Dihydroquercetin betaine co-crystal, and preparation method and application thereof

Abstract

The application provides a dihydroquercetin betaine co-crystal as well as a preparation method and application thereof. A structural formula of the dihydroquercetin betaine co-crystal is shown as formula I. The dihydroquercetin betaine co-crystal has relatively excellent antioxidant performance and water solubility.

Description

Technical Field

[0001] This application relates to the field of eutectic manufacturing technology, and more specifically, to a dihydroquercetin betaine eutectic, its preparation method, and its application. Background Technology

[0002] Dihydroquercetin (also known as piperidine) has five phenolic hydroxyl groups in its molecular structure, making it a natural antioxidant that can effectively remove free radicals and toxins from the body. It possesses a broad spectrum of biological and pharmacological activities, including anti-inflammatory, antibacterial, anti-radiation, anti-cancer, antiviral, immune-regulating, melanin-clearing, and microcirculation-improving effects, making it a valuable raw material for the production of food, pharmaceuticals, cosmetics, and skincare products. However, dihydroquercetin is relatively scarce (found only in small amounts in yew, paulownia, larch, privet, and wild black cherry), hindering its large-scale application. Furthermore, its poor water solubility (resulting in low bioavailability and poor compatibility) limits its promotion and application. Therefore, effectively improving its antioxidant properties (enhanced antioxidant properties allow even small amounts of dihydroquercetin to achieve superior antioxidant effects) and water solubility (improved water solubility enables the preparation of more diverse dosage forms and products, while also improving bioavailability) is a major challenge. Summary of the Invention

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

[0004] The embodiments of this application are implemented as follows:

[0005] In a first aspect, embodiments of this application provide a dihydroquercetin betaine eutectic, the structural formula of which is shown in Formula I:

[0006]

[0007] In the above technical solution, the amino group in betaine has strong electronegativity and forms an inner salt structure. Dihydroquercetin has functional groups such as a benzene ring and five phenolic hydroxyl groups, which allows 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 crystal lattice. After dihydroquercetin and betaine form a eutectic, the crystal arrangement is different from that of dihydroquercetin alone. This results in improved antioxidant properties and water solubility of the dihydroquercetin-betaine eutectic containing the same mass of dihydroquercetin, provided that the mass of dihydroquercetin is constant.

[0008] In some alternative embodiments, the molecular formula of the dihydroquercetin betaine eutectic is C2. 20 H23 NO9, and in the dihydroquercetin-betaine eutectic, the molar ratio of dihydroquercetin to betaine is 1:1.

[0009] In some alternative embodiments, the dihydroquercetin betaine eutectic is triclinic with space group P1 and cell parameters of [missing information]. α=73.9200(10)°, β=88.0180(10)°, γ=69.3930(10)°, Z=2, cell volume

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

[0011] Secondly, embodiments of this application provide a method for preparing dihydroquercetin-betaine eutectic as provided in the first aspect embodiment, comprising the following steps:

[0012] S1. Dihydroquercetin, betaine, and an organic solvent are mixed to obtain a mixed solution; S2. The mixed solution is stirred at 55–85°C to obtain a precursor solution containing dihydroquercetin-betaine eutectic; S3. The precursor solution is subjected to crystallization treatment, solid-liquid separation treatment, and drying treatment in sequence to obtain dihydroquercetin-betaine eutectic.

[0013] By following the above process, a dihydroquercetin-betaine eutectic as provided in the first aspect embodiment can be prepared. The stirring reaction is carried out at 55-85°C to provide a suitable driving force so that dihydroquercetin and betaine can react and react relatively completely. 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 betaine and dihydroquercetin form hydrogen bonds and other interaction forces, thereby combining together to form a eutectic that is regularly arranged and stably exists in the same crystal lattice.

[0014] In some alternative embodiments, the step of placing the mixed solution at 55–85°C and stirring for 2–4 hours, or / and at a stirring speed of 300–800 rpm, is carried out.

[0015] In the above technical solution, the reaction time and stirring speed are limited to the above range to provide more suitable reaction conditions, which helps dihydroquercetin and betaine to react fully, thereby improving the utilization rate of raw materials, product yield and purity.

[0016] In some alternative embodiments, the molar ratio of dihydroquercetin to betaine in the mixed solution 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).

