Dihydroquercetin caffeine co-crystal and preparation method and application thereof

Abstract

This application provides a dihydroquercetin-caffeine cocrystal, its preparation method, and its application, belonging to the field of cocrystal manufacturing technology. The structural formula of the dihydroquercetin-caffeine cocrystal is shown in Formula I. This dihydroquercetin-caffeine cocrystal has excellent water solubility, thus giving it ideal compatibility and bioavailability.

Description

Technical Field

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

[0002] Dihydroquercetin is a natural flavonoid compound widely found in larch, buckwheat, and citrus fruits, possessing a variety of biological activities. Due to its unique molecular structure, it can efficiently scavenge free radicals and mitigate oxidative stress-induced cell damage, earning it the title of "antioxidant star." Scientific research shows that dihydroquercetin not only has potent antioxidant capabilities but may also help maintain cardiovascular health, alleviate post-exercise fatigue, and support the skin's resistance to environmental stressors such as ultraviolet radiation by inhibiting the release of inflammatory factors and regulating metabolic pathways. However, dihydroquercetin has poor solubility, being only slightly soluble in water, resulting in poor compatibility (it can only be used with certain polyols and polar oils, the presence of which makes the formulation thicker and more viscous, making it difficult to apply to different dosage forms). Its absorption rate in the human body is also low (animal experiments show its oral bioavailability is less than 10%). Therefore, effectively improving the water solubility of dihydroquercetin, thereby enhancing its compatibility and bioavailability in the human body, is a major challenge currently facing the market. Summary of the Invention

[0003] The purpose of this application is to provide a dihydroquercetin-caffeine cocrystal, its preparation method and application, wherein the dihydroquercetin-caffeine cocrystal has excellent water solubility, thereby giving it ideal compatibility and bioavailability.

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

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

[0006]

[0007] In the aforementioned technical solution, on the one hand, the hydroxyl group in dihydroquercetin can act as a hydrogen bond donor, and the N and double-bonded oxygen in caffeine can act as hydrogen bond acceptors, allowing dihydroquercetin and caffeine to bind via hydrogen bonds. On the other hand, dihydroquercetin and caffeine have relatively parallel unsaturated cyclic structures, enabling them to bind through PI-PI stacking interactions. Through the combined effect of these two aspects, dihydroquercetin and caffeine can form a crystalline material that is regularly arranged and stably exists in the same crystal lattice through intermolecular forces. After dihydroquercetin and caffeine form a eutectic, the crystal arrangement differs from that of dihydroquercetin and caffeine alone, resulting in a significant increase in the water solubility of the dihydroquercetin-caffeine eutectic containing the same mass of dihydroquercetin, given a constant mass of dihydroquercetin.

[0008] In some alternative embodiments, the molecular formula of the dihydroquercetin-caffeine cocrystal is C0. 23 H 22 N4O9, and in the dihydroquercetin-caffeine eutectic, the molar ratio of dihydroquercetin to caffeine is 1:1.

[0009] In some alternative embodiments, the dihydroquercetin-caffeine eutectic is orthorhombic with space group P212121 and cell parameters of [missing information]. α = 90°, β = 90°, γ = 90°, Z = 4, cell volume

[0010] In some alternative implementations, the water solubility of the dihydroquercetin-caffeine cocrystal is greater than 12 mg / mL.

[0011] In the above technical solution, the water solubility of the dihydroquercetin-caffeine cocrystal is higher than 12 mg / mL (based on the molar ratio of dihydroquercetin and caffeine in the cocrystal, it can be seen that the water solubility of dihydroquercetin in the cocrystal is higher than 7 mg / mL), while the water solubility of dihydroquercetin monomer is only 0.4-0.6 mg / mL, indicating that the formation of a cocrystal between dihydroquercetin and caffeine significantly improves the water solubility of dihydroquercetin.

[0012] Secondly, embodiments of this application provide a method for preparing dihydroquercetin-caffeine cocrystal as provided in the first aspect embodiment, comprising the following steps:

[0013] S1. Dihydroquercetin, caffeine, and an organic solvent are mixed to obtain a premix; S2. The premix is ​​ball-milled to allow the dihydroquercetin and caffeine in the premix to react and obtain a precursor containing dihydroquercetin-caffeine cocrystal; S3. The precursor is dried to obtain dihydroquercetin-caffeine cocrystal.

[0014] In the above technical solution, ball milling is used to react dihydroquercetin with caffeine to form a eutectic. Since ball milling can usually be carried out at room temperature and pressure, it does not require harsh reaction conditions such as high temperature, high pressure, vacuum or special atmosphere. This not only reduces the requirements for equipment and operational risks, but also makes the entire preparation process easier to control and safer. At the same time, compared with other methods of preparing eutectic, such as high temperature, high pressure or complex chemical reactions, ball milling has the advantage of lower energy consumption, making it particularly suitable for large-scale industrial production.

[0015] In some alternative embodiments, the step of mixing dihydroquercetin, caffeine, and an organic solvent to obtain a premix includes: mixing dihydroquercetin and caffeine to obtain a premix precursor; and then mixing the premix precursor with an organic solvent to obtain the premix.

[0016] In the above technical solution, dihydroquercetin and caffeine are first mixed, and then the mixed system is mixed with an organic solvent. This allows the two raw materials to come into contact with the organic solvent simultaneously in a uniformly mixed state, thereby effectively reducing the risk of dissolution loss when a single raw material comes into contact with a large amount of organic solvent. This helps to improve the utilization rate of raw materials, product yield and purity.

