Dihydroquercetin betaine eutectic crystal as well as preparation method and application thereof

Abstract

The invention provides a dihydroquercetin betaine eutectic crystal and a preparation method and application thereof, the structural formula of the dihydroquercetin betaine eutectic crystal is as shown in formula I. The dihydroquercetin betaine eutectic crystal has excellent oxidation resistance and water solubility.

Description

Technical Field

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

[0002] Dihydroquercetin (also known as Taxol) has five phenolic hydroxyl groups in its molecular structure. Therefore, it can be used as a natural antioxidant to effectively remove free radicals and toxins in the human body. It has a broad spectrum of biological and pharmacological activities such as anti-inflammatory, antibacterial, anti-radiation, anti-cancer, antiviral, immune regulation, melanin removal, and microcirculation improvement. It is a precious raw material for the production of food, medicine, cosmetics, or skin care products. However, the source of dihydroquercetin is relatively scarce (only a small amount exists in yew, yellow fir, larch, striata, and wild black cherry), which makes it difficult to apply it on a large scale. In addition, its poor water solubility (extremely low solubility leads to low bioavailability and poor compatibility) has limited its promotion and application. Therefore, if its antioxidant activity is effectively improved (after the antioxidant activity is improved, a small amount of dihydroquercetin can also exert a relatively excellent antioxidant effect) and water solubility (after the water solubility is improved, a more diverse range of dosage forms and products can be prepared, and at the same time, its bioavailability can be improved), it is a major problem currently faced. Summary of the Invention

[0003] The purpose of this application is to provide a dihydroquercetin betaine cocrystal and its preparation method and application, wherein the dihydroquercetin betaine cocrystal has both excellent antioxidant properties and water solubility.

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

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

[0006]

[0007] In the above technical solution, the amino group in betaine has a strong electronegativity and forms an inner salt structure by itself. Dihydroquercetin has functional groups such as a benzene ring and five phenolic hydroxyl groups, which enable dihydroquercetin 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 dihydroquercetin and betaine form a eutectic, the crystal arrangement is different from that of dihydroquercetin alone, so that when the mass of dihydroquercetin is constant, the antioxidant and water solubility of the dihydroquercetin betaine eutectic containing the same mass of dihydroquercetin are improved.

[0008] In some alternative embodiments, the molecular formula of the dihydroquercetin betaine cocrystal is C 20 H23 NO9, and in the dihydroquercetin-betaine cocrystal, the molar ratio of dihydroquercetin to betaine is 1:1.

[0009] In some optional embodiments, the dihydroquercetin betaine cocrystal is a triclinic system with a space group of P1 and a unit cell parameter of α=73.9200(10)°,β=88.0180(10)°,γ=69.3930(10)°,Z=2,unit cell volume

[0010] In some optional embodiments, the X-ray powder diffraction pattern of the dihydroquercetin betaine cocrystal has characteristic peaks at 2θ angles of 17.52°±0.2°, 22.07°±0.2°, 24.14°±0.2° and 47.41°±0.2°.

[0011] In a second aspect, the present application provides a method for preparing a dihydroquercetin betaine cocrystal as provided in the first aspect, comprising the following steps:

[0012] S1: mixing dihydroquercetin, betaine and an organic solvent to obtain a mixed solution; S2: placing the mixed solution at 55-85° C. for stirring and reacting to obtain a precursor solution containing dihydroquercetin-betaine cocrystals; S3: sequentially performing crystallization treatment, solid-liquid separation treatment and drying treatment on the precursor solution to obtain dihydroquercetin-betaine cocrystals.

[0013] According to the above process, a dihydroquercetin-betaine cocrystal as provided in the embodiment of the first aspect can be prepared, wherein the stirring reaction is carried out at 55-85° C. to provide a more suitable driving force so that dihydroquercetin 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 in dihydroquercetin attract each other, so that hydrogen bonds and other interaction forces are generated between betaine and dihydroquercetin, thereby combining them together, thereby forming a cocrystal that is regularly arranged and stably exists in the same crystal lattice.

[0014] In some optional embodiments, in the step of placing the mixed solution at 55-85° C. for stirring reaction, the reaction time is 2-4 h, or / and the stirring speed is 300-800 rpm.

[0015] In the above technical solution, the reaction time and the stirring speed are respectively limited within the above ranges to provide more suitable reaction conditions, thereby facilitating the full reaction of dihydroquercetin and betaine, thereby improving the raw material utilization, product yield and purity.

[0016] In some optional embodiments, in the mixed solution, the molar ratio of dihydroquercetin to betaine is 1:1, or / and the ratio of the sum of the mass of dihydroquercetin and betaine to the mass of the organic solvent is 1:(5-20).

[0017] In the above technical solution, the molar ratio of dihydroquercetin to betaine in the mixed solution is limited to the above range, and a eutectic product with a molar ratio closer to 1:1 can be obtained; and the mass ratio of the sum of the mass of dihydroquercetin and betaine in the mixed solution to the mass of the organic solvent is limited to the above range, which can dissolve and disperse the reaction raw materials more quickly and thoroughly.

[0018] In some optional embodiments, the organic solvent is selected from at least one of ethanol, ether, methanol and ethanol aqueous solution; optionally, the organic solvent is selected from ethanol aqueous solution; optionally, the water content in the ethanol aqueous solution is not higher than 10 wt%.

[0019] In the above technical solution, a wide variety of organic solvents are applicable to the embodiments of the present application, providing a wide range of feasible implementation options, thereby facilitating the promotion and application of the technical solutions provided by the embodiments of the present application. Furthermore, the use of an ethanol aqueous solution as an organic solvent can more easily dissolve dihydroquercetin and betaine, thereby helping to reduce the amount of solvent used and save costs. Furthermore, limiting the water content in the ethanol aqueous solution to the above range can more easily dissolve dihydroquercetin and betaine.