[0017] In the above technical solution, limiting the molar ratio of dihydroquercetin to betaine in the mixed solution to the above range can yield a eutectic product with a molar ratio closer to 1:1; limiting the mass ratio of the sum of the masses of dihydroquercetin and betaine in the mixed solution to the mass ratio of the organic solvent to the above range 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, diethyl ether, methanol, and aqueous ethanol solution; optionally, the organic solvent is selected from aqueous ethanol solution; optionally, the water content in the aqueous ethanol solution is not higher than 10 wt%.

[0019] In the above technical solutions, the embodiments of this application are applicable to a wide variety of organic solvents, providing more feasible implementation schemes, thereby facilitating the promotion and application of the technical solutions provided in the embodiments of this application. Furthermore, using an aqueous ethanol solution as the organic solvent can more easily dissolve dihydroquercetin and betaine, thus helping to reduce the amount of solvent used and save costs; furthermore, limiting the water content in the aqueous ethanol solution to the above-mentioned range makes it even easier to dissolve dihydroquercetin and betaine.

[0020] In some alternative embodiments, after the step of obtaining the precursor solution and before the step of crystallization treatment, a step of hot filtration of the precursor solution is included; and / or, in the crystallization treatment step, the treatment temperature is 0–30°C.

[0021] In the above technical solution, the addition of a hot filtration step can remove insoluble impurities in the mixed system before the eutectic product precipitates, thereby improving the purity of the obtained eutectic product; in the crystallization treatment step, limiting the temperature within the above range helps to balance the crystallization rate and the uniformity of grain size.

[0022] Thirdly, embodiments of this application provide the application of dihydroquercetin betaine eutectic as provided in the first aspect embodiment in the preparation of food, pharmaceuticals, cosmetics or skin care products. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of a single crystal of the dihydroquercetin betaine eutectic obtained in Example 1 of this application;

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

[0026] Figure 3 This is an infrared comparison image of the dihydroquercetin betaine eutectic obtained in Example 1 of this application with various monomers;

[0027] Figure 4 These are SEM comparison images of the dihydroquercetin betaine eutectic obtained in Example 1 of this application and various monomers;

[0028] Figure 5 These are XRD comparison images of the dihydroquercetin betaine eutectic, the physical mixture of dihydroquercetin betaine, and various monomers obtained in Example 1 of this application;

[0029] Figure 6 This is a DSC comparison chart of the dihydroquercetin betaine eutectic obtained in Example 1 of this application with various monomers;

[0030] Figure 7 This is the TG spectrum of the dihydroquercetin betaine eutectic obtained in Example 1 of this application;

[0031] Figure 8 This is a comparison of the dissolution curves of the dihydroquercetin betaine eutectic obtained in Example 1 of this application and dihydroquercetin.

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

[0033] Figure 10 This is a comparison diagram of mitochondrial ROS of the dihydroquercetin betaine cocrystal obtained in Example 1 of this application and dihydroquercetin.

[0034] Figure 11 This is a comparison diagram of the mitochondrial morphology of the dihydroquercetin-betaine cocrystal obtained in Example 1 of this application and dihydroquercetin. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0036] It should be noted that the terms "and / or" in this application, such as "feature 1 and / or feature 2", all refer to the three cases of "feature 1" alone, "feature 2" alone, and "feature 1" plus "feature 2".

[0037] In addition, in the description of this application, unless otherwise stated, "one or more" means two or more; the range of "numerical value a to numerical value b" includes the two endpoints "a" and "b"; and "unit of measurement" in "numerical value a to numerical value b + unit of measurement" represents the "unit of measurement" of both "numerical value a" and "numerical value b".

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

[0039] However, the formation of eutectics is unpredictable. Even two compounds with extremely similar structures may react with the same ligand to form completely different products. For example, salicylic acid and nicotinamide form a eutectic, while 4-methoxysalicylic acid and nicotinamide form an ionic salt. This means that even a slight difference in a single substituent can lead to entirely different products when combined with the same ligand. Therefore, selecting a suitable ligand capable of forming a eutectic for improving the performance of the modified material is extremely difficult.

[0040] Based on this, the inventors have made an innovative discovery that by using betaine as a binding ligand, dihydroquercetin can bind with betaine to form a eutectic, and this eutectic product has both excellent antioxidant properties and water solubility.

[0041] The following is a detailed description of a dihydroquercetin-betaine eutectic, its preparation method, and its application, based on embodiments of this application.