[0017] In some alternative embodiments, the mass ratio of the sum of dihydroquercetin and caffeine to the mass of the organic solvent in the premix is ​​1:(0.15 to 0.3), or / and the molar ratio of dihydroquercetin to caffeine is 1:1.

[0018] In the above technical solution, limiting the ratio of the sum of the masses of dihydroquercetin and caffeine to the mass of the organic solvent within the aforementioned range serves several purposes. Firstly, an appropriate amount of organic solvent provides suitable lubrication, ensuring good flowability of the material during ball milling and reducing energy loss due to frictional resistance. Simultaneously, it enhances the impact and grinding effect of the grinding media on the material, allowing dihydroquercetin and caffeine to fully contact and react. Secondly, an appropriate amount of organic solvent also reduces the risk of raw material loss due to dissolution in the solvent, thereby improving raw material utilization, product yield, and purity. Furthermore, limiting the molar ratio of dihydroquercetin to caffeine within the aforementioned range helps the two raw materials react fully and form a eutectic product with a molar ratio closer to 1:1.

[0019] In some alternative embodiments, the step of ball milling the premix includes mixing the premix and grinding balls and then ball milling them at a frequency of 20 to 30 Hz for 15 to 30 times, wherein the time for a single ball milling is 60 to 120 s and the interval between ball milling is 30 to 60 s.

[0020] In the above technical solution, limiting the frequency, cycle, and time of each ball milling cycle within the above range helps dihydroquercetin and caffeine to fully contact and react, thereby improving raw material utilization, product yield, and purity. Limiting the interval between two adjacent ball milling cycles within the above range can effectively reduce the material temperature after each ball milling, thereby better maintaining the physicochemical stability of the materials (raw materials and products).

[0021] In some alternative implementations, the mass ratio of the premix to the grinding balls is 1:(4 to 10).

[0022] In the above technical solution, limiting the mass ratio of premix to grinding balls within the above range helps the materials to fully contact and react under the action of the grinding balls, thereby improving the utilization rate of raw materials, the yield of products, and the purity.

[0023] Thirdly, embodiments of this application provide the application of dihydroquercetin-caffeine eutectic as provided in the first aspect embodiment in the preparation of food, pharmaceuticals, cosmetics, or skin care products. Attached Figure Description

[0024] 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.

[0025] Figure 1 This is a schematic diagram of a single crystal of the dihydroquercetin-caffeine eutectic obtained in Example 1 of this application;

[0026] Figure 2 This is the 1H NMR spectrum of the dihydroquercetin-caffeine cocrystal obtained in Example 1 of this application;

[0027] Figure 3 This is an infrared comparison image of the dihydroquercetin-caffeine cocrystal obtained in Example 1 of this application with various monomers;

[0028] Figure 4 These are SEM comparison images of the dihydroquercetin-caffeine cocrystal obtained in Example 1 of this application and various monomers;

[0029] Figure 5 This is a theoretical calculation diagram of the weak interaction of dihydroquercetin-caffeine eutectic provided in the embodiments of this application;

[0030] Figure 6 This is a comparison of the dissolution curves of the dihydroquercetin-caffeine cocrystal obtained in Example 1 of this application and dihydroquercetin.

[0031] Figure 7 This is a comparison chart of the bioavailability of dihydroquercetin-caffeine cocrystal and dihydroquercetin obtained in Example 1 of this application;

[0032] Figure 8 This is a comparison image of mitochondrial ROS fluorescence of dihydroquercetin-caffeine cocrystal, dihydroquercetin, and caffeine obtained in Example 1 of this application.

[0033] Figure 9 This is a fluorescence comparison image of the mitochondrial morphology of dihydroquercetin-caffeine cocrystal, dihydroquercetin, and caffeine obtained in Example 1 of this application.

[0034] Figure 10 This is a comparison diagram of the average fluorescence area of ​​the mitochondrial morphology of the dihydroquercetin-caffeine cocrystal, dihydroquercetin, and caffeine obtained in Example 1 of this application. 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 caffeine as a binding ligand, dihydroquercetin can bind with caffeine to form a co-crystal, and this co-crystal product has excellent water solubility.

[0041] The following is a detailed description of a dihydroquercetin-caffeine 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-caffeine cocrystal, the structural formula of which is shown in Formula I:

[0043]

[0044] In this application, on the one hand, the hydroxyl group in dihydroquercetin can act as a hydrogen bond donor, and the N and double oxygen bonds in caffeine can act as hydrogen bond acceptors, allowing dihydroquercetin and caffeine to bind via hydrogen bonds. On the other hand, dihydroquercetin and caffeine have relatively parallel unsaturated cyclic structures, allowing them to bind via PI-PI stacking interactions. Through the combined effect of these two aspects, dihydroquercetin and caffeine can form a crystalline material that is regularly arranged and stably exists in the same crystal lattice through intermolecular forces. After dihydroquercetin and caffeine form a eutectic, the crystal arrangement is different from that of dihydroquercetin and caffeine alone, resulting in a significant increase in the water solubility of the dihydroquercetin-caffeine eutectic containing the same mass of dihydroquercetin, given a constant mass of dihydroquercetin.

[0045] To better understand the efficacy of the dihydroquercetin-caffeine cocrystal, the physicochemical properties of the two monomers are explained here.

[0046] Dihydroquercetin is a flavonoid compound, usually a pale yellow crystalline powder, soluble in organic solvents such as methanol and ethanol, and widely found in various plants. Chemically, dihydroquercetin has a characteristic skeleton of two benzene rings linked by oxygen atoms and multiple phenolic hydroxyl groups, giving it a variety of biological activities. (1) It has antioxidant properties, effectively scavenging free radicals, reducing oxidative damage, and protecting cells from oxidative stress; (2) In terms of anti-inflammation, it can inhibit the production of inflammatory factors and alleviate inflammatory responses; (3) It also has various pharmacological effects such as antiviral, antibacterial, anti-allergic, cardiovascular protection, and potential antitumor effects, thus making it widely applicable in the fields of medicine, health products, and cosmetics.

