Baicalein-copper nano microgel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano material synthesis, and particularly relates to baicalein-copper nano microgel as well as a preparation method and application thereof. According to the invention, copper ions and baicalein are coordinated to form nanowires, and the nanowires are coated with hyaluronic acid (HA) to prepare the nano microgel. The method is carried out at normal temperature, is simple in process and high in yield, and does not need toxic solvents; the obtained microgel has high stability (gastric acid resistance), targeted delivery (CD44 receptor mediation) and multiple biological activities (oxidation resistance and inflammation resistance). Animal experiments show that the pharmaceutical composition can significantly relieve ulcerative colitis, the dosage is as low as 5 mg / kg, the pharmaceutical composition is non-toxic, and a new strategy is provided for treatment of inflammatory diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomaterial synthesis, and particularly relates to a baicalein-copper nanomicrogel as well as a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application and should not necessarily be regarded as acknowledging or implicitly suggesting that this information constitutes prior art known to those skilled in the art.

[0003] As a natural polyphenol antioxidant, baicalein has pharmacological activities such as antioxidant, anti-inflammatory, antibacterial and free radical scavenging, but its poor water solubility, low bioavailability, insufficient chemical stability and short half-life seriously limit its clinical application.

[0004] Metal polyphenol materials are a kind of metal-organic materials constructed by metal ions and natural polyphenol ligands, and the physical and chemical properties and biological functions thereof can be regulated by selecting appropriate metal ions and phenolic ligands. At present, transition metal-phenolic materials exhibit rich structures and natural enzyme-like catalytic activities, that is, they can be used as a kind of excellent antioxidant nanoenzyme material to effectively scavenge active oxygen, active nitrogen and other species in the body. This kind of material has flexible composition adjustability, biodegradability, and great potential in disease treatment. However, the existing metal polyphenol materials can improve the drug delivery efficiency, but the synthesis often requires high temperature and high pressure, the steps are complex, the targeting is poor, and the activity is easily lost under the complex biological oxidation and reduction environment in the body.

[0005] Therefore, it is urgent to develop an efficient, stable and targeted baicalein delivery system to solve the problems of poor water solubility, low bioavailability and insufficient stability of baicalein, so as to better exert the medicinal value of baicalein and improve the practical application significance. SUMMARY

[0006] In view of the needs of the prior art, the purpose of the present application is to provide a baicalein-copper nanomicrogel as well as a preparation method and application thereof. In the present application, transition metal copper (Cu) is used as a coordination metal, NaOH is used as an acid-base regulator, and a surfactant (such as PVP) is added. Nanowires are formed by coordination of copper ions and baicalein, and then nanomicrogels are prepared by coating with hyaluronic acid (HA). The method is carried out at room temperature, and has the advantages of simple process, high yield and no need for toxic solvents. The obtained microgels have high stability (resistance to gastric acid), targeted delivery (CD44 receptor-mediated) and multiple biological activities (antioxidant and anti-inflammatory). At the same time, animal experiments show that it can significantly relieve ulcerative colitis, with a low dose of 5 mg / kg and no toxicity, providing a new strategy for the treatment of inflammatory diseases.

[0007] Specifically, the present application provides the following technical solutions. In a first aspect, the present application provides a baicalein-copper nanogel, which comprises a nanowire core formed by coordination of baicalein and copper and a hyaluronic acid coating layer covering the nanowire core; the mass ratio of baicalein to copper in the nanowire core is 1-2:1; and the mass ratio of the nanowire core to hyaluronic acid is 0.5-2.5:1.

[0008] The mass ratio range of baicalein to copper is set to ensure the formation of mixed-valence copper (Cu + / Cu 2+ ), provide electron transfer active centers, and enhance the catalytic efficiency; and the mass ratio range of the nanowire core to hyaluronic acid is used to balance the protection of the stability of the core and the degree of exposure of the active sites.

[0009] Preferably, the copper in the nanowire core is mixed-valence Cu + / Cu 2+ , and the nanowire core is formed by coordination of copper ions and baicalein.

[0010] Preferably, the hydration radius of the nanowire core is 90-98 nm, and the hydration radius of the baicalein-copper nanogel is 100-120 nm.

[0011] Further preferably, the hydration radius of the nanowire core is 96 nm, and the hydration radius of the baicalein-copper nanogel is 110 nm.

[0012] Preferably, the baicalein-copper nanogel has a stability ≥24 hours in a simulated gastrointestinal fluid at pH 1.5 and pH 6.8 and does not dissociate or aggregate.

[0013] In a second aspect, the present application provides a preparation method of the above-mentioned baicalein-copper nanogel, which comprises the following steps: mixing a copper salt and a surfactant in a mixed solvent, adding a NaOH solution as a regulator for stirring reaction, then adding a baicalein solution to the reaction solution, adjusting the pH, and then performing room temperature reaction to obtain a baicalein-copper nanowire dispersion, and then mixing a hyaluronic acid aqueous solution for mixing reaction to obtain the baicalein-copper nanogel.

[0014] Preferably, the copper salt is selected from one of CuSO4·5H2O and CuSO4; the surfactant is selected from one or more of polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA); and the mass ratio of the copper salt to the surfactant is 0.2-1:1.

[0015] Preferably, the mixed solvent is obtained by mixing water and an organic solvent, the organic solvent is selected from one or more of methanol and ethanol, and the volume ratio of the water to the organic solvent is 1-5:1.

[0016] Preferably, the concentration of the adjusting agent NaOH solution is 0.5-1.0 M, and the pH is adjusted to 8-12.