[0042] In a first aspect, embodiments of this application provide a dihydroquercetin betaine eutectic, the structural formula of which is shown in Formula I:

[0043]

[0044] In this application, the amino group in betaine has strong electronegativity and forms an inner salt structure. Dihydroquercetin has functional groups such as a benzene ring and five phenolic hydroxyl groups, enabling 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 crystal lattice. After dihydroquercetin and betaine form a eutectic, the crystal arrangement is different from that of dihydroquercetin alone. This results in improved antioxidant properties and water solubility of the dihydroquercetin-betaine eutectic containing the same mass of dihydroquercetin, given a fixed mass of dihydroquercetin. Furthermore, the embodiment of this application prepares dihydroquercetin into a stable and unique eutectic form, providing a new application 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, belonging to the quaternary ammonium base class. Betaine is widely found in both plants and animals. Specifically, in plants, wolfberry and legumes contain betaine, with molasses from beets being the main source. In animals, octopus, cuttlefish, shrimp, and other mollusks, as well as the liver, spleen, and amniotic fluid of vertebrates (including humans), all contain betaine. Betaine possesses various physiological functions, including regulating osmotic pressure, protecting cell structure, and promoting lipid metabolism, and is widely used in the pharmaceutical and food industries. Therefore, in this application, betaine is co-crystallized with dihydroquercetin. This not only enhances the antioxidant properties and water solubility of dihydroquercetin but also allows the co-crystallized product to possess the multiple physiological functions of betaine.

[0046] As an example, the molecular formula of the dihydroquercetin-betaine eutectic is C2. 20 H 23 NO9, and in the dihydroquercetin-betaine eutectic, the molar ratio of dihydroquercetin to betaine is 1:1.

[0047] As an example, the dihydroquercetin-betaine eutectic is triclinic with space group P1 and cell parameters of [missing information]. α = 73.9200(10)°, β = 88.0180(10)°, γ = 69.3930(10)°, Z = 2, cell volume

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

[0049] Secondly, embodiments of this application provide a method for preparing dihydroquercetin-betaine eutectic as provided in the first aspect embodiment, comprising the following steps:

[0050] S1. Dihydroquercetin, betaine, and an organic solvent are mixed to obtain a mixed solution; S2. The mixed solution is placed 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 any range between two of them) and stirred to obtain a precursor solution containing dihydroquercetin-betaine eutectic; S3. The precursor solution is subjected to crystallization treatment, solid-liquid separation treatment, and drying treatment in sequence to obtain dihydroquercetin-betaine eutectic.

[0051] In this application, by preparing according to the above process, a dihydroquercetin-betaine eutectic as provided in the first aspect embodiment can be obtained. The stirring reaction at 55-85°C is to provide a suitable driving force so that dihydroquercetin and betaine can react and react relatively completely. 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 betaine and dihydroquercetin generate hydrogen bonds and other interaction forces to combine together, thereby forming a eutectic that is regularly arranged and stably exists in the same crystal lattice.

[0052] It should be noted that there are no restrictions on the method of mixing dihydroquercetin, betaine, and organic solvent. For example, it can be a one-step mixing method or a stepwise mixing method (for example, first mix dihydroquercetin and organic solvent to obtain a premix; then mix the premix with betaine). The specific method can be adapted 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 2–4 h, for example, but not limited to any one of 2 h, 2.5 h, 3 h, 3.5 h and 4 h or any range between two; and / or the stirring speed is 300–800 rpm, for example, but not limited to any one of 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm and 800 rpm or any range between two.

[0054] In this embodiment, the reaction time and stirring speed are limited to the above-mentioned ranges to provide more suitable reaction conditions, which helps dihydroquercetin and betaine to react fully, thereby improving the utilization rate of raw materials, product yield and purity.

[0055] As an example, in the mixed solution, the molar ratio of dihydroquercetin to 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 to 20), for example, but not limited to any one of the mass ratios of 1:5, 1:10, 1:15 and 1:20 or any range between the two.

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

[0057] It should be noted that there are no restrictions on the type of organic solvent, and it can be selected and set in accordance with the conventional methods in this field.

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

[0059] In this embodiment, the types of organic solvents applicable to the present application are numerous, providing a wide range of feasible implementation schemes, thereby facilitating the promotion and application of the technical solutions provided in the present application embodiments.

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

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

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

[0063] In this embodiment, limiting the water content in the ethanol aqueous solution to the above range makes it easier to dissolve dihydroquercetin and betaine.

[0064] As an example, after obtaining the precursor solution and before the crystallization process, a step of hot filtration of the precursor solution is included.

[0065] It should be noted that "hot filtration" refers to filtering the solution system while it is still at a relatively high temperature (close to the temperature of the stirring reaction) after the stirring reaction is completed. Under this temperature condition, the eutectic product will not precipitate from the solution system. Then, the solution system with a relatively high temperature is directly filtered.