[0047] Caffeine is a naturally occurring alkaloid widely found in various plants such as coffee beans, tea leaves, and cocoa beans, and it also possesses a variety of biological activities. (1) It can inhibit the inhibitory effect of adenosine on the central nervous system by blocking adenosine receptors in the brain, thereby stimulating the nerves, effectively relieving fatigue, improving attention and alertness, and helping people maintain mental alertness in situations requiring concentration; (2) It can promote the decomposition of adipose tissue, increase the release of free fatty acids, provide more energy sources for muscle movement, and enhance muscle contraction, improve muscle endurance and athletic performance. It is often added to sports nutrition foods to help improve athletic performance; (3) It can also stimulate the central nervous system to release some neurotransmitters, such as dopamine and norepinephrine, thereby improving mood and making people feel happy after consuming caffeine-containing foods. In addition, caffeine also has important applications in the medical field, such as in the treatment of headaches, migraines and other diseases.

[0048] As an example, the molecular formula of the dihydroquercetin-caffeine eutectic is C2. 23 H 22 N4O9, and in the dihydroquercetin-caffeine eutectic, the molar ratio of dihydroquercetin to caffeine is 1:1.

[0049] As an example, the dihydroquercetin-caffeine eutectic is an orthorhombic crystal system with space group P212121 and cell parameters of [missing information]. α = 90°, β = 90°, γ = 90°, Z = 4, cell volume

[0050] As an example, the water solubility of dihydroquercetin caffeine cocrystal is higher than 12 mg / mL.

[0051] In this embodiment, the water solubility of the dihydroquercetin-caffeine cocrystal is higher than 12 mg / mL (based on the molar ratio of dihydroquercetin to caffeine in the cocrystal, it can be determined that the water solubility of dihydroquercetin in the cocrystal is higher than 7 mg / mL), while the water solubility of the dihydroquercetin monomer is only 0.4–0.6 mg / mL, indicating that the formation of a cocrystal between dihydroquercetin and caffeine significantly improves the water solubility of dihydroquercetin.

[0052] Secondly, embodiments of this application provide a method for preparing dihydroquercetin-caffeine cocrystal as provided in the first aspect embodiment, comprising the following steps:

[0053] S1. Dihydroquercetin, caffeine, and an organic solvent are mixed to obtain a premix; S2. The premix is ​​ball-milled to allow the dihydroquercetin and caffeine in the premix to react and obtain a precursor containing dihydroquercetin-caffeine cocrystal; S3. The precursor is dried to obtain dihydroquercetin-caffeine cocrystal.

[0054] In this application, ball milling is used to react dihydroquercetin with caffeine to form a eutectic. Since ball milling can usually be carried out at room temperature and pressure, it does not require harsh reaction conditions such as high temperature, high pressure, vacuum or special atmosphere. This not only reduces the requirements for equipment and operational risks, but also makes the entire preparation process easier to control and safer. At the same time, compared with other methods for preparing eutectic, such as high temperature, high pressure or complex chemical reactions, ball milling has the advantage of lower energy consumption, making it particularly suitable for large-scale industrial production.

[0055] As an example, the step of mixing dihydroquercetin, caffeine, and an organic solvent to obtain a premix includes: mixing dihydroquercetin and caffeine to obtain a premix precursor; and then mixing the premix precursor with an organic solvent to obtain the premix.

[0056] In this embodiment, dihydroquercetin and caffeine are first mixed, and then the mixture is mixed with an organic solvent. This allows the two raw materials to come into contact with the organic solvent simultaneously in a uniformly mixed state, thereby effectively reducing the risk of dissolution loss when a single raw material comes into contact with a large amount of organic solvent. This helps to improve the utilization rate of raw materials, product yield, and purity.

[0057] As an example, in the premix, the ratio of the sum of the masses of dihydroquercetin and caffeine to the mass of the organic solvent is 1:(0.15 to 0.3), for example, but not limited to, any one or any two of the following mass ratios: 1:0.15, 1:0.18, 1:0.20, 1:0.22, 1:0.24, 1:0.26, 1:0.28, and 1:0.3.

[0058] In this embodiment, the ratio of the sum of the masses of dihydroquercetin and caffeine to the mass of the organic solvent is limited to the above-mentioned range. On the one hand, an appropriate amount of organic solvent can provide suitable lubricity, enabling the material to have good fluidity during ball milling and reducing energy loss due to frictional resistance. At the same time, it can also improve the impact and grinding effect of the grinding media on the material, so that dihydroquercetin and caffeine can fully contact and react. On the other hand, an appropriate amount of organic solvent can also reduce the risk of raw material dissolution and loss in the solvent, thereby improving the raw material utilization rate, product yield, and purity.

[0059] 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.

[0060] As an example, the organic solvent is selected from at least one of methanol, ethanol, diethyl ether, acetonitrile, and acetone.

[0061] As an example, the molar ratio of dihydroquercetin to caffeine is 1:1.

[0062] In this embodiment, limiting the molar ratio of dihydroquercetin and caffeine to the above range helps the two raw materials to react fully and form a eutectic product with a molar ratio closer to 1:1.