[0017] Preferably, the temperature of the stirring reaction is 25-50℃, and the time is 0.5-3 h.

[0018] Preferably, the baicalein solution is prepared by dissolving baicalein in an organic solution selected from one or more of methanol, ethanol and acetone, and the concentration of the baicalein solution is 10-60 mg / mL.

[0019] Preferably, the pH is adjusted to 10-12, the room temperature reaction is performed for 12-24 h, and then the product is dispersed in deionized water by centrifugation and washing, to obtain a baicalein-copper nanowire dispersion; wherein the centrifugation is performed at a speed of 8000-12000 rpm for 10-15 min. Too low pH inhibits the coordination reaction, and too high pH causes copper ions to preferentially form copper hydroxide precipitate.

[0020] Preferably, the volume ratio of the baicalein-copper nanowire dispersion to the hyaluronic acid aqueous solution is 2-10:1, preferably 5:1; the concentration of the hyaluronic acid aqueous solution is 2.5-10 mg / mL, and the concentration of the baicalein-copper nanowire dispersion is 1-5 mg / mL; and the mixing reaction is performed at a temperature of 25-35℃ for 12-24 h. The concentration is set in relation to the mass of the components, and the concentration is controlled to achieve the regulation of the structure and the exposure of the active sites.

[0021] In a third aspect, the application provides a use of the baicalein-copper nanomicrogel of the first aspect in the preparation of a medicament for treating inflammatory diseases.

[0022] Preferably, the inflammatory diseases include ulcerative colitis, Crohn's disease and periodontitis.

[0023] The above one or more technical solutions of the application have the following beneficial effects: (1) The baicalein-copper nanomicrogel prepared by the simple room temperature preparation process of the application solves the problems of poor water solubility and insufficient stability of baicalein, and at the same time endows it with high biocompatibility, targeted delivery capability and multiple biological activities (antioxidation, anti-inflammation). It exhibits the advantages of low dosage, high efficiency and safety in the treatment of inflammatory diseases (such as ulcerative colitis), and provides an innovative strategy for natural drug delivery systems; Specifically embodied as: ①High structural stability: no aggregation or dissociation in simulated gastric juice (pH 1.5) and intestinal juice (pH 6.8) for ≥24 hours, i.e. resistant to gastric acid and trypsin degradation, ensuring the effectiveness of oral administration; ②Low cytotoxicity (biocompatibility): cell survival rate > 70% when the concentration of copper ions is ≤100 μg / mL for L929 fibroblasts; cell survival rate ≈ 100% when the concentration of copper ions is ≤20 μg / mL for RAW264.7 macrophages; and no in vivo toxicity reaction is observed when the dose is as low as 5 mg / kg in animal experiments; ③Strong antioxidant property: significant ability to scavenge ABTS and DPPH free radicals; and ability to protect cells from oxidative stress damage; ④Highly efficient anti-inflammatory property: significantly inhibits the secretion of pro-inflammatory factors (TNF-α, IL-1β, IL-6), and the effect is better than that of single baicalein, single copper ions, and the physical mixing group; and the level of inflammatory factors is reduced by more than 50% in lipopolysaccharide-activated macrophages.

[0024] ⑤Targeted delivery capability: mediated targeting by hyaluronic acid (HA), and the HA coating layer targets the inflammation site (such as the lesion of ulcerative colitis) through the CD44 receptor, thereby increasing the enrichment of the drug in the diseased tissue and enhancing the therapeutic effect.

[0025] (2) The synthesis method has the characteristics of short process flow, green and safe raw materials, high yield, good stability, and the like, and does not require additional conditions such as high temperature, high pressure or heating, but can synthesize baicalein-copper nanomicrogel by stirring at room temperature.

[0026] (3) The nanowire structure prepared by the application has good water dispersibility, high stability, uniform morphology and the like. The coordination ratio of baicalein and copper is adjusted to ensure the formation of mixed valence copper; the appropriateness of the HA coating layer is adjusted to balance protection and site exposure; the pH (10-12) of the coordination reaction is controlled to maintain the structural stability; and the uniformity of the nanowire morphology is maintained to provide a high catalytic specific surface area.

[0027] (4) The baicalein-copper nanomicrogel synthesized by the application has good biocompatibility, antioxidant property, anti-inflammatory and antibacterial properties, and can exhibit good therapeutic effect in various chronic inflammatory diseases including inflammatory bowel disease, etc. In a DSS-induced mouse model, after 4 days of intragastric administration at a dose of 5 mg / kg, the colon length is restored from 5.2 cm in the model group to 7.0 cm (close to the normal group), significantly improving the symptoms of colon hyperemia, edema and hemorrhage. BRIEF DESCRIPTION OF DRAWINGS

[0028] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the schematic embodiments of the present application and the description thereof are used to explain the present application, and do not constitute an improper limitation on the present application.