[0066] In this embodiment, the addition of a hot filtration step can remove insoluble impurities in the mixed system before the eutectic product precipitates, thereby improving the purity of the obtained eutectic product.

[0067] Understandably, lower crystallization temperatures result in higher efficiency, making them suitable for large-scale mass production; higher crystallization temperatures result in slower rates, but produce more uniform grain sizes. Therefore, the crystallization temperature can be adjusted adaptively according to actual needs during the crystallization process.

[0068] As an example, in the crystallization process, the processing temperature is 0 to 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 any range between two of them.

[0069] In this embodiment, limiting the temperature within the above-mentioned range during the crystallization process helps to balance the crystallization rate and the uniformity of 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 adjusted according to the actual situation.

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

[0072] It should be noted that the specific drying method is not limited in the drying process; for example, vacuum drying can be used. During vacuum drying, the drying temperature is 50–55°C, for example, but not limited to any one of 50°C, 51°C, 52°C, 53°C, 54°C, and 55°C, or any range between two of these temperatures. The drying time is 12–48 hours, for example, but not limited to any one of 12 hours, 15 hours, 30 hours, 40 hours, and 48 hours, or any range between two of these times.

[0073] It should be noted that, unless otherwise specified or limited, the processes or steps in the preparation of dihydroquercetin betaine eutectic can be set according to conventional methods in this field.

[0074] Thirdly, embodiments of this application provide the application of dihydroquercetin betaine eutectic as provided in the first aspect embodiment in the preparation of food, pharmaceuticals, cosmetics or skin care products.

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

[0076] Example 1

[0077] This application provides a method for preparing dihydroquercetin betaine eutectic, comprising the following steps:

[0078] S1 cleans the chamber of the reactor with ethanol and dries it with compressed air. Then, 72.2g of dihydroquercetin and 27.8g of betaine are added to the reactor, followed by 1.5kg of anhydrous ethanol. The agitator is turned on and stirred at 500rpm. At the same time, the heating unit is started and the temperature is set to 80℃ until a clear mixed solution is obtained.

[0079] S2 maintains the internal temperature of the reaction chamber at 80℃ and stirs the reaction at 500rpm for 4h to obtain a precursor solution containing dihydroquercetin betaine eutectic.

[0080] S3 filters the precursor solution while hot to obtain a filtrate containing dihydroquercetin betaine eutectic.

[0081] S4 was cooled to 20℃ and kept at that temperature for 10 hours to allow crystallization. Then, solid-liquid separation was performed, and the resulting solid was dried in a vacuum drying oven at 50℃ for 24 hours, yielding 89.42g of product with a yield of 88.9%. Simultaneously, the melting range (i.e., the temperature range from the initial melting point to complete melting of the pure compound) was determined to be 219.45–221.64℃ (where the melting point of dihydroquercetin monomer is close to 240℃, and the melting point of betaine monomer is 301–305℃).

[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℃, indicating that the obtained product is a eutectic with a new structure.

[0083] Example 2

[0084] This application provides a method for preparing dihydroquercetin betaine eutectic, comprising the following steps:

[0085] S1 cleans the chamber of the reactor with ethanol and dries it with compressed air. Then, 72.2g of dihydroquercetin and 27.8g of betaine are added to the reactor, followed by 1kg of methanol. The agitator is turned on and stirred at 700rpm. At the same time, the heating unit is started and the temperature is set to 80℃ until a clear mixed solution is obtained.

[0086] S2 maintains the internal temperature of the reaction chamber at 80℃ and stirs the reaction at 700rpm for 3h to obtain a precursor solution containing dihydroquercetin betaine eutectic.

[0087] S3 filters the precursor solution while hot to obtain a filtrate containing dihydroquercetin betaine eutectic.

[0088] S4 The filtrate was cooled to 5°C and kept at that temperature for 12 hours to allow crystallization. Then, solid-liquid separation was performed, and the resulting solid was dried in a vacuum drying oven at 50°C for 20 hours, yielding 87.96g of product with a yield of 87.96%. The melting range (i.e., the temperature range from the beginning of melting to complete melting of the pure compound) of the product was measured to be 218.62–221.15°C (as can be seen from the comparison between Example 1 and Example 2, 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] This application provides a method for preparing dihydroquercetin betaine eutectic, comprising the following steps:

[0091] S1 cleans the chamber of the reactor with ethanol and dries it with compressed air. Then, 72.2g of dihydroquercetin and 27.8g of betaine are added to the reactor, followed by 0.5kg of ethanol with a water content of 5%. The agitator is turned on and stirred at 500rpm. At the same time, the heating unit is started and the temperature is set to 85℃ until a clear mixed solution is obtained.