[0063] As an example, the step of ball milling the premix includes: mixing the premix and grinding balls and then ball milling them at a frequency of 20–30 Hz (e.g., but not limited to frequencies of any one or any two of 20 Hz, 21 Hz, 22 Hz, 23 Hz, 24 Hz, 25 Hz, 26 Hz, 27 Hz, 28 Hz, 29 Hz, and 30 Hz) for 15–30 times (e.g., but not limited to ball milling 15, 18, 20, 22, 24, 26, 28, and 15 times). The range of 30 times or any two of the following values, wherein the time for a single ball milling process is 60 to 120 s, for example, but not limited to, any two of 60 s, 70 s, 80 s, 90 s, 100 s, 110 s and 120 s; and the interval time for ball milling processes is 30 to 60 s, for example, but not limited to, any two of 30 s, 35 s, 40 s, 45 s, 50 s, 55 s and 60 s.

[0064] It should be noted that the interval time refers to the waiting period after one grinding cycle is completed before the next grinding cycle begins. The waiting time is the interval time.

[0065] In this embodiment, limiting the frequency, cycle, and time of each ball milling cycle within the aforementioned range helps dihydroquercetin and caffeine to fully contact and react, thereby improving raw material utilization, product yield, and purity. Limiting the interval between two adjacent ball milling cycles within the aforementioned range can effectively reduce the material temperature after each ball milling, thereby better maintaining the physicochemical stability of the materials (raw materials and products).

[0066] As an example, the mass ratio of the premix to the grinding balls is 1:(4 to 10), for example, but not limited to, any one of the mass ratios of 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10, or any range between the two.

[0067] In this embodiment, limiting the mass ratio of premix to grinding balls within the above range helps the materials to fully contact and react under the action of the grinding balls, thereby improving the utilization rate of raw materials, the yield of products, and the purity.

[0068] 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.

[0069] It should be noted that, unless otherwise specified or limited, the processes or steps in the preparation of dihydroquercetin-caffeine cocrystal can be set according to conventional methods in the field.

[0070] Thirdly, embodiments of this application provide the application of dihydroquercetin-caffeine eutectic as provided in the first aspect embodiment in the preparation of food, pharmaceuticals, cosmetics, or skin care products.

[0071] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0072] Example 1

[0073] This application provides a method for preparing dihydroquercetin-caffeine cocrystal, comprising the following steps:

[0074] S1 mixes 12.2g of dihydroquercetin and 7.8g of caffeine evenly to obtain a premixed precursor.

[0075] S2 mixes the above premixed precursor with 4g of ethanol to obtain the premix.

[0076] S3 mixes the above premix with 100g of grinding balls and then ball-mills it 30 times at a frequency of 20Hz. The time for each ball milling process is 120s, and the interval between ball milling processes is 60s, so that the dihydroquercetin and caffeine in the premix react to obtain a precursor containing dihydroquercetin-caffeine eutectic.

[0077] The precursor obtained in S4 was dried in a vacuum drying oven for 24 hours at a temperature of 50°C, yielding 18.36 g of product with a yield of 91.8%.

[0078] Example 2

[0079] This application provides a method for preparing dihydroquercetin-caffeine cocrystal, comprising the following steps:

[0080] S1 mixes 12.2g of dihydroquercetin and 7.8g of caffeine evenly to obtain a premixed precursor.

[0081] S2 mixes the above premixed precursor with 3g of acetone to obtain the premix.

[0082] S3 mixes the above premix with 115g of grinding balls and then ball-mills it 30 times at a frequency of 30Hz. The time for each ball milling process is 60s and the interval between ball milling processes is 30s, so that the dihydroquercetin and caffeine in the premix react to obtain a precursor containing dihydroquercetin-caffeine eutectic.

[0083] The precursor obtained in S4 was dried in a vacuum drying oven for 24 hours at a temperature of 50°C, yielding 18.07 g of product with a yield of 90.4%.

[0084] Example 3

[0085] This application provides a method for preparing dihydroquercetin-caffeine cocrystal, comprising the following steps:

[0086] S1 mixes 12.2g of dihydroquercetin and 7.8g of caffeine evenly to obtain a premixed precursor.

[0087] S2 mixes the above premixed precursor with 6g of ethanol to obtain the premix.

[0088] S3 mixes the above premix with 130g of grinding balls and then ball-mills it 30 times at a frequency of 20Hz. The time for each ball milling process is 100s and the interval between ball milling processes is 60s, so that the dihydroquercetin and caffeine in the premix react to obtain a precursor containing dihydroquercetin-caffeine eutectic.

[0089] The precursor obtained in S4 was dried in a vacuum drying oven for 24 hours at a temperature of 50°C, yielding 17.92 g of product with a yield of 89.6%.

[0090] Example 4

[0091] This application provides a method for preparing dihydroquercetin-caffeine cocrystal, which differs from Example 3 only in that: in step S2, the above-mentioned premixed precursor is mixed with 10g of ethanol to obtain a premix. The final product yield is 12.62g, with a yield of 63.1%, and the product exhibits poor crystal uniformity.

[0092] The results of Examples 3 and 4 show that limiting the amount of organic solvent within a suitable range helps reduce the risk of raw material loss due to dissolution in the solvent, thereby improving raw material utilization and product yield.

[0093] Experimental Example 1

[0094] Qualitative analysis of dihydroquercetin-caffeine eutectic

[0095] Test method: X-ray single-crystal diffraction was performed on the dihydroquercetin-caffeine eutectic obtained in Example 1. The specific test results are shown in Table 1, and the crystal schematic diagram is shown in [reference needed]. Figure 1 .

[0096] Table 1

[0097]

[0098]

[0099] Experimental Example 2

[0100] 1H NMR spectrum ( 1 H-NMR characterization

[0101] Test method: The dihydroquercetin-caffeine cocrystal obtained in Example 1 was subjected to 1H NMR spectroscopy. 1 Characterization was performed using H-NMR, and deuterated DMSO was used as the test solvent in the experiment.