[0029] Figure 1Synthetic route of baicalein-copper nanowire structure prepared in Example 1 of the present application; Figure 2 Transmission electron micrograph of baicalein-copper nanowire structure, baicalein-copper nanogel prepared in Example 1 of the present application and baicalein-copper nanogel prepared in Example 6 of the present application; Figure 3 Hydrated radius graph of baicalein-copper nanogel prepared in Example 1 of the present application; Figure 4 Transmission electron micrograph of baicalein-copper nanowire structure prepared in Comparative Examples 1-2 of the present application; Figure 5 Transmission electron micrograph of baicalein-copper nanocomposite prepared in Comparative Example 3 of the present application and structural state graph of baicalein-copper nanocomposite prepared in Comparative Example 4 of the present application; Figure 6 Transmission electron micrograph of baicalein-copper nanocomposite prepared in Comparative Examples 5-7 of the present application; Figure 7 X-ray photoelectron spectrogram of baicalein-copper nanogel prepared in Example 1 of the present application; Figure 8 Stability test of baicalein-copper nanogel prepared in Example 1 of the present application in simulated gastrointestinal fluid; Figure 9 Peroxidase-like activity detection graph of baicalein-copper nanogel prepared in Example 1 of the present application at different concentrations; Figure 10 ABTS scavenging graph of baicalein-copper nanogel prepared in Example 1 of the present application at different concentrations •+ UV spectrogram; Figure 11 UV spectrogram of DPPH scavenging of baicalein-copper nanogel prepared in Example 1 of the present application at different concentrations; Figure 12 Intracellular reactive oxygen species detection confocal graph and cell live and dead staining graph of baicalein-copper nanogel prepared in Example 1 of the present application, single copper salt, single baicalein and physical mixed group; wherein a) is the intracellular reactive oxygen species detection confocal graph, and b) is the cell live and dead staining graph; Figure 13 Immune activity regulation test graph of baicalein-copper nanogel prepared in Example 1 of the present application; Figure 14 L929 mouse fibroblast toxicity test graph of baicalein-copper nanogel prepared in Example 1 of the present application; Figure 15 RAW264.7 mouse macrophage toxicity test graph of baicalein-copper nanogel prepared in Example 1 of the present application; Figure 16 Figure of the treatment effect of baicalein-copper nanogel prepared for Example 1 of the present application on ulcerative colitis; Figure 17 Histogram of the colon length statistics of baicalein-copper nanogel prepared for Example 1 of the present application in the treatment of ulcerative colitis. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0031] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples.

[0032] Example 1 The present embodiment provides a baicalein-copper nanogel, and a preparation method thereof includes the following steps: (1) Preparation of copper salt-containing suspension A Compound (40 mg), PVP (80 mg) were weighed, and a mixed solution of 10 mL of water and methanol (water:methanol = 5:1, by volume) was stirred and dissolved. The solution was light blue. The pH was adjusted to 10 using NaOH (1 M), and the solution gradually appeared as light blue flocculation. After continuous stirring for 3 h, the solution became a uniform suspension A. (2) Preparation of baicalein-copper nanowires

[0033] Baicalein (60 mg) was dissolved in 5 mL of methanol to obtain solution B, which was light yellow-green. Then it was added dropwise to solution A, and the color gradually changed from light blue to tan. After stirring for half an hour, NaOH (1 M) was added to adjust the pH to 12, and the solution gradually became clear. After stirring for 12 h, centrifugation and washing were performed, and the solution was redispersed with deionized water to obtain a baicalein-copper nanowire aqueous dispersion C. The chemical reaction process is shown in Figure 1 Figure 2

[0034] (3) Preparation of baicalein-copper nanogel ​​​The 5 mL baicalein-copper dispersion solution C (1 mg / mL) was placed in a 25 mL open beaker, 1 mL HA (2.5 mg / mL) aqueous solution was added and stirred to mix, the solution was yellow-brown, and was placed in a shaking bed at room temperature for 12 h of reaction in the dark. After the reaction was completed, the precipitate was collected by using a high-speed centrifuge (9000 rpm), and was washed with ultrapure water, and finally dispersed in ultrapure water to obtain the baicalein-copper nanogel. Transmission electron microscopy test of the final structure found that the nanowire structure was still maintained (scale 100 nm), as shown in Figure 2 Fig. 2b.

[0035] In addition, as shown in Figure 3 , by testing the hydration radius of the baicalein-copper nanowire structure and the baicalein-copper nanogel, the results show that the hydration radius of the nanowire core (baicalein-copper nanowire structure) is 96 nm, and the hydration radius of the baicalein-copper nanogel is 110 nm, that is, compared with the baicalein-copper nanowire structure, the hydration radius of the nanowire increases after being coated with HA, which indirectly indicates that the HA is successfully modified on the surface of the nanowire.

[0036] Example 2 The baicalein-copper nanogel provided by the embodiment, and the preparation method thereof, comprises the following steps: The difference between this embodiment and embodiment 1 is that the mass ratio of baicalein to copper salt is 1.2:1, and the specific preparation steps are as follows: (1) Preparation of copper salt-containing suspension A Compound (50 mg) and PVP (80 mg) were weighed, 10 mL of a mixture of water and methanol (water:methanol = 5:1, by volume) was added, and the solution was stirred and dissolved, and the solution was light blue. The pH was adjusted to 10 using NaOH (1 M), and the solution gradually appeared as light blue flocculation. After continuous stirring for 3 h, the solution became a uniform suspension A.

[0037] (2) Preparation of baicalein-copper nanowire Baicalein (60 mg) was dissolved in 5 mL of methanol to obtain solution B, and the solution was light yellow-green. Then it was added dropwise to solution A, and the color gradually changed from light blue to tan. After stirring for half an hour, NaOH (1 M) was added to adjust the pH to 12, and the solution gradually became clear. After stirring for 12 h, the solution was centrifuged and washed, and then redispersed in deionized water to obtain a baicalein-copper nanowire aqueous dispersion C.