[0092] S2 maintains the internal temperature of the reaction chamber at 85℃ and stirs the reaction at 500rpm for 2h to obtain a precursor solution containing dihydroquercetin betaine eutectic.

[0093] S3 filters the precursor solution while hot to obtain a filtrate containing dihydroquercetin betaine eutectic.

[0094] S4 The filtrate was cooled to 0℃ and kept at that temperature for 10 hours to allow crystallization. Then, solid-liquid separation was performed, and the resulting solid was dried in a vacuum drying oven at 50℃ for 24 hours, yielding 88.75g of product with a yield of 88.75%. The melting range (i.e., the temperature range from the beginning of melting to complete melting of the pure compound) of the product was measured to be 220.36~222.2℃ (as can be seen from the comparison between Example 1 and Example 2, 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, as can be seen from the comparison of Examples 1 to 3, when an aqueous ethanol solution is used as an organic solvent, the amount of organic solvent used is significantly reduced, which helps to reduce solvent costs.

[0096] Comparative Example 1

[0097] This application provides a method for preparing dihydroquercetin betaine eutectic, which differs from Example 1 only in that the temperature of the reaction vessel is set to 50°C in steps S1 and S2.

[0098] 85.35 g of product was obtained, with a yield of 85.35%. Simultaneously, the melting range (i.e., the temperature range from the initial melting point to complete melting of the pure compound) of the product was measured to be 240.03–304.98 °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 was too low, making it difficult for dihydroquercetin and betaine to react and form a eutectic).

[0099] Experimental Example 1

[0100] Qualitative analysis of dihydroquercetin-betaine eutectic

[0101] Test method: X-ray single-crystal diffraction was performed on the dihydroquercetin-betaine eutectic obtained in Example 1. Specific test results are shown in Table 1, and a schematic diagram of the crystal is shown below. Figure 1 .

[0102] Table 1

[0103]

[0104]

[0105] Experimental Example 2

[0106] 1H NMR spectrum ( 1 H-NMR characterization

[0107] Test method: The dihydroquercetin-betaine cocrystal obtained in Example 1 was subjected to 1H NMR spectroscopy. 1 Characterization was performed using H-NMR, and MeOD was used as the test solvent in the experiment.

[0108] See Figure 2 The 1H NMR spectrum clearly shows 7 hydrogen atoms of dihydroquercetin and 11 hydrogen atoms of betaine, with the remainder being peaks of deuterated reagents. No obvious impurity peaks were observed, indicating that dihydroquercetin and betaine exist in a 1:1 molar ratio in the dihydroquercetin-betaine eutectic.

[0109] Experimental Example 3

[0110] Infrared spectroscopy characterization

[0111] Test methods: Infrared spectroscopy was performed on dihydroquercetin, betaine, and the dihydroquercetin-betaine cocrystal obtained in Example 1. The test parameter was transmittance, and the test wavenumber was 400 cm⁻¹.-1 ~4000cm -1 The test mode is ATR.

[0112] See Figure 3 The infrared spectrum of the dihydroquercetin-betaine cocrystal shows different absorption peaks from those of betaine and dihydroquercetin, indicating that it is not a simple superposition of the characteristic peaks of the two precursors. The OH group in dihydroquercetin exhibits stretching at 3420 cm⁻¹. -1 A characteristic peak is observed at this location; in the eutectic, the OH group of dihydroquercetin acts as a hydrogen bond donor, and its OH group undergoes a stretching red shift to 3057 cm⁻¹. -1 At the location; the C=O in betaine is stretched to 1614 cm. -1 A characteristic peak is observed at this location; in the eutectic, the C=O group of betaine acts as a hydrogen bond acceptor, and its C=O group undergoes a stretching blue shift to 1625 cm⁻¹. -1 The location indicates that dihydroquercetin and betaine formed a eutectic, which is a new structure.

[0113] Test Example 4

[0114] Morphological characterization (SEM)

[0115] Test methods: The dihydroquercetin, betaine, and the dihydroquercetin-betaine cocrystal obtained in Example 1 were characterized by scanning electron microscopy.