[0102] See Figure 2 The 1H NMR spectrum clearly shows 12 hydrogen atoms of dihydroquercetin and 10 hydrogen atoms of caffeine. The remaining peaks are those of deuterated reagent and residual solvent. No obvious impurity peaks were observed, indicating that dihydroquercetin and caffeine exist in a 1:1 molar ratio in the dihydroquercetin-caffeine cocrystal.

[0103] Experimental Example 3

[0104] Infrared spectroscopy characterization

[0105] Test methods: Infrared spectroscopy was performed on dihydroquercetin, caffeine, and the dihydroquercetin-caffeine 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.

[0106] See Figure 3 The infrared spectrum of the dihydroquercetin-caffeine cocrystal shows different absorption peaks from those of caffeine and dihydroquercetin, indicating that it is not a simple superposition of the characteristic peaks of the two precursors, and that the peaks at 3000 cm⁻¹ are distinct. -1 ~3500cm -1 Within the specified range, the hydrogen bond association peak of the dihydroquercetin-caffeine cocrystal becomes significantly larger and broader, indicating that dihydroquercetin and caffeine form a cocrystal structure.

[0107] Test Example 4

[0108] Morphological characterization (SEM)

[0109] Test methods: Dihydroquercetin, caffeine, and the dihydroquercetin-caffeine cocrystal obtained in Example 1 were characterized by scanning electron microscopy.

[0110] See Figure 4 Dihydroquercetin solid particles are columnar crystals, caffeic acid is needle-shaped crystals, and dihydroquercetin-caffeine eutectic solid particles are blocky crystals. The microstructure of the eutectic is significantly different from that of the monomer.

[0111] Experimental Example 5

[0112] Weak interaction theory calculations

[0113] Test method: Weak interaction simulation was performed on the structure of the dihydroquercetin-caffeine cocrystal.

[0114] See Figure 5 We can see that weak interactions such as PI-PI stacking and hydrogen bonds are widely present in the dihydroquercetin-caffeine eutectic, and the two rely on these weak interactions to form a new crystal structure.

[0115] Experimental Example 6

[0116] Water solubility test

[0117] Test method: Dihydroquercetin and the dihydroquercetin-caffeine cocrystal obtained in Experiment 1 were dissolved in water. The solutions were collected at 5 min, 10 min, 20 min, 45 min, 60 min, 90 min, 120 min and 180 min, filtered, and the content of dihydroquercetin was detected by high performance liquid chromatography.

[0118] See Figure 6After dissolution, the concentration of the dihydroquercetin-caffeine cocrystal gradually increased and then stabilized. At 180 min, the solubility of the dihydroquercetin-caffeine cocrystal was 12.06 mg / mL (based on the molar ratio of dihydroquercetin to caffeine in the cocrystal, the water solubility of dihydroquercetin in the cocrystal was calculated to be 7.36 mg / mL), while the solubility of the dihydroquercetin monomer was only 0.45 mg / mL. These results indicate that the formation of a cocrystal between dihydroquercetin and caffeine significantly improves the solubility of dihydroquercetin, thus contributing to its bioavailability.

[0119] Experimental Example 7

[0120] Bioavailability test

[0121] Experimental materials and methods:

[0122] Test substance:

[0123] Dihydroquercetin-caffeine cocrystal aqueous solution (based on a dihydroquercetin content of 0.2%) (10% ethanol), and dihydroquercetin content of 0.2% aqueous solution (10% ethanol).

[0124] Experimental methods:

[0125] 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.

[0126] (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.

[0127] (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.

[0128] (3) Drug administration: Turn on the instrument in advance and adjust the water bath temperature to 32±1℃. Add 1.0mL 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 1.13cm². 2 .

[0129] (4) Infiltration: Set the stirring speed to 350 rpm.

[0130] (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.

[0131] (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.

[0132] Table 2

[0133]

[0134] See Table 2 and Figure 7 Two hours after processing, the cumulative permeation per unit area of ​​the dihydroquercetin-caffeine cocrystal was higher than that of dihydroquercetin alone, indicating that the dihydroquercetin-caffeine cocrystal was absorbed more quickly. After 24 hours, the cumulative permeation per unit area of ​​the dihydroquercetin-caffeine cocrystal was 3.07 times that of dihydroquercetin. After 24 hours, the retention rate of the dihydroquercetin-caffeine cocrystal in the sample was also significantly higher than that of dihydroquercetin, approximately three times higher. Based on the data in Table 2, the bioavailability of the dihydroquercetin-caffeine cocrystal was calculated to be three times that of the dihydroquercetin monomer, demonstrating that the dihydroquercetin-caffeine cocrystal is beneficial for the absorption and utilization of dihydroquercetin.

[0135] Experimental Example 8

[0136] Toxicity testing

[0137] (1) Experimental Principle

[0138] Based on human skin-derived fibroblasts (Fbs), cell viability was detected using the CCK-8 (Cell Counting Kit-8) method to evaluate the cytotoxicity of the samples and screen for appropriate efficacy detection concentrations.

