displacement parameters of phenylethylresorcinol betaine cocrystal

[0127]

[0128] It should be noted that the anisotropic atomic displacement factor power is: -2π 2 [h 2 a* 2 U 11 +2hka*b*U 12 +…].

[0129] Table 5 Bond lengths of phenylethylresorcinol betaine cocrystal

[0130]

[0131] Table 6 Chemical bond angles of phenylethylresorcinol betaine cocrystal

[0132]

[0133] Table 7 Hydrogen bonding parameters of phenylethylresorcinol betaine cocrystal

[0134]

[0135] 1 1-X,1-Y,-Z; 2 1-X,1 / 2+Y,-1 / 2-Z.

[0136] Table 8 Torsion angle parameters of phenylethylresorcinol betaine cocrystal

[0137]

[0138] Table 9 Hydrogen atom coordinates of phenylethylresorcinol betaine cocrystal (Å×10 4 ) and the isotropic atomic displacement parameters (Å 2 ×10 3 )

[0139]

[0140] Test Example 3

[0141] H NMR spectroscopy ( 1 H-NMR) characterization

[0142] Test method:

[0143] The phenylethylresorcinol betaine 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.

[0144] See Figure 2 From the H NMR spectrum, we can clearly find 12 hydrogen atoms of phenylethylresorcinol and 11 hydrogen atoms on betaine. 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 phenylethylresorcinol-betaine cocrystal, phenylethylresorcinol and betaine exist in a molar ratio of 1:1.

[0145] Test Example 4

[0146] C NMR spectroscopy ( 13 C-NMR) characterization

[0147] Test method:

[0148] The phenylethylresorcinol betaine cocrystal obtained in Example 1 was subjected to C NMR spectroscopy ( 13 C-NMR) characterization, and MeOD solvent was selected as the test solvent.

[0149] See Figure 3 The test results show that phenethylresorcinol and betaine in the phenethylresorcinol-betaine eutectic are present in a molar ratio of 1:1.

[0150] Test Example 5

[0151] Infrared spectroscopy characterization

[0152] Test method:

[0153] The phenethylresorcinol betaine 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.

[0154] See Figure 4 The infrared spectrum of phenethylresorcinol-betaine cocrystal has different absorption peaks from betaine and phenethylresorcinol, and is not a simple superposition of the characteristic peaks of the two precursors, indicating that phenethylresorcinol and betaine form a cocrystal, which is a new structure.

[0155] Test Example 6

[0156] Morphological characterization

[0157] Test method:

[0158] Phenethylresorcinol, betaine, and the phenethylresorcinol betaine co-crystals obtained in Example 1 and Example 4 were characterized by scanning electron microscopy.

[0159] See Figure 5 、 Figure 6 and Figure 7 The morphology of the phenethylresorcinol betaine cocrystal is blocky, which is completely different from the morphology of phenethylresorcinol and betaine. The particle size is about 10~50 μm, and the particle uniformity is good.

[0160] See Figure 7 and Figure 8 Compared with stirred crystallization, the particles of phenylethylresorcinol betaine cocrystals obtained by static crystallization are larger, the surface is rougher and the particle uniformity is poorer, indicating that the stirred crystallization method is better.

[0161] Test Example 7

[0162] X-ray powder diffraction (XRD) test

[0163] Test method:

[0164] The phenylethylresorcinol betaine 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°.

[0165] See Figure 9 ,The results showed that the phenethylresorcinol betaine cocrystal had characteristic peaks at 2θ angles of approximately 5.29°±0.2°, 18.41°±0.2°, 20.81°±0.2°, 21.59°±0.2°, 26.08°±0.2°, and 35.85°±0.2°.

[0166] Test Example 8

[0167] Water solubility test

[0168] Test method:

[0169] The phenethylresorcinol betaine cocrystals obtained in Examples 1 to 3 were dissolved in water to prepare a supersaturated solution under magnetic stirring. After sampling and filtration, the content of phenethylresorcinol in the phenethylresorcinol betaine cocrystals was detected by high performance liquid chromatography, and the results are summarized in Table 10.

[0170] Table 10

[0171]

[0172] Referring to Table 10, the water solubility of phenethylresorcinol in the phenethylresorcinol-betaine cocrystal is greater than 550 ppm, which is much higher than the water solubility of phenethylresorcinol itself (less than 10 ppm, data from the ACD database), indicating that the water solubility of phenethylresorcinol is greatly improved after the cocrystal is formed between phenethylresorcinol and betaine.