[0116] See Figure 4 Dihydroquercetin solid particles are columnar crystals, betaine solid particles are clumps of solid without obvious crystal form, and dihydroquercetin-betaine eutectic solid particles are blocky crystals. The microstructure of the eutectic is significantly different from that of the monomer.

[0117] Experimental Example 5

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

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

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

[0121] Experimental Example 6

[0122] Differential Scanning Calorimetry (DSC) Test

[0123] Test methods: Differential scanning calorimetry (DSC) was performed on dihydroquercetin, betaine, and the dihydroquercetin-betaine eutectic obtained in Example 1.

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

[0125] Experimental Example 7

[0126] Thermogravimetric analysis (TG) test

[0127] Test method: Thermogravimetric analysis (TG) was performed on the dihydroquercetin betaine eutectic obtained in Example 1.

[0128] See Figure 7 The thermal decomposition temperature of dihydroquercetin betaine eutectic is above 200℃, and it has good thermal stability, indicating that it is not easily decomposed during processing and is easy to prepare into various products for application.

[0129] Experimental Example 8

[0130] Water solubility test

[0131] Test method: Dihydroquercetin and the dihydroquercetin-betaine cocrystal obtained in Experiment 1 were dissolved in water. 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 achieves high solubility within a short time, reaching its highest solubility at 10 minutes, which is higher than the water solubility of the monomeric dihydroquercetin. This indicates that the water solubility of the dihydroquercetin-betaine cocrystal is enhanced after it forms a cocrystal with betaine, suggesting that the dihydroquercetin-betaine cocrystal is absorbed quickly in the human body. Furthermore, as time progresses, it reaches a basic dissolution equilibrium at 30 minutes, and the solubility of the dihydroquercetin-betaine cocrystal remains higher than that of the monomeric dihydroquercetin, implying that the dihydroquercetin-betaine cocrystal has a higher absorption rate in the human body.

[0133] Experimental Example 9

[0134] Bioavailability test

[0135] Experimental materials and methods:

[0136] Test substance:

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

[0138] Experimental methods:

[0139] Using a pig skin model, the skin penetration amount at different times was detected to evaluate the transdermal delivery efficiency of the main components in the sample.

[0140] (1) Microscopic examination: Under a dissecting microscope, select undamaged pig skin, cut 3 pieces of skin of the same 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 pool with the stratum corneum facing the administration chamber and the dermis facing the receiving chamber. Add 15 mL of sodium chloride solution (10% ethanol) to the receiving chamber and remove air bubbles to ensure that there are no air 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 1.0 mL of the drug to the drug administration chamber, seal it with sealing film and aluminum foil to prevent liquid evaporation. The effective penetration area is 1.13 cm². 2 .

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

[0144] (5) Sampling: At time points of 2h, 4h, 6h, 22h, 23h, and 24h, 1mL of subcutaneous receiving fluid was pipetted into a 5.0mL EP tube, and then 1.0mL of sodium chloride (10% ethanol) solution was added to the receiving cell using a pipette. Skin samples taken 24h later were cut into small pieces, placed in a 5mL centrifuge tube, soaked overnight with extraction solvent, sonicated for 30min, and then filtered into a sample vial.

[0145] (6) Detection: All the above samples were filtered through a 0.22μm aqueous membrane and then detected by HPLC. The permeation per unit area was calculated and the test results were summarized in Table 2.

[0146] Table 2

[0147]

[0148] See Table 2 and Figure 9The cumulative permeation per unit area of ​​the dihydroquercetin-betaine cocrystallization was significantly higher than that of dihydroquercetin after 6 hours, and 3.23 times higher after 24 hours. After 24 hours, the retention in the cocrystallization shell of the dihydroquercetin-betaine cocrystallization was also significantly higher than that of dihydroquercetin, approximately three times higher. Based on the data in Table 2, the bioavailability of the samples was calculated to be 6.47% for the dihydroquercetin-betaine cocrystallization and 2.11% for dihydroquercetin, indicating that the bioavailability of the dihydroquercetin-betaine cocrystallization was superior to that of the dihydroquercetin monomer.

[0149] Experimental Example 10

[0150] ABTS + Free radical scavenging test

[0151] (1) Experimental Principle

[0152] In the presence of an oxidizing agent, ABTS will be oxidized to ABTS. + Free radicals will cause the solution to turn green and exhibit strong absorption at a wavelength of 734 nm in the ultraviolet light. When an antioxidant is added to the system, ABTS... + The amount of ABTS produced will decrease, the solution color will lighten, gradually changing from dark green to light green, and the absorbance at 734 nm will decrease. This is used to determine the ABTS content of the substance. + Free radical scavenging rate.