[0139] (2) Experimental steps

[0140] Under aseptic conditions, caffeine, dihydroquercetin-caffeine cocrystal, physically mixed dihydroquercetin-caffeine (in the same proportion as the dihydroquercetin-caffeine cocrystal), and dihydroquercetin were dissolved in anhydrous ethanol and diluted with serum-free medium to seven concentration gradients: 1, 0.5, 0.2, 0.1, 0.05, 0.02, and 0.01 mg / mL. The ethanol concentration for each sample was 1%. Fbs cells were seeded in 96-well plates and cultured overnight at 95% humidity, 5% CO2, and 37°C. Samples were added the following day according to Table 3. Twenty-four hours after sample addition, cell metabolic activity was detected using the CCK-8 assay. The CV90, the sample concentration at which cell viability was 90%, was calculated. The formula for calculating cell viability is as follows:

[0141]

[0142] Table 3

[0143]

[0144]

[0145] (3) Experimental Results

[0146] According to statistics, based on Fbs cells, the co-crystal CV of dihydroquercetin and caffeine 90 =0.6494 mg / mL, CV of physically mixed dihydroquercetin caffeine 90 =0.1355 mg / mL, caffeine CV 90 =0.2977 mg / mL, CV of dihydroquercetin 90 =0.1087 mg / mL. The results showed that the toxicity of the dihydroquercetin-caffeine cocrystal in cells was lower than that of the physically mixed dihydroquercetin-caffeine, caffeine, and dihydroquercetin, proving that the formation of the cocrystal reduced the cytotoxicity of the raw materials and improved biocompatibility.

[0147] Experimental Example 9

[0148] ABTS + Free radical scavenging test

[0149] (1) Experimental Principle

[0150] 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.

[0151] (2) Experimental materials: the dihydroquercetin-caffeine co-crystal, dihydroquercetin, caffeine, and L-ascorbic acid obtained in Example 1 (positive control group).

[0152] (3) Experimental procedures

[0153] For each sample in (2), set up sample tubes (A S ), sample backgrounds (A b ), and sample blank tubes (A0). For each test concentration of each sample, 3 parallel tubes need to be set up for the sample tubes (A S ), and 3 parallel tubes also need to be set up for the sample blank tubes (A0). Add 0.2 mL of the same concentration of sample solution to the sample tubes (A S ) and the sample backgrounds (A b ), and add 0.2 mL of PBS buffer to the sample blank tubes (A0). Add 0.8 mL of ABTS S working solution to the sample tubes (A + ) and the sample blank tubes (A0), and add 0.8 mL of PBS buffer to the sample backgrounds (A b ). React in the dark for 6 min, transfer the solutions in each reaction tube into 1-cm cuvettes, and measure the absorbance values corresponding to the sample tubes (A S ), sample backgrounds (A b ), and sample blank tubes (A0) at 734 nm respectively. Then calculate the ABTS + radical scavenging rate according to the test results, and summarize the test results in Table 4.

[0154] Among them, the calculation formula for the ABTS + radical scavenging rate is as follows:

[0155]

[0156] Table 4

[0157]

[0158]

[0159] Statistical method: Analysis was performed using the t-test method, with a significance level of α = 0.05; P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference; P < 0.001 indicates an extremely significant difference.

[0160] Referring to Table 4, the dihydroquercetin-caffeine co-crystal, caffeine, and dihydroquercetin all have the effect of scavenging ABTS + radicals. At the same molar concentration, the dihydroquercetin-caffeine co-crystal has a better effect on ABTS+ The free radical scavenging effect is better than that of dihydroquercetin and caffeine, proving that the dihydroquercetin-caffeine cocrystal has better antioxidant capacity.

[0161] Experimental Example 10

[0162] Mitochondrial ROS detection based on human skin-derived fibroblasts

[0163] (1) Experimental Principle

[0164] 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.

[0165] (2) Experimental steps

[0166] 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 according to Table 5, 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.

[0167] Table 5

[0168]

[0169] 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 to calculate the mitochondrial ROS reduction rate. The results are then summarized in Table 6; the calculation formula is as follows:

[0170]

[0171] 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.

[0172] Table 6

[0173] Group Average fluorescence intensity Decrease rate (vs. negative control group) Blank control group 22.228±2.134 / negative control group 64.935±0.473## / Positive control group 19.736±1.481** 69.6％ 0.526mM dihydroquercetin-caffeine eutectic 30.617±1.733** 52.8％ 0.526mM caffeine 35.110±1.424** 45.9％ 0.526mM dihydroquercetin 38.738±0.972** 40.3％ 0.329mM dihydroquercetin-caffeine eutectic 33.255±1.680** 48.8％ 0.329mM caffeine 42.586±2.364** 34.4％ 0.329mM dihydroquercetin 43.671±1.948** 32.7％

[0174] Compared with the blank control group, ##: P<0.01; compared with the negative control group, **: P<0.01.

[0175] See Table 6 and Figure 8 (The red area in the figure represents MitoSOX-labeled mitochondrial ROS, with a scale bar of 100 μm.) 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 cells of the 0.526mM dihydroquercetin-caffeine cocrystal group, caffeine group, and dihydroquercetin group was significantly decreased (P<0.01), with decrease rates of 52.8%, 45.9%, and 40.3%, respectively. Compared with the negative control group, the mean fluorescence intensity of mitochondrial ROS in cells of the 0.329mM dihydroquercetin-caffeine cocrystal group, caffeine group, and dihydroquercetin group was significantly decreased (P<0.01), with decrease rates of 48.8%, 34.4%, and 32.7%, respectively. At the same molar concentration, the dihydroquercetin-caffeine cocrystal was more effective than caffeine and dihydroquercetin alone, demonstrating that the two cocrystals have a synergistic effect, reducing mitochondrial ROS and thus providing antioxidant protection for mitochondria.

[0176] Experimental Example 11

[0177] Mitochondrial morphology detection based on human skin-derived fibroblasts

[0178] (1) Experimental Principle

[0179] 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 judged by the average fluorescence intensity after Mitotracker Green staining: the greater the average fluorescence intensity, the greater the number of mitochondria. The larger the average fluorescence area, the larger the average mitochondrial volume, indicating more severe mitochondrial swelling.