[0038] (3) Preparation of baicalein-copper nanogel 5 mL of baicalin-copper dispersion C (1 mg / mL) was placed in a 25 mL open beaker, and 1 mL of HA (2.5 mg / mL) aqueous solution was added. After stirring and mixing, the solution turned yellowish-brown. The mixture was then placed in a shaker at room temperature and allowed to react in the dark for 12 h. After the reaction was complete, the precipitate was collected by centrifugation using a high-speed centrifuge (9000 rpm), washed with ultrapure water, and finally dispersed in ultrapure water to obtain the final product.

[0039] Example 3 This embodiment provides a baicalin-copper nanogel, the preparation method of which includes the following steps: The difference between this embodiment and Embodiment 1 is that: baicalin and copper salt ( The mass ratio of the two components is 2:1, and the specific preparation steps are as follows: (1) Preparation of suspension A containing copper salt Weigh the compound (30 mg) and PVP (80 mg) were added to a mixture of 10 mL of water and methanol (water:methanol = 5:1, volume ratio). After stirring and dissolving, the solution was light blue. The pH was adjusted to 10 using NaOH (1 M), and light blue flocculent matter gradually appeared in the solution. After stirring continuously for 3 h, the solution became a homogeneous suspension A.

[0040] (2) Preparation of baicalin-copper nanowires Baicalein (60 mg) was dissolved in 5 mL of methanol and sonicated to obtain solution B, which was pale yellow-green. This solution was then added dropwise to solution A, and the color gradually changed from pale blue to brownish-yellow. After stirring for half an hour, NaOH (1 M) was added to adjust the pH to 12, and the solution gradually became clear. After stirring for 12 hours, the solution was centrifuged, washed, and redispersed with deionized water to obtain baicalein-copper nanowire aqueous dispersion C.

[0041] (3) Preparation of baicalin-copper nanogels 5 mL of baicalin-copper dispersion C (1 mg / mL) was placed in a 25 mL open beaker, and 1 mL of HA (2.5 mg / mL) aqueous solution was added. After stirring and mixing, the solution turned yellowish-brown. The mixture was then placed in a shaker at room temperature and allowed to react in the dark for 12 h. After the reaction was complete, the precipitate was collected by centrifugation using a high-speed centrifuge (9000 rpm), washed with ultrapure water, and finally dispersed in ultrapure water to obtain the final product.

[0042] Example 4 This embodiment provides a baicalin-copper nanogel, the preparation method of which includes the following steps: The difference between this embodiment and Example 1 is that the mass ratio of baicalin-copper nanowires to hyaluronic acid (HA) is 1:1. The specific preparation steps are as follows: (1) Preparation of the suspension A containing copper salt The compounds were weighed (40 mg), PVP (80 mg) were added into 10 mL of a mixture of water and methanol (water: methanol = 5:1, by volume) and stirred to dissolve. The solution was light blue. The pH was adjusted to 10 using NaOH (1 M). Light blue floccules appeared in the solution. After 3 h of continuous stirring, the solution became a uniform suspension A.

[0043] (2) Preparation of baicalein-copper nanowires Baicalein (60 mg) was dissolved in 5 mL of methanol to obtain solution B, which was light yellow-green. Solution B was then added dropwise into solution A. The color of the solution changed from light blue to tan. After 0.5 h of stirring, the pH was adjusted to 12 using NaOH (1 M). The solution gradually became clear. After 12 h of stirring, centrifugation and washing, and redispersion in deionized water, baicalein-copper nanowire aqueous dispersion C was obtained.

[0044] (3) Preparation of baicalein-copper nanogels 5 mL of baicalein-copper dispersion C (1 mg / mL) was placed in a 25 mL open beaker. 2 mL of HA (2.5 mg / mL) aqueous solution was added and stirred to mix. The solution was yellow-brown. The solution was placed in a shaking table at room temperature and reacted for 12 h in the dark. After the reaction was completed, the precipitate was collected by high-speed centrifugation (9000 rpm) and washed with ultrapure water. Finally, the baicalein-copper nanogels were dispersed in ultrapure water.

[0045] Example 5 The embodiment provides a baicalein-copper nanogel, and a preparation method thereof, which comprises the following steps: The embodiment differs from embodiment 1 in that the mass ratio of baicalein-copper nanowires to hyaluronic acid (HA) is 2.5:1. The specific preparation steps are as follows: (1) Preparation of the suspension A containing copper salt The compounds were weighed (40 mg), PVP (80 mg) were added into 10 mL of a mixture of water and methanol (water: methanol = 5:1, by volume) and stirred to dissolve. The solution was light blue. The pH was adjusted to 10 using NaOH (1 M). Light blue floccules appeared in the solution. After 3 h of continuous stirring, the solution became a uniform suspension A.

[0046] (2) Preparation of baicalein-copper nanowires Baicalein (60 mg) was dissolved in 5 mL methanol to obtain solution B, which was light yellow green. Then it was added dropwise to solution A, and the color gradually changed from light blue to tan. After stirring for half an hour, NaOH (1 M) was added to adjust the pH to 12, and the solution gradually became clear. After stirring for 12 h, centrifugal washing was performed, and redispersion was performed using deionized water to obtain a baicalein-copper nanowire water dispersion C.

[0047] (3) Preparation of baicalein-copper nanogel 5 mL of baicalein-copper dispersion C (1 mg / mL) was placed in a 25 mL open beaker, 0.8 mL of HA (2.5 mg / mL) aqueous solution was added, and the solution was stirred and mixed to be yellow brown. After reaction at room temperature for 12 h, the precipitate was collected by high-speed centrifuge (9000 rpm), washed with ultrapure water, and finally dispersed in ultrapure water.