[0173] Test Example 9

[0174] Transdermal delivery performance testing

[0175] Test substances: 1% aqueous solution of phenethylresorcinol betaine eutectic obtained in Example 1 (10% propylene glycol), 0.65% aqueous solution of phenethylresorcinol (10% propylene glycol), saturated aqueous solution of phenethylresorcinol betaine eutectic obtained in Example 1, and saturated aqueous solution of phenethylresorcinol.

[0176] Test method:

[0177] A porcine skin model was used to detect the skin permeation at different times and to evaluate the transdermal delivery efficiency of the main components in the samples.

[0178] (1) Microscopic examination: Select undamaged pig skin under a dissecting microscope, cut skin of the same size, wash it once with sodium chloride solution, and absorb the surface moisture with filter paper.

[0179] (2) Fix the skin: Fix the skin on the Franz diffusion cell with the stratum corneum facing the administration chamber and the dermis facing the receiving chamber. Add 15 mL of sodium chloride solution (10% ethanol) into the receiving chamber and remove bubbles to ensure that there are no bubbles between the dermis and the receiving solution.

[0180] (3) Drug administration: Turn on the instrument in advance and adjust the water bath temperature to 37±1℃. Add 1.0 mL of drug into the drug administration chamber, seal it with sealing film and tin foil to prevent liquid evaporation. The effective penetration area is 1.13 cm 2 .

[0181] (4) Infiltration: Set the stirring speed to 350 rpm / min.

[0182] (5) Sampling: At 3, 6, 21, and 24 h, 10 mL of subcutaneous receiving fluid was pipetted into a 5.0 mL EP tube and then 1.0 mL of sodium chloride solution (10% ethanol) was added to the receiving pool using a pipette.

[0183] (6) Detection: The above samples were filtered through a 0.22 μm organic membrane and then tested by HPLC to calculate the permeation per unit area. The results were then statistically reported in Table 11. At the same time, a graph was drawn based on the statistical results.

[0184] Table 11

[0185]

[0186] See Table 11 and Figure 10 , whether in saturated aqueous solution or unsaturated aqueous solution, the cumulative permeation amount of phenylethylresorcinol betaine cocrystal is much higher than that of phenylethylresorcinol.

[0187] Test Example 10

[0188] Detection of Tyrosinase Activity Inhibitory Ability

[0189] (1) Experimental principle: In the biosynthesis of skin melanin, tyrosinase is the key enzyme, which acts on dopa to form dopaquinone, and the latter spontaneously undergoes a series of reactions to finally form melanin. In the Na2HPO4-citric acid buffer solution with pH 6.8, tyrosinase can catalyze the conversion of dopa into dopaquinone, and the absorbance value can be measured at 475 nm with a spectrophotometer. Raw materials with inhibitory effects on tyrosinase activity can reduce the conversion of dopa into dopaquinone, thereby reducing the absorbance value. According to the change of the absorbance value, the inhibitory effect of the raw material on tyrosinase activity can be evaluated.

[0190] (2) Experimental materials: The phenethyl resorcinol betaine eutectic obtained in Example 1, phenethyl resorcinol alone, and kojic acid (positive control group).

[0191] (3) Test method: T / SHRH 015-2018 "Cosmetics - Test Method for Tyrosinase Activity Inhibition", and then the test results are respectively statistically analyzed in Tables 12 and 13. At the same time, draw a graph according to the statistical results.

[0192] Table 12

[0193]

[0194] * The data in the table are mean ± relative deviation.

[0195] * Statistical method: Analyzed by the t-test method, the test 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.

[0196] Table 13

[0197]

[0198] * The data in the table are mean ± relative deviation.

[0199] * Statistical method: Analyzed by the t-test method, the test 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.

[0200] Refer to Table 12 and Table 13, Figure 11 and Figure 12, the half inhibition rate concentration IC of phenylethylresorcinol betaine cocrystal on tyrosinase (bisphenol) activity 50 =0.040 mg / mL (95% confidence interval IC50: 0.037 mg / mL~0.044 mg / mL), which has whitening effect. The half-maximal inhibition rate concentration IC of phenylethyl resorcinol on tyrosinase (bisphenol) activity 50 =0.037 mg / mL (95% confidence interval IC50: 0.034 mg / mL-0.040 mg / mL), demonstrating skin whitening efficacy. This indicates that the phenylethylresorcinol betaine cocrystal and phenylethylresorcinol have similar inhibitory abilities against tyrosinase (bisphenol) activity. However, at the same test concentration, the phenylethylresorcinol betaine cocrystal contains less phenylethylresorcinol, indicating that the phenylethylresorcinol betaine cocrystal can also exhibit a strong whitening effect at lower dosages and with less irritation.