[0020] In some optional embodiments, after the step of obtaining the precursor solution and before the step of performing the crystallization treatment, the step of filtering the precursor solution while hot is further included; or / and, in the step of crystallization treatment, the treatment temperature is 0 to 30°C.

[0021] In the above technical solution, adding the step of hot filtration can remove insoluble impurities in the mixed system before the eutectic product is precipitated, thereby improving the purity of the obtained eutectic product; in the crystallization treatment step, limiting the temperature to the above range helps to take into account both the crystallization rate and the uniformity of the grain size.

[0022] In a third aspect, the embodiments of the present application provide a use of the dihydroquercetin betaine co-crystal provided in the embodiment of the first aspect in the preparation of food, medicine, cosmetics or skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 Schematic diagram of a single crystal of dihydroquercetin betaine cocrystal obtained in Example 1 of the present application;

[0025] Figure 2 This is the H NMR spectrum of the dihydroquercetin betaine cocrystal obtained in Example 1 of the present application;

[0026] Figure 3 This is an infrared comparison of the dihydroquercetin betaine co-crystal obtained in Example 1 of the present application and various monomers;

[0027] Figure 4 This is a SEM comparison of the dihydroquercetin betaine cocrystal obtained in Example 1 of the present application and various monomers;

[0028] Figure 5 This is a comparison of the XRD patterns of the dihydroquercetin betaine cocrystal, the dihydroquercetin betaine physical mixture, and various monomers obtained in Example 1 of the present application;

[0029] Figure 6 This is a DSC comparison chart of the dihydroquercetin betaine cocrystal obtained in Example 1 of the present application and various monomers;

[0030] Figure 7 This is the TG graph of the dihydroquercetin betaine cocrystal obtained in Example 1 of the present application;

[0031] Figure 8 This is a comparison of the dissolution curves of the dihydroquercetin betaine cocrystal and dihydroquercetin obtained in Example 1 of the present application;

[0032] Figure 9 This is a comparison chart of the bioavailability of dihydroquercetin betaine cocrystal and dihydroquercetin obtained in Example 1 of the present application;

[0033] Figure 10 This is a comparison chart of mitochondrial ROS of dihydroquercetin betaine cocrystal and dihydroquercetin obtained in Example 1 of the present application;

[0034] Figure 11 This is a comparison of the mitochondrial morphology of the dihydroquercetin betaine co-crystal and dihydroquercetin obtained in Example 1 of the present application. DETAILED DESCRIPTION

[0035] 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.

[0036] 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”.

[0037] In addition, in the description of this application, unless otherwise specified, the meaning of "multiple" in "one or more" refers to two or more; the range of "value a to value b" includes the two end values "a" and "b", and the "unit of measurement" in "value a to value b+unit of measurement" represents the "unit of measurement" of both "value a" and "value b".

[0038] Co-crystal technology is an innovative development strategy to improve the physical and chemical properties of active ingredients. This technology does not change the chemical structure of the original compound, but optimizes its physical and chemical properties through physical means, such as improving solubility, enhancing stability and improving bioavailability. This method can play a synergistic therapeutic and toxicity-reducing and efficacy-enhancing role.

[0039] However, cocrystal formation is unpredictable. Even two structurally similar compounds reacting with the same ligand can form completely different products. For example, salicylic acid and niacinamide form a cocrystal, while 4-methoxysalicylic acid and niacinamide form an ionic salt. This means that even a slight difference in a single substituent can result in completely different products when combined with the same ligand. Therefore, selecting the right ligand capable of forming a cocrystal to improve the performance of the target compound is extremely difficult.

[0040] On this basis, the inventors discovered through innovative research that by using betaine as a binding ligand, dihydroquercetin can combine with betaine to form a co-crystal, and the co-crystal product has both excellent antioxidant properties and water solubility.

[0041] The following is a detailed description of a dihydroquercetin betaine co-crystal according to an embodiment of the present application, its preparation method, and its application.

[0042] In a first aspect, the present invention provides a dihydroquercetin betaine co-crystal. The structural formula of the dihydroquercetin betaine co-crystal is shown in Formula I:

[0043]

[0044] In the present application, the amino group in betaine has a strong electronegativity and forms an inner salt structure by itself. Dihydroquercetin has functional groups such as a benzene ring and five phenolic hydroxyl groups, which enable dihydroquercetin 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 stably exists in the same lattice. After dihydroquercetin and betaine form a eutectic, the crystal arrangement is different from that of dihydroquercetin alone, so that when the mass of dihydroquercetin is constant, the antioxidant and water solubility of the dihydroquercetin betaine eutectic containing the same mass of dihydroquercetin are improved; in addition, in the embodiment of the present application, dihydroquercetin is prepared into a stable and unique structural eutectic form, which also provides a new application form for dihydroquercetin.

[0045] It should be noted that betaine is an alkaloid with the chemical name N,N,N-trimethylglycine. Its chemical structure is similar to that of amino acids and it belongs to the class of quaternary ammonium bases. Betaine is widely present in plants and animals. Specifically, in plants, wolfberry and legumes contain betaine, of which beet molasses is the main source of betaine. In animals, mollusks such as octopus, cuttlefish, and shrimp, as well as the liver, spleen, and amniotic fluid of vertebrates (including humans) contain betaine. Betaine has multiple physiological functions such as regulating osmotic pressure, protecting cell structure, and promoting fat metabolism, and is widely used in the fields of medicine and food. Therefore, in this application, betaine is combined with dihydroquercetin to form a cocrystal, which can not only enhance the antioxidant and water solubility of dihydroquercetin, but also enable the cocrystal product to have both the multiple physiological functions of betaine.