[0048] Example 6 The embodiment provides a baicalein-copper nanogel, and a preparation method thereof, which comprises the following steps: (1) Preparation of copper salt-containing suspension A The compounds (40 mg) and PVA (80 mg) were weighed, 10 mL of a mixture of water and methanol (water:methanol = 5:1, by volume) was added, and the solution was stirred and dissolved to be light blue. NaOH (1 M) was used to adjust the pH to 10, and the solution gradually appeared as light blue flocculation. After continuous stirring for 3 h, the solution became a uniform suspension A.

[0049] (2) Preparation of baicalein-copper nanowire Baicalein (60 mg) was dissolved in 5 mL methanol to obtain solution B, which was light yellow green. Then it was added dropwise to solution A, and the color gradually changed from light blue to tan. After stirring for half an hour, NaOH (1 M) was added to adjust the pH to 12, and the solution gradually became clear. After stirring for 12 h, centrifugal washing was performed, and redispersion was performed using deionized water to obtain a baicalein-copper nanowire water dispersion C.

[0050] (3) Preparation of baicalein-copper nanogel 5 mL of baicalein-copper dispersion C (1 mg / mL) was placed in a 25 mL open beaker, and 1 mL of HA (2.5 mg / mL) aqueous solution was added. After stirring and mixing, the solution turned brown. The mixture was then placed in a shaker at room temperature and allowed to react in the dark for 12 h. After the reaction was complete, the precipitate was collected by centrifugation (9000 rpm), washed with ultrapure water, and finally dispersed in ultrapure water to obtain the final product. Transmission electron microscopy revealed the final structure to be an intertwined nanowire network (scale bar 100 nm). Figure 2 As shown in c.

[0051] Comparative Example 1 This comparative example provides a baicalin-copper nanocomposite, the preparation method of which includes the following steps: The difference between this comparative example and Example 1 is that: baicalin and copper salt ( The mass ratio of the two components is 2.125:1, and the specific preparation steps are as follows: (1) Preparation of suspension A containing copper salt Weigh the compound (40 mg) and PVP (80 mg) were added to a mixture of 10 mL of water and methanol (water:methanol = 5:1, volume ratio). After stirring and dissolving, the solution was light blue. The pH was adjusted to 10 using NaOH (1 M), and light blue flocculent matter gradually appeared in the solution. After stirring continuously for 3 h, the solution became a homogeneous suspension A.

[0052] (2) Preparation of baicalin-copper nanowires Baicalein (85 mg) was dissolved in 5 mL of methanol and sonicated to obtain solution B, which was yellowish-green. This solution was then added dropwise to solution A, and the color rapidly changed from light blue to brownish-yellow. After stirring for half an hour, NaOH (1 M) was added to adjust the pH to 12, resulting in a dark brown solution with flocculent precipitate. After stirring for 12 hours, the solution was centrifuged, washed, and redispersed with deionized water to obtain baicalein-copper nanostructure aqueous dispersion C. The obtained baicalein-copper nanostructure is shown below. Figure 4 As shown in Figure a (scale bar 100 nm), it can be seen that the structure does not present a nanowire structure, but rather a network cross-linked material.

[0053] (3) Preparation of baicalin-copper nanocomposite 5 mL of baicalin-copper nanostructure aqueous dispersion C (1 mg / mL) was placed in a 25 mL open beaker, and 1 mL of HA (2.5 mg / mL) aqueous solution was added. After stirring and mixing, the solution turned yellowish-brown. The mixture was then placed in a shaker at room temperature and allowed to react in the dark for 12 h. After the reaction was complete, the precipitate was collected by centrifugation using a high-speed centrifuge (9000 rpm), washed with ultrapure water, and finally dispersed in ultrapure water to obtain the final product.

[0054] Comparative Example 2 This comparative example provides a baicalein-copper nanocomposite, and the preparation method comprises the following steps: The difference between this comparative example and Example 1 is that the mass ratio of baicalein to copper salt (CuSO4·5H2O) is 0.5:1, and the specific preparation steps are as follows: (1) Preparation of copper salt-containing suspension A Take compound (40 mg), PVP (80 mg), and add 10 mL of a mixture of water and methanol (water:methanol = 5:1, by volume). Stir and dissolve the solution, which is light blue. Adjust the pH to 10 using NaOH (1 M), and the solution gradually forms light blue flocculation. Continue stirring for 3 h, and the solution becomes a uniform suspension A.

[0055] (2) Preparation of baicalein-copper nanowires Dissolve baicalein (20 mg) in 5 mL of methanol to obtain solution B, which is yellowish. Then add it dropwise to solution A, and the color changes from light blue to light brown. Stir for half an hour, then adjust the pH to 12 using NaOH (1 M), and the solution gradually becomes clear. Stir for 12 h, then centrifuge and wash, and disperse in deionized water to obtain baicalein-copper nanostructure aqueous dispersion C. The obtained baicalein-copper nanostructure is shown in FIG. b (scale 100 nm), and it can be seen that the structure is not a nanowire structure, but a flaky or massive structure. Figure 4

[0056] (3) Preparation of baicalein-copper nanocomposite Place 5 mL of baicalein-copper nanostructure aqueous dispersion C (1 mg / mL) in a 25 mL open beaker, add 1 mL of HA (2.5 mg / mL) aqueous solution, stir and mix well, and the solution is yellowish brown. Place it in a shaking incubator at room temperature and avoid light for 12 h. After the reaction is completed, use a high-speed centrifuge (9000 rpm) to collect the precipitate, wash it with ultrapure water, and finally disperse it in ultrapure water to obtain the baicalein-copper nanocomposite.