[0201] Test Example 11

[0202] Detection of tyrosinase activity inhibition ability in human melanocytes

[0203] (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.

[0204] (2) Experimental materials: phenethylresorcinol betaine eutectic obtained in Example 1, a physical mixture of phenethylresorcinol betaine at a molar ratio of 1:1, phenethylresorcinol and kojic acid (positive control).

[0205] (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 cells were cultured for 3 consecutive 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 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 statistically summarized in Tables 14 and 15. At the same time, plots were drawn based on the statistical results.

[0206]

[0207] Table 14

[0208]

[0209] Note: # indicates that the difference is statistically significant compared with the blank control group (NC) (p < 0.05);

[0210] *Indicates that the difference is statistically significant compared with the model control group (M) (p<0.05).

[0211] Table 15

[0212]

[0213] See Tables 14, 15 and Figure 13Compared with the blank control group (NC), the tyrosinase activity of the model control group (M) was significantly increased (p<0.05); compared with the model control group, the relative content of tyrosinase activity of the positive control group (PC) 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 in the sample "phenylethylresorcinol betaine cocrystal" at the test concentrations of 0.00075 mg / mL and 0.00045 mg / mL was significantly reduced (p<0.05), decreasing by 23.59% and 17.85%, respectively, indicating an inhibitory effect on tyrosinase activity; the relative content of tyrosinase activity in the sample "phenylethylresorcinol betaine physical mixture" at the test concentrations of 0.00075 mg / mL and 0.00045 mg / mL was significantly reduced (p<0.05), decreasing by 21.50% and 11.16%, respectively, indicating an inhibitory effect on tyrosinase activity; the relative content of tyrosinase activity in the sample "phenylethylresorcinol" at the test concentrations of 0.0005 mg / mL and 0.0003 mg / mL was significantly reduced (p<0.05), decreasing by 17.41% and 12.62%, respectively, indicating an inhibitory effect on tyrosinase activity. The significant differences between the test substance groups with the same concentration showed that the phenethylresorcinol betaine cocrystal had a significant difference from phenethylresorcinol at a concentration of 0.00075 mg / mL; the phenethylresorcinol betaine cocrystal had a significant difference from the physical mixture of phenethylresorcinol betaine and phenethylresorcinol at a concentration of 0.00045 mg / mL, indicating that the phenethylresorcinol betaine cocrystal had the best effect in inhibiting tyrosinase activity.

[0214] Test Example 12

[0215] Detection of human melanocyte melanin production inhibition ability

[0216] (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.

[0217] (2) Experimental materials: phenethylresorcinol betaine eutectic obtained in Example 1, a physical mixture of phenethylresorcinol betaine at a molar ratio of 1:1, and kojic acid (positive control).

[0218] (3) Test method:

[0219] 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 ± standard deviation. Differences were considered statistically significant if p < 0.05. The results are summarized in Tables 16 and 17, and plotted based on the results.

[0220]

[0221] Table 16

[0222]

[0223] Note: # indicates that the difference is statistically significant compared with the blank control group (NC) (p < 0.05);

[0224] *Indicates that the difference is statistically significant compared with the model control group (M) (p<0.05).

[0225] Table 17

[0226]

[0227] See Table 16, Table 17 and Figure 14 Compared with the blank control (NC), the relative melanin content in the model control (M) group was significantly increased (p < 0.05). Compared with the model control group, the relative melanin content in the positive control (PC) group was significantly decreased (p < 0.05), indicating successful model establishment. Compared with the model control group, the relative melanin content in the "phenylethylresorcinol betaine cocrystal" sample at a test concentration of 0.00025 mg / mL was significantly reduced (p < 0.05), by 19.66%, indicating an inhibitory effect on melanin production. The relative melanin content in the "phenylethylresorcinol betaine physical mixture" sample at a test concentration of 0.00025 mg / mL was significantly reduced (p < 0.05), by 13.10%, indicating an inhibitory effect on melanin production. Significant differences were observed between the phenylethylresorcinol betaine cocrystal and the phenylethylresorcinol betaine physical mixture and phenylethylresorcinol, indicating that the phenylethylresorcinol betaine cocrystal exhibited a more potent inhibitory effect on melanin production.