[0046] As an example, the molecular formula of dihydroquercetin betaine cocrystal is C 20 H 23 NO9, and in the dihydroquercetin-betaine cocrystal, the molar ratio of dihydroquercetin to betaine is 1:1.

[0047] As an example, the dihydroquercetin betaine cocrystal is a triclinic system with a space group of P1 and unit cell parameters of α=73.9200(10)°,β=88.0180(10)°,γ=69.3930(10)°,Z=2,unit cell volume

[0048] As an example, the X-ray powder diffraction pattern of the dihydroquercetin betaine cocrystal has characteristic peaks at 2θ angles of 17.52°±0.2°, 22.07°±0.2°, 24.14°±0.2° and 47.41°±0.2°.

[0049] In a second aspect, the present application provides a method for preparing a dihydroquercetin betaine cocrystal as provided in the first aspect, comprising the following steps:

[0050] S1: mixing dihydroquercetin, betaine and an organic solvent to obtain a mixed solution; S2: placing the mixed solution at 55-85°C (for example, but not limited to, any one of 55°C, 60°C, 65°C, 70°C, 75°C, 80°C and 85°C, or a range between any two thereof) for stirring and reacting to obtain a precursor solution containing dihydroquercetin-betaine cocrystal; S3: sequentially performing crystallization treatment, solid-liquid separation treatment and drying treatment on the precursor solution to obtain dihydroquercetin-betaine cocrystal.

[0051] In the present application, according to the above process, a dihydroquercetin betaine co-crystal as provided in the embodiment of the first aspect can be prepared, wherein the stirring reaction is carried out at 55-85°C to provide a more suitable driving force so that dihydroquercetin 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 dihydroquercetin attract each other, so that hydrogen bonds and other interaction forces are generated between betaine and dihydroquercetin, thereby combining them together, thereby forming a co-crystal that is regularly arranged and stably exists in the same crystal lattice.

[0052] It should be noted that the manner of mixing dihydroquercetin, betaine and the organic solvent is not limited, for example, it can be a one-step mixing manner or a step-by-step mixing manner (for example, first mixing dihydroquercetin and the organic solvent to obtain a premix; and then mixing the premix with betaine), and the specific method can be adaptively adjusted according to actual needs.

[0053] As an example, in the step of placing the mixed solution at 55-85°C for stirring reaction, the reaction time is 2-4h, for example, but not limited to, any one of 2h, 2.5h, 3h, 3.5h and 4h, or a range of values between any two; or / and, the stirring speed is 300-800rpm, for example, but not limited to, any one of 300rpm, 400rpm, 500rpm, 600rpm, 700rpm and 800rpm, or a range of values between any two.

[0054] In this embodiment, the reaction time and the stirring speed are respectively limited within the above ranges to provide more suitable reaction conditions, thereby facilitating the full reaction of dihydroquercetin and betaine, thereby improving the raw material utilization rate, product yield and purity.

[0055] As an example, in the mixed solution, the molar ratio of dihydroquercetin and betaine is 1:1, or / and, the ratio of the sum of the masses of dihydroquercetin and betaine to the mass of the organic solvent is 1:(5-20), for example, but not limited to, the mass ratio is any one of 1:5, 1:10, 1:15 and 1:20 or a range between any two of them.

[0056] In this embodiment, the molar ratio of dihydroquercetin to betaine in the mixed solution is limited to the above range, and a eutectic product with a molar ratio closer to 1:1 can be obtained; and the mass ratio of the sum of the masses of dihydroquercetin and betaine in the mixed solution to the mass of the organic solvent is limited to the above range, so that the reaction raw materials can be dissolved and dispersed more quickly and thoroughly.

[0057] It should be noted that the type of organic solvent is not limited and can be selected according to routine practices in the art.

[0058] As an example, the organic solvent is selected from at least one of ethanol, ether, methanol and ethanol aqueous solution.

[0059] In this embodiment, there are many types of organic solvents that can be applied to the embodiments of the present application, and more feasible implementation plans can be provided, thereby facilitating the promotion and application of the technical solutions provided by the embodiments of the present application.

[0060] As an example, the organic solvent is selected from ethanol aqueous solution.

[0061] In this embodiment, an ethanol aqueous solution is used as the organic solvent, which can dissolve dihydroquercetin and betaine relatively easily, thereby helping to reduce the amount of solvent used and save costs.

[0062] As an example, the water content in the ethanol aqueous solution is not higher than 10 wt%, for example, it can be any one of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% or a range between any two of them.

[0063] In this embodiment, by limiting the water content in the ethanol aqueous solution to the above range, dihydroquercetin and betaine can be dissolved more easily.

[0064] As an example, after the step of obtaining the precursor solution and before the step of performing the crystallization treatment, the step of filtering the precursor solution while hot is also included.

[0065] It should be noted that "hot filtration" means that after the stirring reaction is completed, the entire solution system still maintains a relatively high temperature (close to the temperature of the stirring reaction). Under this temperature condition, the eutectic product will not precipitate from the solution system, and then the solution system with a relatively high temperature is directly filtered.

[0066] In this embodiment, the step of hot filtration is added to remove insoluble impurities in the mixed system before the eutectic product is precipitated, thereby improving the purity of the obtained eutectic product.