[0057] Comparative Example 3 This comparative example provides a baicalein-copper nanocomposite, and the preparation method comprises the following steps: The difference between this comparative example and Example 1 is that the mass ratio of baicalein-copper nanowires to hyaluronic acid (HA) is 5:1, and the specific preparation steps are as follows: (1) Preparation of copper salt-containing suspension A Take compound (40 mg), PVP (80 mg), and add 10 mL of a mixture of water and methanol (water:methanol = 5:1, by volume). Stir and dissolve the solution, which is light blue. Adjust the pH to 10 using NaOH (1 M), and the solution gradually forms light blue flocculation. Continue stirring for 3 h, and the solution becomes a uniform suspension A. ​​​(40 mg), PVP (80 mg) were added into 10 mL of a mixture of water and methanol (water: methanol = 5:1, volume ratio) and stirred to dissolve, and the solution was light blue. The pH was adjusted to 10 using NaOH (1 M), and the solution gradually appeared as light blue flocculation. After 3 h of continuous stirring, the solution became a uniform suspension A.

[0058] (2) Preparation of baicalein-copper nanowires Baicalein (60 mg) was dissolved in 5 mL of methanol to obtain solution B, which was light yellow-green. Then, it was added dropwise into solution A, and the color gradually changed from light blue to tan. After stirring for half an hour, the pH was adjusted to 12 using NaOH (1 M), and the solution gradually became clear. After 12 h of stirring reaction, centrifugation and washing, and redispersion using deionized water, baicalein-copper nanowire aqueous dispersion C was obtained.

[0059] (3) Preparation of baicalein-copper nanocomposites 5 mL of baicalein-copper dispersion C (1 mg / mL) was placed in a 25 mL open beaker, 0.4 mL of HA (2.5 mg / mL) aqueous solution was added, and the solution was stirred and mixed to be reddish brown. After 12 h of reaction under room temperature and shaking in the dark, the precipitate was collected using a high-speed centrifuge (9000 rpm), washed using ultrapure water, and finally dispersed in ultrapure water. The final structure was found to be not uniform nanowires, but a coexistence of particles and nanowires (scale 20 nm), as shown in FIG. 1a. Figure 5

[0060] Comparative Example 4 This comparative example provides a baicalein-copper nanocomposite, and the preparation method thereof includes the following steps: The difference between this comparative example and Example 1 is that the mass ratio of baicalein-copper nanowires to hyaluronic acid (HA) is 0.25:1, and the specific preparation steps are as follows: (1) Preparation of copper salt-containing suspension A Compound (40 mg), PVP (80 mg) were added into 10 mL of a mixture of water and methanol (water: methanol = 5:1, volume ratio) and stirred to dissolve, and the solution was light blue. The pH was adjusted to 10 using NaOH (1 M), and the solution gradually appeared as light blue flocculation. After 3 h of continuous stirring, the solution became a uniform suspension A.

[0061] (2) Preparation of baicalein-copper nanowires ​Baicalein (60 mg) was dissolved in 5 mL of methanol and sonicated to obtain solution B, which was pale yellow-green. This solution was then added dropwise to solution A, and the color gradually changed from pale blue to brownish-yellow. After stirring for half an hour, NaOH (1 M) was added to adjust the pH to 12, and the solution gradually became clear. After stirring for 12 hours, the solution was centrifuged, washed, and redispersed with deionized water to obtain baicalein-copper nanowire aqueous dispersion C.

[0062] (3) Preparation of baicalin-copper nanocomposite 5 mL of baicalein-copper dispersion C (1 mg / mL) was placed in a 25 mL open beaker, and 1 mL of HA (20 mg / mL) aqueous solution was added. After stirring and mixing, the solution became a yellowish-brown jelly-like substance. The mixture was placed at room temperature and shaken in the dark for 12 h. Finally, it could not be washed and re-dispersed by ultrasonication, and its structural state was as follows. Figure 5 As shown in b.

[0063] Comparative Example 5 This comparative example provides a baicalin-copper nanocomposite, the preparation method of which includes the following steps: The difference between this comparative example and Example 1 is that NaOH was not added to adjust the pH of the reaction solution.

[0064] Weigh the compound (40 mg) and PVP (80 mg) were added to a mixture of 10 mL of water and methanol (water:methanol = 5:1, volume ratio) and stirred until dissolved, resulting in a pale blue solution. Subsequently, baicalin (60 mg) was dissolved in 5 mL of methanol and sonicated until dissolved, then added dropwise to the aforementioned copper salt solution. After stirring and reacting for 12 h, the mixture was centrifuged, washed, and redispersed with deionized water.

[0065] 5 mL of the above dispersion was placed in a 25 mL open beaker, and 1 mL of HA (2.5 mg / mL) aqueous solution was added. After stirring and mixing, the mixture was placed in a shaker at room temperature and reacted in the dark for 12 h. After the reaction was completed, the mixture was centrifuged, washed, and sonicated for further dispersion. Transmission electron microscopy (TEM) characterized its morphology as irregular flakes, without forming a uniform nanowire structure (scale bar 50 nm). Figure 6 As shown in Figure a.

[0066] Comparative Example 6 This comparative example provides a baicalin-copper nanocomposite, the preparation method of which includes the following steps: The difference between this comparative example and Example 1 is that in step (2), the pH is adjusted to 8.

[0067] (1) Preparation of suspension A containing copper salt Weigh the compound (40 mg), PVP (80 mg) were added into 10 mL of a mixture of water and methanol (water: methanol = 5: 1, volume ratio) and stirred to dissolve, and the solution was light blue. The pH was adjusted to 10 using NaOH (1 M), and the solution gradually appeared as light blue flocculation. After 3 h of continuous stirring, the solution became a uniform suspension A.