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

[0154] (3) Experimental steps

[0155] For each sample in (2), a sample tube (A) is set up. S ), Sample background (A) b ), sample blank tube (A0), and sample tube (A1) for each test concentration of each sample. S Three parallel tubes are required, and three parallel tubes are also required for the sample blank tube (A0). In the sample tube (A... S ) and sample background (A b Add 0.2 mL of the same concentration of sample solution to each of the sample tubes (A1, A2, and A3), and add 0.2 mL of PBS buffer to the blank sample tube (A0). S Add 0.8 mL of ABTS to both the sample tube (A0) and the blank sample tube (A0). + Working solution, sample background (A) b Add 0.8 mL of PBS buffer, react in the dark for 6 min, then transfer the solution from each reaction tube into a 1 cm cuvette, and test the sample tubes (A) at 734 nm. S ), Sample background (A)b ) 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 calculation formula of the ABTS + radical scavenging rate is as follows:

[0157]

[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 significant difference; P < 0.01 indicates very significant difference; P < 0.001 indicates 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 + scavenging effect on ABTS radicals than dihydroquercetin, proving that the formation of eutectic by dihydroquercetin and betaine has better antioxidant capacity.

[0162] Test Example 11

[0163] 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] Based on human fibroblast cells, dihydroquercetin-betaine co-crystal CV 90 =0.2296 mg / mL, CV of dihydroquercetin 90 =0.1087 mg / mL. Based on human keratinocytes, the CV of the dihydroquercetin-betaine co-crystal 90 =0.1350 mg / mL, CV of dihydroquercetin 90 =0.0431 mg / mL. In different cell types, the toxicity of the dihydroquercetin-betaine cocrystal was lower than that of dihydroquercetin alone, demonstrating that the cytotoxicity of the cocrystal formed by dihydroquercetin and betaine is even lower.

[0170] Experimental Example 12

[0171] Mitochondrial ROS detection based on human skin-derived fibroblasts

[0172] (1) Experimental Principle

[0173] Based on human skin-derived fibroblasts (Fbs), this study evaluates the protective effect of samples on mitochondria from the perspective of mitochondrial ROS production. MitoSOX Red is a live-cell fluorescent probe specifically targeting mitochondria and possessing cell membrane permeability. After entering the mitochondria, MitoSOX Red is oxidized by reactive oxygen species (ROS). The oxidized MitoSOX Red then binds to nucleic acids within the mitochondria / nucleus, producing strong red fluorescence. MitoSOX Red can serve as a fluorescent indicator to specifically detect the ROS content within mitochondria; the stronger the fluorescence, the higher the ROS content in the mitochondria.

[0174] (2) Experimental steps

[0175] Fbs cells were seeded in 24-well plates and cultured overnight at 95% humidity, 5% CO2, and 37°C. The following day, UVA irradiation was performed as described in Table 4, with an irradiation dose of 4.8 J / cm². 2 After UVA irradiation, each well was replaced with a culture medium containing the test substance (i.e., the sample set as needed), and incubated for 24 hours under saturated humidity, 5% CO2, and 37°C.

[0176] Table 4

[0177]

[0178]

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

[0180]

[0181] All data are expressed as mean ± standard deviation. The t-test was used to compare the results between groups. P < 0.05 was considered to be statistically significant, and P < 0.01 was considered to be highly statistically significant.

[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 was effective in this experiment. 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 was 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 was significantly decreased (P<0.01), with decrease rates of 40.3%, 32.7%, and 26.4%, respectively. At the same molar concentration, the cocrystallization of dihydroquercetin and betaine showed better results than that of dihydroquercetin alone, demonstrating that the cocrystallization of dihydroquercetin and betaine can reduce mitochondrial ROS, thereby playing an antioxidant role in protecting mitochondria.