[0067] It is understood that the lower the crystallization temperature, the higher the efficiency, which is suitable for large-scale batch production; while the higher the crystallization temperature, the slower the rate, but the more uniform the grain size. Therefore, during the crystallization process, the treatment temperature can be adaptively adjusted according to actual needs.

[0068] As an example, in the crystallization treatment step, the treatment temperature is 0-30°C, for example but not limited to any one of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C and 30°C or a range between any two of them.

[0069] In this embodiment, in the step of crystallization treatment, limiting the temperature to the above range helps to achieve both the crystallization rate and the uniformity of the grain size.

[0070] It should be noted that the duration of the crystallization treatment is not limited, for example, it can be 2 to 15 hours, and can be adaptively adjusted according to actual conditions.

[0071] It should be noted that in the solid-liquid separation step, the specific separation method is not limited, for example, filtration can be used.

[0072] It should be noted that, in the drying step, the specific drying method is not limited, for example, vacuum drying can be used; wherein, during the vacuum drying process, 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 12-48h, for example, but not limited to, the drying time is any one of 12h, 15h, 30h, 40h and 48h, or a range between any two of them.

[0073] It should be noted that any process or step not specifically described or limited in the preparation of dihydroquercetin-betaine cocrystals may be performed according to conventional methods in the art.

[0074] In a third aspect, the embodiments of the present application provide a use of the dihydroquercetin betaine co-crystal provided in the embodiment of the first aspect in the preparation of food, medicine, cosmetics or skin care products.

[0075] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0076] Example 1

[0077] The present invention provides a method for preparing a dihydroquercetin-betaine cocrystal, comprising the following steps:

[0078] S1 cleaned the cavity of the reactor with ethanol and dried it with compressed air, then added 72.2 g of dihydroquercetin and 27.8 g of betaine to the reactor, and then added 1.5 kg of anhydrous ethanol to the reactor, turned on the stirring paddle and stirred at a speed of 500 rpm. At the same time, started the heating unit and set the temperature to 80°C until a clear mixed solution was obtained.

[0079] S2: maintaining the internal temperature of the reaction chamber at 80° C. and stirring the reaction at 500 rpm for 4 h to obtain a precursor solution containing dihydroquercetin-betaine cocrystal.

[0080] S3: filtering the precursor solution while hot to obtain a filtrate containing dihydroquercetin betaine cocrystal.

[0081] S4: The filtrate is cooled to 20°C and kept warm for crystallization for 10 hours; solid-liquid separation is then performed, and the obtained solid is dried in a vacuum drying oven, wherein the drying time is 24 hours and the drying temperature is 50°C, to obtain 89.42 g of product with a yield of 88.9%; at the same time, the melting range of the product (i.e., the melting point range, which refers to the temperature range from the beginning of melting to complete melting of the pure compound) is tested to be 219.45-221.64°C (wherein, the melting point of dihydroquercetin monomer is close to 240°C, and the melting point of betaine monomer is 301-305°C).

[0082] It should be noted that, by comparing the melting range of the product with the melting points of dihydroquercetin monomer and betaine monomer, it can be seen that the melting point of the product is significantly different from the melting points of the two monomers and is located at around 220°C, indicating that the obtained product is a eutectic with a new structure.

[0083] Example 2

[0084] The present invention provides a method for preparing a dihydroquercetin-betaine cocrystal, comprising the following steps:

[0085] S1 cleaned the cavity of the reactor with ethanol and dried it with compressed air, then added 72.2 g of dihydroquercetin and 27.8 g of betaine to the reactor, and then added 1 kg of methanol to the reactor, turned on the stirring paddle and stirred at a speed of 700 rpm. At the same time, started the heating unit and set the temperature to 80°C until a clear mixed solution was obtained.

[0086] S2: maintaining the internal temperature of the reaction chamber at 80° C. and stirring the reaction at 700 rpm for 3 h to obtain a precursor solution containing dihydroquercetin-betaine cocrystal.

[0087] S3: filtering the precursor solution while hot to obtain a filtrate containing dihydroquercetin betaine cocrystal.

[0088] S4: The filtrate is cooled to 5°C and kept warm for crystallization for 12 hours; solid-liquid separation is then performed, and the obtained solid is dried in a vacuum drying oven, wherein the drying time is 20 hours and the drying temperature is 50°C, to obtain 87.96 g of product with a yield of 87.96%; at the same time, the melting range of the product (i.e., the melting point range, which refers to the temperature range from the beginning of melting to complete melting of the pure compound) is measured to be 218.62 to 221.15°C (from the comparison of Example 1 and Example 2, it can be seen that the melting ranges of the products obtained by the two are basically the same, indicating that the product prepared in Example 2 is also a eutectic).

[0089] Example 3

[0090] The present invention provides a method for preparing a dihydroquercetin-betaine cocrystal, comprising the following steps:

[0091] S1 cleaned the cavity of the reactor with ethanol and dried it with compressed air, then added 72.2 g of dihydroquercetin and 27.8 g of betaine to the reactor, and then added 0.5 kg of ethanol with a water content of 5% to the reactor, turned on the stirring paddle and stirred at a speed of 500 rpm. At the same time, started the heating unit and set the temperature to 85°C until a clear mixed solution was obtained.

[0092] S2: maintaining the internal temperature of the reaction chamber at 85° C. and stirring the reaction at 500 rpm for 2 h to obtain a precursor solution containing dihydroquercetin-betaine cocrystal.

[0093] S3: filtering the precursor solution while hot to obtain a filtrate containing dihydroquercetin betaine cocrystal.