[0068] (2) Preparation of baicalein-copper nanowires Baicalein (60 mg) was dissolved in 5 mL of methanol to obtain solution B, which was light yellow-green. Then, it was added dropwise into solution A, and the color gradually changed from light blue to tan. After stirring for half an hour, the pH was adjusted to 8 using NaOH (1 M), and the solution was stirred for 12 h. After centrifugation and washing, and redispersion using deionized water, baicalein-copper aqueous dispersion C was obtained.

[0069] (3) Preparation of baicalein-copper nanocomposites 5 mL of baicalein-copper dispersion C (1 mg / mL) was placed in a 25 mL open beaker, 1 mL of HA (2.5 mg / mL) aqueous solution was added, and the solution was stirred and mixed to be brown. After 12 h of reaction at room temperature under shaking and light protection, the precipitate was collected using a high-speed centrifuge (9000 rpm), washed using ultrapure water, and finally dispersed in ultrapure water. The final structure is shown in FIG. 1b (scale bar 50 nm). As can be seen, no nanowires were formed, but rather extremely small and poorly crystalline nanoparticles. Figure 6

[0070] Comparative Example 7 This comparative example provides a baicalein-copper nanocomposite, and the preparation method thereof comprises the following steps: The difference between this comparative example and Example 1 is that in step (2), the pH is adjusted to 14.

[0071] (1) Preparation of copper salt-containing suspension A The compound (40 mg), PVP (80 mg) were added into 10 mL of a mixture of water and methanol (water: methanol = 5: 1, volume ratio) and stirred to dissolve, and the solution was light blue. The pH was adjusted to 10 using NaOH (1 M), and the solution gradually appeared as light blue flocculation. After 3 h of continuous stirring, the solution became a uniform suspension A.

[0072] (2) Preparation of baicalein-copper nanowires ​Baicalein (60 mg) was dissolved in 5 mL methanol to obtain solution B, which was a light yellow-green solution. Then it was added dropwise to solution A, and the color gradually changed from light blue to tan. After stirring for half an hour, NaOH (1 M) was added to adjust the pH to 14. After stirring for 12 h, centrifugal washing was performed, and re-dispersion was performed using deionized water to obtain a baicalein-copper water dispersion C.

[0073] (3) Preparation of a baicalein-copper nanocomposite 5 mL of baicalein-copper dispersion C (1 mg / mL) was placed in a 25 mL open beaker, 1 mL of HA (2.5 mg / mL) aqueous solution was added, and the solution was stirred and mixed to be brown. It was placed in a shaking bed at room temperature and reacted for 12 h in the dark. After the reaction was completed, the precipitate was collected by using a high-speed centrifuge (9000 rpm), washed with ultrapure water, and finally dispersed in ultrapure water. The final structure is shown in Figure 6 c (scale 50 nm), and it can be seen that a large number of irregular agglomerated structures are present.

[0074] Comparative Example 8 This comparative example provides a baicalein-copper nanocomposite, and the preparation method thereof comprises the following steps: The compound (40 mg) was weighed, 10 mL of a mixture of water and methanol (water:methanol = 5:1, by volume) was added, and the solution was stirred and dissolved to be light blue. Then baicalein (60 mg) was dissolved in 5 mL methanol and added dropwise to the copper salt solution, and the solution was stirred and reacted for 12 h. After centrifugal washing, re-dispersion was performed using deionized water to obtain a physical mixture of baicalein and copper salt.

[0075] Experimental Example 1 This experimental example performs structure and performance characterization on the baicalein-copper nanogel prepared in Example 1. As shown in Figure 7 a, the synthesized baicalein-copper nanogel contains Cu, O, N, and C elements. As shown in Figure 7 b, after baicalein is coordinated with copper, the electron cloud density is redistributed, and the peaks at 932.8 / 953.8 and 931.2 / 951.4 eV in the fine spectrum of copper ions correspond to Cu 2+ and Cu + (mixed valence copper), which proves that baicalein is successfully coordinated with copper ions, and the valence state of the copper ions contained in the nanowire is Cu 2+ and Cu + mixed valence, which is beneficial to the neutralization of active oxygen, nitrogen, and other free radicals in the subsequent antioxidant activity.

[0076] As shown in Figure 8As shown, the transmission electron microscopy (TEM) images show that the baicalein-copper nanogel synthesized by the embodiments of the present application can still exist stably in the gastric simulation solution (pH 1.5) and the intestinal simulation solution (pH 6.8) without dissociation or aggregation. The test conditions are as follows: to evaluate the gastrointestinal stability, 200 μL of the baicalein-copper nanogel solution was added to 4 mL of the simulated gastric solution (pH = 1.5) containing pepsin (3.2 mg / mL), and after 24 h, the supernatant was collected by centrifugation at 9000 r / min for 10 min. Similarly, another 200 μL of the nanogel solution was mixed with 4 mL of the simulated intestinal solution (pH = 6.8) containing trypsin (10 mg / mL), and after 24 h, the supernatant was collected by centrifugation at 9000 r / min for 10 min. Then, the gastrointestinal stability of the baicalein-copper nanogel was evaluated by transmission electron microscopy (TEM).