[0180] (2) Experimental steps

[0181] 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 according to Table 7, 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.

[0182] Table 7

[0183]

[0184] 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. The average fluorescence intensity and average fluorescence area after Mitotracker Green FM staining were analyzed using ImageJ software. The rate of increase in average fluorescence intensity and the rate of decrease in average area after Mitotracker staining were calculated. The results are then statistically presented in Tables 8 and 9, respectively. The calculation formulas are as follows:

[0185]

[0186] 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.

[0187] Table 8

[0188] Group Average fluorescence intensity Elevation rate (vs negative control group) Blank control group 55.501±0.787 / negative control group 24.952±2.503## / Positive control group 51.579±4.177** 106.7％ 0.526mM dihydroquercetin-caffeine eutectic 40.691±3.021** 63.1％ 0.526mM caffeine 35.183±2.621** 41.0％ 0.526mM dihydroquercetin 34.739±3.388* 39.2％ 0.329mM dihydroquercetin-caffeine eutectic 34.941±2.469** 40.0％ 0.329mM caffeine 31.970±2.738* 28.1％ 0.329mM dihydroquercetin 31.293±1.738* 25.4％

[0189] Compared with the blank control group, ##: P<0.01; compared with the negative control group, **: P<0.01.

[0190] Table 9

[0191]

[0192]

[0193] Compared with the blank control group, ##: P<0.01; compared with the negative control group, **: P<0.01.

[0194] See Table 8 and Figure 9 (The green areas in the figure represent mitochondria labeled with Mitotracker Green FM, with a scale bar of 100 μm.) 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 in this experiment was effective. Compared with the negative control group, the mean fluorescence intensity of mitochondria in the 0.526 mM dihydroquercetin-caffeine cocrystal group, caffeine group, and dihydroquercetin group was significantly increased (P<0.01), with increase rates of 63.1%, 41.0%, and 39.2%, respectively. Compared with the negative control group, the mean fluorescence intensity of mitochondria in the 0.329 mM dihydroquercetin-caffeine cocrystal group, caffeine group, and dihydroquercetin group was significantly increased (P<0.01), with increase rates of 40.0%, 28.1%, and 25.4%, respectively.

[0195] See Table 9 and Figure 10 Compared with the blank control group, the mean fluorescence area of ​​mitochondria 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 area of ​​mitochondria 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 area of ​​mitochondria in the 0.526mM dihydroquercetin-caffeine cocrystal group, caffeine group, and dihydroquercetin group was extremely significantly decreased (P<0.01), with decrease rates of 42.8%, 36.2%, and 12.4%, respectively. Compared with the negative control group, the mean fluorescence area of ​​mitochondria in the 0.329mM dihydroquercetin-caffeine cocrystal group and dihydroquercetin group was extremely significantly decreased (P<0.01), with decrease rates of 34.0% and 20.7%, respectively. The mean fluorescence area of ​​mitochondria in the 0.329mM caffeine group did not show a decrease.

[0196] From Table 8, Table 9, Figure 9 and Figure 10 It is known that, at the same molar concentration, the co-crystal effect of dihydroquercetin and caffeine is better than that of caffeine and dihydroquercetin. It can increase the number of mitochondria, reduce mitochondrial swelling, and has the effect of protecting mitochondrial function.

[0197] Experimental Example 12

[0198] Anti-inflammatory and soothing efficacy test

[0199] (1) Experimental Principle

[0200] IL-8 is an important chemokine that attracts inflammatory cells, such as neutrophils, to the site of inflammation. Lowering IL-8 levels can reduce the infiltration and aggregation of neutrophils at the site of inflammation, thereby alleviating the inflammatory response. Excessive neutrophil infiltration can lead to tissue damage. IL-8 can also induce neutrophils to release enzymes such as elastase, which can degrade tight junction proteins in tissues and impair the barrier function of epithelial cells. Lowering IL-8 levels helps protect the integrity and function of tissue structure and reduces the damage caused by inflammation. TNF-α is a potent pro-inflammatory cytokine that can activate various inflammatory cells and signaling pathways, triggering a series of inflammatory cascade responses. Reducing TNF-α can effectively block the initiation and spread of the inflammatory response, preventing further inflammation. TNF-α can induce apoptosis and necrosis in certain situations, causing toxic damage to tissue cells. By reducing TNF-α levels, its cytotoxic effects can be reduced, protecting cell survival and normal function, and promoting tissue repair and regeneration. This experiment used LPS-induced human keratinocytes as an in vitro inflammatory cell model and used an ELISA kit to detect the inflammatory factors (IL-8 and TNF-α) secreted in the cell supernatant to evaluate the anti-inflammatory and soothing effects of the test samples.

[0201] (2) Experimental steps

[0202] Cells were cultured using standard methods and seeded into 12-well plates, then incubated for 18–24 h. The plates were removed, and the original culture medium in each well was discarded. DMEM culture medium was added to the negative control (NC), DMEM culture medium containing LPS was added to the model control group (M), DMEM culture medium containing LPS and different concentrations of the test sample was added to each well of the sample group (TA), and DMEM culture medium containing LPS and dexamethasone was added to each well of the positive control group (PC). Each group was divided into three replicates, and the cells were incubated for 24 ± 1 h. Experimental groupings are shown in Table 10. The supernatant was collected and stored at -80℃. Cytokines were measured using an ELISA kit. The differences in IL-8 and TNF-α between different groups are summarized in Tables 11 and 12, respectively.