[0094] S4: The filtrate is cooled to 0°C and kept warm for crystallization for 10 hours; solid-liquid separation is then performed, and the obtained solid is dried in a vacuum drying oven, wherein the drying time is 24 hours and the drying temperature is 50°C, to obtain 88.75 g of product with a yield of 88.75%; at the same time, the melting range of the product (i.e., the melting point range, which refers to the temperature range from the beginning of melting to complete melting of the pure compound) is measured to be 220.36 to 222.2°C (from the comparison of Example 1 and Example 2, it can be seen that the melting ranges of the products obtained by the two are basically the same, indicating that the product prepared in Example 2 is also a eutectic).

[0095] It should be noted that, from the comparison of Examples 1 to 3, it can be seen that when ethanol aqueous solution is used as the organic solvent, the amount of organic solvent used is significantly reduced, which helps to reduce the solvent cost.

[0096] Comparative Example 1

[0097] This embodiment of the present application provides a method for preparing dihydroquercetin-betaine cocrystal, which differs from Example 1 only in that in step S1 and step S2, the temperature of the reactor is set to 50°C.

[0098] 85.35 g of product was obtained with a yield of 85.35%; at the same time, the melting range of the product (i.e., the melting point range, which refers to the temperature range from the beginning of melting to complete melting of the pure compound) was measured to be 240.03 to 304.98 ° C (i.e., the lower and upper limits of the melting point of the product in Comparative Example 1 correspond to the melting points of dihydroquercetin and betaine, respectively, indicating that the product is not a eutectic but a mixed solution of dihydroquercetin and betaine. In other words, the reaction temperature is too low, which makes it difficult for dihydroquercetin and betaine to react to form a eutectic).

[0099] Test Example 1

[0100] Qualitative Analysis of Dihydroquercetin-Betaine Cocrystal

[0101] Test method: The dihydroquercetin betaine cocrystal obtained in Example 1 was subjected to X-ray single crystal diffraction test. The specific test results are shown in Table 1, and the crystal schematic diagram is shown in Figure 1 .

[0102] Table 1

[0103]

[0104]

[0105] Test Example 2

[0106] H NMR spectroscopy ( 1 H-NMR) characterization

[0107] Testing method: The dihydroquercetin betaine cocrystal obtained in Example 1 was subjected to nuclear magnetic resonance spectroscopy ( 1 H-NMR) characterization, and MeOD was used as the test solvent.

[0108] See Figure 2 From the H NMR spectrum, we can clearly find 7 hydrogen atoms of dihydroquercetin and 11 hydrogen atoms on betaine, and the rest are deuterated reagent peaks. No obvious impurity peaks are seen, which shows that in the dihydroquercetin-betaine cocrystal, dihydroquercetin and betaine exist in a molar ratio of 1:1.

[0109] Test Example 3

[0110] Infrared spectroscopy characterization

[0111] Test method: Dihydroquercetin, betaine, and the dihydroquercetin betaine cocrystal obtained in Example 1 were 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.

[0112] See Figure 3 The infrared spectrum of dihydroquercetin-betaine cocrystal has different absorption peaks from betaine and dihydroquercetin, which is not a simple superposition of the characteristic peaks of the two precursors. The OH stretching peak in dihydroquercetin is at 3420 cm -1 There is a characteristic peak at the position. In the cocrystal, the OH of dihydroquercetin acts as a hydrogen bond donor, and its OH stretching red-shifts to 3057 cm -1 Position; C=O stretching in betaine at 1614cm -1 There is a characteristic peak at the position. In the eutectic, the C=O of betaine acts as a hydrogen bond acceptor, and its C=O stretching blue shifts to 1625cm -1 Position, indicating that dihydroquercetin and betaine formed a cocrystal, which is a new structure.

[0113] Test Example 4

[0114] Morphological characterization (SEM)

[0115] Testing method: Dihydroquercetin, betaine, and the dihydroquercetin-betaine cocrystal obtained in Example 1 were characterized by scanning electron microscopy.

[0116] See Figure 4 The solid particles of dihydroquercetin are columnar crystals, the solid particles of betaine are agglomerated solids with no obvious crystal form, and the solid particles of dihydroquercetin-betaine eutectic are block crystals. The microscopic morphology of the eutectic is significantly different from that of the monomer.

[0117] Test Example 5

[0118] X-ray powder diffraction (XRD) test

[0119] Test method: Powder X-ray diffraction tests were performed on the dihydroquercetin-betaine cocrystal, dihydroquercetin-betaine mixture, betaine, and dihydroquercetin prepared in Experimental Example 1 of the present application.

[0120] See Figure 5 The peaks in the powder X-ray diffraction spectrum of the dihydroquercetin betaine mixture are basically the superposition of the peaks of dihydroquercetin and betaine monomers, while the peaks in the powder X-ray diffraction spectrum of the dihydroquercetin betaine cocrystal are not a simple superposition of the peaks of dihydroquercetin and betaine monomers. Specifically, the dihydroquercetin betaine cocrystal has characteristic peaks at 2θ angles of 17.52°, 22.07°, 24.14°, and 47.41°. This result indicates that the dihydroquercetin prepared in Example 1 and betaine formed a new cocrystal.

[0121] Test Example 6

[0122] Differential Scanning Calorimetry (DSC) Test

[0123] Test method: Differential scanning calorimetry (DSC) tests were performed on dihydroquercetin, betaine, and the dihydroquercetin-betaine cocrystal obtained in Example 1.

[0124] See Figure 6 The endothermic peak of dihydroquercetin-betaine cocrystal (220°C) is significantly different from that of dihydroquercetin (241°C) and betaine (306°C), indicating that the melting point of the cocrystal product prepared in Example 1 is significantly different from the melting points of the two monomers and is basically consistent with the test results in the Examples section.