[0228] Test Example 13

[0229] Irritation test

[0230] (1) Experimental materials: 1% emulsion of phenylethylresorcinol betaine eutectic; negative control: blank + filter disc; number of subjects: 30.

[0231] (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 flexor 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 18, and summarize the test results in Table 19.

[0232] Table 18

[0233]

[0234] Table 19

[0235]

[0236] As shown in Table 19, due to the use of the same-body self-comparison method, no irritation was observed in the negative control group, which consisted of 30 areas. This demonstrates the validity of the experiment and the reliability of the results. The phenylethylresorcinol betaine cocrystal group showed no irritation at 0.5, 24, or 48 hours, demonstrating the mild and low irritation properties of the phenylethylresorcinol betaine cocrystal.

[0237] 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 betaine eutectic, characterized in that: The structural formula of the phenethylresorcinol betaine co-crystal is shown in Formula I: Formula I; The X-ray powder diffraction pattern of the phenethylresorcinol betaine cocrystal has characteristic peaks at 2θ angles of 5.29°±0.2°, 18.41°±0.2°, 20.81°±0.2°, 21.59°±0.2°, 26.08°±0.2° and 35.85°±0.2°.

2. The phenethylresorcinol betaine eutectic according to claim 1, characterized in that The molecular formula of the phenethylresorcinol betaine eutectic is C 19 H 25 NO4, and in the phenethylresorcinol betaine eutectic, the molar ratio of the phenethylresorcinol to the betaine is 1:

1.

3. The phenethylresorcinol betaine eutectic according to claim 1, wherein The phenethylresorcinol betaine eutectic is a monoclinic crystal with a space group of P21 / c, unit cell parameters of a=17.6546(9) Å, b=9.1649(5)Å, c=11.6028(6) Å, α=90°, β=104.814(5)°, γ=90°, Z=4, and unit cell volume V=1814.96(17) Å. 3 .

4. A method for preparing the phenethylresorcinol betaine eutectic as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Under an inert atmosphere and in the dark, dissolving phenylethylresorcinol and betaine in an organic solvent to obtain a mixed solution; S2 stirring the mixed solution at a first preset temperature, wherein the first preset temperature is 50-80° C., so that the phenylethylresorcinol and the betaine react to obtain a precursor solution containing the phenylethylresorcinol-betaine cocrystal; S3: performing cooling and crystallization treatment, solid-liquid separation treatment, and drying treatment on the precursor solution in sequence to obtain the phenethylresorcinol betaine eutectic.

5. The preparation method according to claim 4, characterized in that The step of dissolving phenylethylresorcinol and betaine in an organic solvent under an inert atmosphere and light-proof conditions to obtain a mixed solution comprises: Under an inert atmosphere and light-proof conditions, the phenethyl resorcinol is first dissolved in the organic solvent to obtain a mixed solution intermediate, and then the betaine is dissolved in the mixed solution intermediate to obtain the mixed solution.

6. The preparation method according to claim 5, characterized in that The step of first dissolving the phenylethyl resorcinol in the organic solvent is performed under ultrasonic conditions.

7. The preparation method according to claim 6, wherein The ultrasonic frequency is 20~60 kHz, and the ultrasonic power is 700~6000 W.

8. The preparation method according to claim 5, characterized in that The step of dissolving the betaine in the mixed solution intermediate is performed at a second preset temperature and under stirring conditions, wherein the second preset temperature is 50-80°C.

9. The preparation method according to any one of claims 4 to 8, characterized in that In the mixed solution, the molar ratio of the phenethyl resorcinol to the betaine is 1:(0.9-1.2), and / or the mass ratio of the phenethyl resorcinol to the organic solvent is 1:(5-10).

10. The preparation method according to any one of claims 4 to 8, characterized in that In the step of stirring the mixed solution at a first preset temperature, the first preset temperature is 70-80° C., and the reaction time is 2-6 h.

11. The preparation method according to any one of claims 4 to 8, characterized in that The step of cooling and crystallizing the precursor solution comprises cooling the precursor solution to 0-10° C. and then maintaining the temperature unchanged to perform crystallization.

12. The preparation method according to claim 11, characterized in that The crystallization treatment is carried out under stirring conditions.

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

14. Use of the phenethylresorcinol betaine cocrystal according to any one of claims 1 to 3 in the preparation of cosmetics or skin care products.

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