[0203] Table 10

[0204]

[0205] (3) Test Results

[0206] Table 11

[0207] Group IL-8 content (pg / mL) Relative content (%) Negative control group (NC) 17.50±1.32* 85.25±6.42* Solvent control group (SC) 17.29±1.56* 84.23±7.60* Model control group (M) 20.52±1.71# 100.00±8.33# Positive control group (PC) 16.56±1.29* 80.67±6.27* 0.16mM dihydroquercetin 17.64±1.66* 85.96±8.11* 0.16mM dihydroquercetin-caffeine eutectic 14.93±0.12* 72.73±0.58* 0.16mM physically mixed dihydroquercetin caffeine 26.31±2.31* 128.18±11.25* 0.16mM caffeine 23.76±1.64* 115.77±7.99*

[0208] # indicates a statistically significant difference compared to the negative control group (p<0.05); * indicates a statistically significant difference compared to the model control group (p<0.05).

[0209] Table 12

[0210]

[0211]

[0212] # indicates a statistically significant difference compared to the negative control group (p<0.05); * indicates a statistically significant difference compared to the model control group (p<0.05).

[0213] Referring to Tables 11 and 12, under the experimental conditions, the relative IL-8 content in the model control group (M) was significantly higher than that in the negative control group (NC) (p<0.05); the relative IL-8 content in the positive control group was significantly lower than that in the model control group (p<0.05), indicating successful model establishment. Similarly, the relative TNF-α content in the model control group (M) was significantly higher than that in the negative control group (NC) (p<0.05); the relative TNF-α content in the positive control group was significantly lower than that in the model control group (p<0.05), indicating successful model establishment.

[0214] At a concentration of 0.16 mM, the relative IL-8 content of the dihydroquercetin sample decreased by 14.04% compared to the model control group, a statistically significant difference (p<0.05). At a concentration of 0.16 mM, the relative IL-8 content of the dihydroquercetin-caffeine cocrystal sample decreased by 27.27% compared to the model control group, a statistically significant difference (p<0.05). At a concentration of 0.16 mM, the relative IL-8 content of the physically mixed dihydroquercetin-caffeine and caffeine sample did not decrease compared to the model control group, indicating no effect on reducing IL-8 content.

[0215] At a concentration of 0.16 mM, the relative TNF-α content of the dihydroquercetin sample decreased by 23.45% compared to the model control group, a statistically significant difference (p<0.05). At a concentration of 0.16 mM, the relative TNF-α content of the dihydroquercetin-caffeine cocrystal sample decreased by 27.74% compared to the model control group, both statistically significant differences (p<0.05). At a concentration of 0.16 mM, the relative TNF-α content of the physically mixed dihydroquercetin-caffeine and caffeine sample decreased by 14.63% compared to the model control group, both statistically significant differences (p<0.05). At a concentration of 0.16 mM, the relative TNF-α content of the caffeine sample did not decrease significantly compared to the model control group, indicating no effect on reducing TNF-α content.

[0216] Comprehensive analysis showed that, at the same test concentration, the dihydroquercetin-caffeine cocrystal significantly reduced the levels of IL-8 and TNF-α, with the best effect, indicating that the cocrystal form had a significant synergistic effect. This further verified the synergistic effect of the dihydroquercetin-caffeine cocrystal, suggesting that the dihydroquercetin-caffeine cocrystal has the potential to reduce the levels of inflammatory factors and improve the inflammatory response.

[0217] 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-caffeine eutectic, characterized in that, The molecular formula of the dihydroquercetin-caffeine cocrystal is C2. 23 H 22 N4O9, and in the dihydroquercetin-caffeine cocrystal, the molar ratio of dihydroquercetin to caffeine is 1:1; the water solubility of the dihydroquercetin-caffeine cocrystal is higher than 12 mg / mL; The dihydroquercetin-caffeine eutectic is an orthorhombic crystal with space group P212121, cell parameters a=8.9476(3) Å, b=9.0782(3) Å, c=30.3065(9) Å, α=90°, β=90°, γ=90°, Z=4, and cell volume V=2461.76(13) Å. 3 ; The structural formula of the dihydroquercetin-caffeine eutectic is shown in Formula I: Formula I.

2. A method for preparing the dihydroquercetin-caffeine eutectic as described in claim 1, characterized in that, Includes the following steps: S1. Dihydroquercetin, caffeine, and an organic solvent are mixed, wherein the molar ratio of dihydroquercetin to caffeine is 1:1, to obtain a premix. S2. The premix is ​​ball-milled to react the dihydroquercetin and caffeine in the premix to obtain a precursor containing the dihydroquercetin-caffeine cocrystal. S3. The precursor is dried to obtain the dihydroquercetin-caffeine eutectic.

3. The preparation method according to claim 2, characterized in that, The step of mixing dihydroquercetin, caffeine, and an organic solvent to obtain a premix includes: Dihydroquercetin and caffeine were mixed to obtain a premixed precursor; The premixed precursor is then mixed with an organic solvent to obtain the premix.

4. The preparation method according to claim 2, characterized in that, In the premix, the ratio of the sum of the masses of dihydroquercetin and caffeine to the mass of the organic solvent is 1:(0.15~0.3).

5. The preparation method according to any one of claims 2 to 4, characterized in that, The step of ball milling the premix includes: mixing the premix with grinding balls and then ball milling at a frequency of 20-30 Hz for 15-30 times, wherein the time for a single ball milling is 60-120 s and the interval between ball milling is 30-60 s.

6. The preparation method according to claim 5, characterized in that, The mass ratio of the premix to the grinding balls is 1:(4~10).

7. The use of the dihydroquercetin-caffeine eutectic as described in claim 1 in the preparation of food, pharmaceuticals, cosmetics or skin care products.

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