[0125] Test Example 7

[0126] Thermogravimetric analysis (TG) test

[0127] Testing method: The dihydroquercetin betaine cocrystal obtained in Example 1 was subjected to thermogravimetric analysis (TG) test.

[0128] See Figure 7 The thermal decomposition temperature of dihydroquercetin betaine cocrystal is higher than 200 °C, and it has good thermal stability, which means that it is not easy to decompose during the processing and is easy to prepare into various products for application.

[0129] Test Example 8

[0130] Water solubility test

[0131] Test method: Dihydroquercetin and the dihydroquercetin betaine cocrystal obtained in Experimental Example 1 were dissolved in water respectively, and the solutions were collected at 5 min, 10 min, 20 min, 30 min, 50 min, 70 min, 100 min, 130 min, and 180 min, filtered, and the content of dihydroquercetin was detected by high performance liquid chromatography.

[0132] See Figure 8 The dihydroquercetin-betaine cocrystal can achieve a high solubility in a short time. The solubility is the highest at 10 minutes and is higher than the water solubility of monomeric dihydroquercetin. That is, after dihydroquercetin and betaine form a cocrystal, its corresponding water solubility is improved, which means that the dihydroquercetin-betaine cocrystal is absorbed faster in the human body; and as time goes on, the dissolution equilibrium is basically reached at 30 minutes. The solubility of the dihydroquercetin-betaine cocrystal is still higher than that of the dihydroquercetin monomer, which means that the dihydroquercetin-betaine cocrystal has a higher absorption rate in the human body.

[0133] Test Example 9

[0134] Bioavailability testing

[0135] Experimental materials and methods:

[0136] Test substance:

[0137] Dihydroquercetin betaine eutectic aqueous solution (based on a dihydroquercetin content of 0.04%) (10% ethanol), dihydroquercetin content of 0.04% aqueous solution (10% ethanol).

[0138] Experimental methods:

[0139] 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.

[0140] (1) Microscopic examination: Select undamaged pig skin under a dissecting microscope, cut three pieces of skin of equal size, wash them once with sodium chloride solution, and dry the surface moisture with filter paper.

[0141] (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.

[0142] (3) Drug administration: Turn on the instrument in advance and adjust the water bath temperature to 32±1℃. Add 1.0mL of drug into the drug administration chamber, seal it with sealing film and tin foil to prevent liquid evaporation. The effective penetration area is 1.13cm 2 .

[0143] (4) Infiltration: Set the stirring speed to 350 rpm.

[0144] (5) Sampling: At 2 h, 4 h, 6 h, 22 h, 23 h, and 24 h, 1 mL of subcutaneous receiving fluid was pipetted into a 5.0 mL EP tube. 1 mL of sodium chloride (10% ethanol) solution was then added to the receiving pool using a pipette. 24 h skin was collected, the pig skin was chopped, and placed in a 5 mL centrifuge tube. The extractant was added and soaked overnight. After sonication for 30 min, the tube was filtered into a sample injection bottle.

[0145] (6) Detection: The above samples were filtered through a 0.22 μm water membrane and then tested by HPLC to calculate the permeation rate per unit area. The test results are then statistically reported in Table 2.

[0146] Table 2

[0147]

[0148] See Table 2 and Figure 9The cumulative permeation per unit area of the dihydroquercetin-betaine cocrystal after 6 hours was significantly higher than that of dihydroquercetin, and 3.23 times that of dihydroquercetin after 24 hours. After 24 hours, the amount of dihydroquercetin-betaine cocrystal retained in the skin was also significantly higher than that of dihydroquercetin, approximately 3 times higher. The bioavailability of the samples was calculated based on the data in Table 2. The bioavailability of the dihydroquercetin-betaine cocrystal was 6.47%, while that of dihydroquercetin was 2.11%, indicating that the bioavailability of the dihydroquercetin-betaine cocrystal was superior to that of dihydroquercetin monomer.

[0149] Test Example 10

[0150] ABTS + Free radical scavenging test

[0151] (1) Experimental principle

[0152] In the presence of an oxidant, ABTS is oxidized to ABTS + Free radicals, the solution will appear green and have strong absorption at the ultraviolet wavelength of 734nm. When antioxidants are added to the system, ABTS + The amount of generated will decrease, the color of the solution will become lighter, gradually changing from dark green to light green, and the absorbance at 734nm will become smaller, so as to determine the ABTS of the substance + Free radical scavenging rate.

[0153] (2) Experimental materials: dihydroquercetin betaine cocrystal obtained in Example 1, dihydroquercetin and L-ascorbic acid (positive control group).

[0154] (3) Experimental steps

[0155] For each sample in (2), set up a sample tube (A S ), sample background (A b ), sample blank tube (A0), sample tubes of each tested concentration of each sample (A S ) need to set up 3 parallel tubes, and the sample blank tube (A0) also need to set up 3 parallel tubes. S ) and sample background (A b ) were added with 0.2 mL of sample solution of the same concentration, and 0.2 mL of PBS buffer was added to the blank sample tube (A0). S ) and the sample blank tube (A0) were each added with 0.8 mL ABTS. + Working solution, sample background (A b ) was added with 0.8 mL of PBS buffer and reacted for 6 min in the dark. The solution in each reaction tube was transferred into a 1 cm cuvette and the sample tube (A) was tested at 734 nm. S ), sample background (Ab ) The absorbance value corresponding to the sample blank tube (A0), and then calculate the ABTS + radical scavenging rate according to the test results, and summarize the test results in Table 3 uniformly.