[0186] Experimental Example 13

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

[0188] (1) Experimental Principle

[0189] Based on human skin-derived fibroblasts (Fbs), this study evaluates the protective effect of samples on mitochondria from the perspective of mitochondrial morphology. MitoTracker Green is a mitochondrial-specific green fluorescent probe that can freely cross the cell membrane and mitochondrial membrane, covalently binding to free sulfhydryl groups in the mitochondrial matrix. It is commonly used for mitochondrial morphology observation and mitochondrial quantity detection. When exposed to ultraviolet radiation, mitochondria in cells swell due to oxidative stress, resulting in an increase in mitochondrial matrix volume and fluorescence area. When the number of mitochondria in the cell increases, GSH synthesis or thioredoxin in the mitochondrial matrix increases, leading to an increase in Mitotracker Green conjugates and an increase in average fluorescence intensity. Therefore, the mitochondrial state can be determined by the average fluorescence intensity after Mitotracker Green staining: the higher the average 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 95% humidity, 5% CO2, and 37°C. The following day, UVA irradiation was performed as described in Table 6, with an irradiation dose of 4.8 J / cm². 2 After UVA irradiation, each well was replaced with a culture medium containing the test substance (i.e., the sample set as needed) and incubated for 24 hours under saturated humidity, 5% CO2, and 37°C.

[0192] Table 6

[0193]

[0194] At the end of the treatment time, referring to the reagent instructions, mitochondrial morphology fluorescent probe MitotrackerGreen FM was added to each well for staining. After staining, images were taken with a fluorescence microscope at 20× objective, and the average fluorescence intensity after Mitotracker Green FM staining was analyzed using ImageJ software. The average fluorescence intensity increase rate after Mitotracker staining was calculated, and the results are summarized in Table 7; the calculation formula is as follows:

[0195]

[0196] All data are expressed as mean ± standard deviation. The t-test was used to compare the results between groups. P < 0.05 was considered to be statistically significant, and P < 0.01 was considered to be highly statistically significant.

[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 was significantly decreased (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 was significantly increased (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 was significantly increased (P<0.01), with increase rates 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 was significantly increased (P<0.05), with increase rates of 39.2% and 25.4%, respectively. At the same molar concentration, the dihydroquercetin-betaine cocrystal was more effective than dihydroquercetin alone, proving that the dihydroquercetin-betaine cocrystal can protect mitochondria.

[0202] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A dihydroquercetin betaine eutectic, characterized in that, The molecular formula of the dihydroquercetin-betaine eutectic is C2. 20 H 23 NO9, and in the dihydroquercetin-betaine eutectic, the molar ratio of dihydroquercetin to betaine is 1:1; the structural formula of the dihydroquercetin-betaine eutectic is shown in Formula I: Formula I; The X-ray powder diffraction pattern of the dihydroquercetin betaine eutectic exhibits characteristic peaks at 2θ angles of 17.52°±0.2°, 22.07°±0.2°, 24.14°±0.2°, and 47.41°±0.2°.

2. The dihydroquercetin-betaine eutectic according to claim 1, characterized in that, The dihydroquercetin-betaine eutectic is triclinic with space group P1 and cell parameters a = 8.5570(2) Å, b = 9.3559(2) Å, c = 13.1312(3) Å, α = 73.9200(10)°, β = 88.0180(10)°, γ = 69.3930(10)°, Z = 2, and cell volume V = 943.19(4) Å. 3 .

3. A method for preparing dihydroquercetin-betaine eutectic as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Dihydroquercetin, betaine, and an organic solvent are mixed to obtain a mixed solution; S2 The mixed solution is stirred at 55~85℃ to obtain a precursor solution containing the dihydroquercetin betaine eutectic; S3 The precursor solution is subjected to crystallization treatment, solid-liquid separation treatment and drying treatment in sequence to obtain the dihydroquercetin betaine eutectic.

4. The preparation method according to claim 3, characterized in that, In the step of placing the mixed solution at 55~85℃ for stirring reaction, the reaction time is 2~4 h, or / and the stirring speed is 300~800 rpm.

5. The preparation method according to claim 3, characterized in that, In the mixed solution, the molar ratio of dihydroquercetin to betaine is 1:1, or / and, and the ratio of the sum of the masses of dihydroquercetin and betaine to the mass of the organic solvent is 1:(5~20).

6. The preparation method according to any one of claims 3 to 5, characterized in that, The organic solvent is selected from at least one of ethanol, diethyl ether, methanol, and aqueous ethanol solution.

7. The preparation method according to claim 6, characterized in that, The organic solvent is selected from aqueous ethanol solution.

8. The preparation method according to claim 6, characterized in that, The water content in the ethanol-water solution is not higher than 10 wt%.

9. The preparation method according to any one of claims 3 to 5, characterized in that, After obtaining the precursor solution and before performing the crystallization treatment, the process further includes a step of hot filtration of the precursor solution. Or / and, in the crystallization treatment step, the treatment temperature is 0~30℃.

10. The use of the dihydroquercetin betaine eutectic as described in claim 1 or 2 in the preparation of cosmetics or skin care products.

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