[0156] Among them, the + formula for calculating the ABTS [[ID=?]]

[0157] [[ID=?]] [[ID=?]] [[ID=?]]

[0158] Table 3

[0159]

[0160] Statistical method: The t-test method was used for analysis, and the test level α = 0.05; P≥0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference; P < 0.001 indicates an extremely significant difference.

[0161] Referring to Table 3, both dihydroquercetin betaine eutectic and dihydroquercetin in Experimental Example 1 have the effect of scavenging ABTS + radicals. Under the same molar concentration (that is, under the condition that the content of dihydroquercetin in the eutectic and aqueous solution is the same), the dihydroquercetin betaine eutectic in Experimental Example 1 has a better + effect on scavenging ABTS

[0162] Test Example 11

[0163] [[ID=?]]Toxicity test

[0164] (1) Experimental principle

[0165] Based on human skin-derived fibroblasts (Fibroblasts, Fbs) and human keratinocytes (HaCaT), the absorbance method was used to detect cell viability, so as to evaluate the cytotoxicity of the sample.

[0166] (2) Experimental steps

[0167] Inoculate different cell suspensions into 96-well cell culture plates for culture, add different concentrations of dihydroquercetin betaine eutectic and dihydroquercetin respectively and continue to culture. Use cell metabolic activity (CCK-8 method) to detect the activity of human skin-derived fibroblasts, use MTT colorimetric method to detect the activity of human keratinocytes, and use GraphPad Prism to calculate the sample concentration when the cell survival rate is 90% as CV 90 .

[0168] (3) Experimental results

[0169] Dihydroquercetin betaine cocrystal CV based on human fibroblast cells 90 =0.2296mg / mL, dihydroquercetin CV 90 =0.1087 mg / mL. Based on human keratinocytes, dihydroquercetin betaine cocrystal CV 90 =0.1350mg / mL, dihydroquercetin CV 90 =0.0431 mg / mL. In different cell types, the toxicity of dihydroquercetin-betaine cocrystals was lower than that of dihydroquercetin, indicating that the cytotoxicity of dihydroquercetin and betaine cocrystals was lower.

[0170] Test Example 12

[0171] Detection of mitochondrial ROS based on human skin-derived fibroblasts

[0172] (1) Experimental principle

[0173] Based on human skin-derived fibroblasts (Fbs), the protective effects of the samples on mitochondria were evaluated in terms of mitochondrial ROS production. MitoSOX Red is a live-cell fluorescent probe that specifically targets mitochondria and has cell membrane permeability. After entering the mitochondria, MitoSOX Red is oxidized by reactive oxygen species. The oxidized MitoSOX Red then binds to nucleic acids within the mitochondria / nucleus, producing strong red fluorescence. MitoSOX Red can be used as a fluorescent indicator to specifically detect the level of reactive oxygen species within the mitochondria. The stronger the fluorescence, the higher the level of reactive oxygen species within the mitochondria.

[0174] (2) Experimental steps

[0175] Fbs cells were seeded in 24-well plates and cultured overnight at 37°C under 95% humidity and 5% CO2. The next day, UVA irradiation was performed as described in Table 4, with an irradiation dose of 4.8 J / cm 2 After UVA irradiation, each well was replaced with a culture medium containing the test substance (i.e., a sample set as needed) and cultured at 37°C for 24 h under saturated humidity, 5% CO2.

[0176] Table 4

[0177]

[0178]

[0179] At the end of the time course, refer to the reagent instructions and stain each well with the mitochondrial ROS fluorescent probe MitoSOX Red. After staining, photographs were taken using a 20× objective lens under a fluorescence microscope. The mean fluorescence intensity after MitoSOX Red staining was analyzed using ImageJ software, and the mitochondrial ROS reduction rate was calculated. The results are summarized in Table 5. The calculation formula is as follows:

[0180]

[0181] All data were expressed as mean ± standard deviation, and the groups were compared using t-test. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0182] Table 5

[0183]

[0184] Compared with the blank control group, ##: P < 0.01; compared with the negative control group, **: P < 0.01.

[0185] See Table 5 and Figure 10 Compared with the blank control group, the mean fluorescence intensity of mitochondrial ROS in the negative control group was significantly increased (P<0.01), indicating that the stimulation conditions in this experiment were effective. Compared with the negative control group, the mean fluorescence intensity of mitochondrial ROS in the positive control group was significantly decreased (P<0.01), indicating that the positive control in this experiment was effective. Compared with the negative control group, the mean fluorescence intensity of mitochondrial ROS in the 0.526mM, 0.329mM, and 0.164mM dihydroquercetin-betaine cocrystal groups significantly decreased (P<0.01), with decrease rates of 59.3%, 54.8%, and 42.7%, respectively. Compared with the negative control group, the mean fluorescence intensity of mitochondrial ROS in the 0.526mM, 0.329mM, and 0.164mM dihydroquercetin groups significantly decreased (P<0.01), with decrease rates of 40.3%, 32.7%, and 26.4%, respectively. At the same molar concentration, the effect of dihydroquercetin betaine cocrystal is better than that of dihydroquercetin, which proves that dihydroquercetin betaine cocrystal can reduce mitochondrial ROS, thereby playing an antioxidant role in protecting mitochondria.

[0186] Test Example 13

[0187] Mitochondrial morphology detection based on human skin-derived fibroblasts

[0188] (1) Experimental principle

[0189] Based on human skin-derived fibroblasts (Fibroblasts, Fbs), the protective effect of the samples on mitochondria was evaluated from the perspective of mitochondrial morphology. MitoTracker Green is a mitochondrial-specific green fluorescent probe that can freely pass through the cell membrane and mitochondrial membrane, and covalently bind to the free thiol groups in the mitochondrial matrix. It is often used for mitochondrial morphology observation and mitochondrial number detection. When irradiated with ultraviolet light, the mitochondria in the cells will swell due to oxidative stress. At this time, the volume of the mitochondrial matrix increases and the fluorescent area increases. When the number of mitochondria in the cell increases, GSH synthesis or thioredoxin in the mitochondrial matrix increases, Mitotracker Green conjugates increase, and the mean fluorescence intensity increases. Therefore, the mitochondrial state can be judged by the mean fluorescence intensity after Mitotracker Green staining: the greater the mean fluorescence intensity, the greater the number of mitochondria.

[0190] (2) Experimental steps

[0191] Fbs cells were seeded in 24-well plates and cultured overnight at 37°C under 95% humidity and 5% CO2. The next day, UVA irradiation was performed as described in Table 6, with an irradiation dose of 4.8 J / cm 2 After UVA irradiation, each well was replaced with a culture medium containing the test substance (i.e., a sample set as needed) and cultured at 37°C for 24 h under saturated humidity, 5% CO2.

[0192] Table 6

[0193]

[0194] At the end of the time course, refer to the reagent instructions and stain each well with Mitotracker Green FM, a fluorescent probe for mitochondrial morphology. After staining, photograph the cells using a 20× objective lens under a fluorescence microscope. The mean fluorescence intensity (MI) after Mitotracker Green FM staining was analyzed using ImageJ software. The MI increase rate after Mitotracker staining was calculated and the results are tabulated in Table 7. The calculation formula is as follows:

[0195]

[0196] All data were expressed as mean ± standard deviation, and the groups were compared using t-test. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0197] Table 7

[0198]

[0199]

[0200] Compared with the blank control group, ##: P < 0.01; compared with the negative control group, **: P < 0.01.

[0201] See Table 7 and Figure 11 Compared with the blank control group, the mean fluorescence intensity of mitochondria in the negative control group decreased significantly (P<0.01), indicating that the stimulation conditions in this experiment were effective. Compared with the negative control group, the mean fluorescence intensity of mitochondria in the positive control group increased significantly (P<0.01), indicating that the positive control was effective. Compared with the negative control group, the mean fluorescence intensity of mitochondria in the 0.526mM and 0.329mM dihydroquercetin-betaine cocrystal groups increased significantly (P<0.01), with increases of 78.5% and 32.1%, respectively. Compared with the negative control group, the mean fluorescence intensity of mitochondria in the 0.526mM and 0.329mM dihydroquercetin groups increased significantly (P<0.05), with increases of 39.2% and 25.4%, respectively. At the same molar concentration, the dihydroquercetin-betaine cocrystal was more effective than dihydroquercetin, demonstrating that the dihydroquercetin-betaine cocrystal can protect mitochondria.

[0202] 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 dihydroquercetin betaine cocrystal, characterized in that The structural formula of the dihydroquercetin betaine co-crystal is shown in Formula I:

2. The dihydroquercetin betaine cocrystal according to claim 1, wherein The molecular formula of the dihydroquercetin betaine cocrystal is C 20 H 23 NO9, and in the dihydroquercetin-betaine cocrystal, the molar ratio of the dihydroquercetin to the betaine is 1:

1.

3. The dihydroquercetin betaine cocrystal according to claim 1, wherein The dihydroquercetin betaine cocrystal is a triclinic system with a space group of P1 and unit cell parameters of α=73.9200(10)°,β=88.0180(10)°,γ=69.3930(10)°,Z=2,unit cell volume 4. The dihydroquercetin betaine cocrystal according to any one of claims 1 to 3, characterized in that The X-ray powder diffraction pattern of the dihydroquercetin betaine cocrystal has characteristic peaks at 2θ angles of 17.52°±0.2°, 22.07°±0.2°, 24.14°±0.2° and 47.41°±0.2°.

5. A method for preparing the dihydroquercetin betaine cocrystal according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: mixing dihydroquercetin, betaine and an organic solvent to obtain a mixed solution; S2 placing the mixed solution at 55-85° C. for stirring and reacting to obtain a precursor solution containing the dihydroquercetin betaine cocrystal; S3: performing crystallization treatment, solid-liquid separation treatment and drying treatment on the precursor solution in sequence to obtain the dihydroquercetin betaine cocrystal.

6. The preparation method according to claim 5, characterized in that In the step of placing the mixed solution at 55-85° C. for stirring reaction, the reaction time is 2-4 hours, or / and the stirring speed is 300-800 rpm.

7. The preparation method according to claim 5, characterized in that In the mixed solution, the molar ratio of the dihydroquercetin to the betaine is 1:1, or / and the ratio of the sum of the mass of the dihydroquercetin and the betaine to the mass of the organic solvent is 1:(5-20).

8. The preparation method according to any one of claims 5 to 7, characterized in that The organic solvent is selected from at least one of ethanol, ether, methanol and ethanol aqueous solution; Optionally, the organic solvent is selected from ethanol aqueous solution; Optionally, the water content in the ethanol aqueous solution is not higher than 10 wt %.

9. The preparation method according to any one of claims 5 to 7, characterized in that After the step of obtaining the precursor solution and before the step of performing the crystallization treatment, the method further includes the step of filtering the precursor solution while it is hot; Or / and, in the step of crystallization treatment, the treatment temperature is 0 to 30°C.

10. Use of the dihydroquercetin betaine cocrystal according to any one of claims 1 to 4 in the preparation of food, medicine, cosmetics or skin care products.

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