[0077] Application Example 1 The present application example provides an exploration of the antioxidant performance of the baicalein-copper nanogel prepared in Examples 1-6 and the baicalein-copper nanocomposite prepared in Comparative Examples 1-8. (1) Peroxidase-like enzyme scavenging experiment ① Test process: the prepared samples were dispersed in ultrapure water to prepare a solution with a concentration of 0.2 μg / mL, then 50 μL of H2O2 (50 mM) and 100 μL of 3,3',5,5'-tetramethylbenzidine solution TMB (10 mg / mL) were added to 2 mL of the above solution at room temperature, and the absorbance value at 652 nm was recorded after 5 min of reaction using a visible-ultraviolet spectrophotometer. The specific values are shown in Tables 1-2: Table 1

[0078] Table 2

[0079] As shown in Tables 1-2, compared with the comparative examples, the baicalein-copper nanogel prepared in Examples 1-6 has a stronger peroxidase-like enzyme scavenging effect; compared with Examples 2-6, the baicalein-copper nanogel prepared in Example 1 has the optimal peroxidase-like enzyme scavenging effect.

[0080] ②Test process: the baicalein-copper nanogel prepared in Example 1 was dispersed in ultrapure water to prepare solutions with concentrations of 0.2, 0.4, and 1.0 μg / mL, respectively, then 100 μL of 3,3',5,5'-tetramethylbenzidine solution (10 mg / mL) and H2O2 (50 mM) were added into 2 mL of the above prepared sample solution at room temperature, and the absorbance value at 652 nm was recorded by a visible-ultraviolet spectrophotometer after 5 min of reaction, and the test results are shown in Table 3: Table 3

[0081] As shown in Table 3 and Table 4, the baicalein-copper nanogel prepared in Example 1 has a gradually decreased absorbance value and an enhanced peroxidase-like effect with the increase of the concentration of the sample solution. Figure 9

[0082] (2) ABTS •+ radical scavenging experiment and DPPH • radical scavenging experiment ① ABTS· + radical scavenging experiment: 1) 1 mL of potassium persulfate solution (37.84 mg / mL, 2.6 mM) was mixed with 5 mL of ABTS solution (3.84 mg / mL, 7.4 mM) in a dark environment for 12 h to prepare an ABTS •+ working solution. Then, the ABTS stock solution was diluted with 80% (v / v) methanol or water or PBS to obtain a working solution with an absorbance of 0.6-1.0. Then, the sample solutions prepared in Examples and Comparative Examples (0.2 μg / mL) were added, respectively, and the reaction was carried out in the dark for 30 min. The absorbance of each sample at 734 nm was measured, and the ABTS •+ radical scavenging rate was calculated, and the test data are shown in Table 4-5: Table 4

[0083] Table 5

[0084] As shown in Table 4-5, compared with the comparative examples, the baicalein-copper nanogel prepared in Examples 1-6 has a stronger ABTS •+ radical scavenging effect; compared with Examples 2-6, the baicalein-copper nanogel prepared in Example 1 has the optimal ABTS •+ radical scavenging effect.

[0085] ​2) 1 mL of potassium persulfate solution (37.84 mg / mL, 2.6 mM) was placed in the dark for 12 h with 5 mL of ABTS solution (3.84 mg / mL, 7.4 mM) to prepare ABTS •+ working solution. Then, the ABTS stock solution was diluted with 80% (v / v) methanol or water or PBS to obtain a working solution with an absorbance of 0.6-1.0. Different concentrations of the baicalein-copper nanogel prepared in Example 1 (0.2, 0.4, 1.0 μg / mL) were added, and the reaction was carried out in the dark for 30 min. The absorbance of each group of samples at 734 nm was measured, and the ABTS •+ radical scavenging rate was calculated. The test data are shown in Table 6. Table 6

[0086] As shown in Table 6 and Figure 10 Table 7, with the increase of the concentration of the sample solution of the baicalein-copper nanogel prepared in Example 1, the ABTS •+ radical scavenging rate gradually increased, and the scavenging effect was enhanced.

[0087] ②DPPH • radical scavenging experiment 1) DPPH (11.8 mg / 2 mL) powder was dissolved in ethanol to obtain a DPPH working solution with an absorption peak at 517 nm, and attention was paid to prepare it immediately before use. The sample solution prepared in the examples and comparative examples (0.2 μg / mL) was added to the DPPH working solution, and the absorbance value at 517 nm was detected after the reaction in the dark for 30 min. The DPPH radical scavenging rate was calculated, and the test data are shown in Tables 7-8. Table 7

[0088] Table 8

[0089] As shown in Tables 7-8, compared with the comparative examples, the baicalein-copper nanogels prepared in Examples 1-6 have stronger DPPH radical scavenging effect; compared with Examples 2-6, the baicalein-copper nanogel prepared in Example 1 has the optimal DPPH radical scavenging effect.

[0090] 2) DPPH (11.8 mg / 2mL) powder was dissolved with ethanol to obtain DPPH working solution with absorption peak at 517 nm, which was prepared immediately before use. Different concentrations of baicalein-copper nanomicrogel (0.2, 0.4, 1.0 μg / mL) solution was added to the DPPH working solution, and the absorbance value at 517 nm was detected after reaction in the dark for 30 min, and the DPPH clearance rate was calculated. The test data are shown in Table 9: Table 9

[0091] As shown in Table 9 and Figure 11 It can be seen that the DPPH free radical clearance rate of the baicalein-copper nanomicrogel prepared in Example 1 gradually increases with the increase of the concentration of the sample solution, and the clearance effect is enhanced accordingly.

[0092] In summary: the absorbance of Example 1 is the lowest (0.117), and the free radical scavenging ability is the strongest. In Comparative Example 1, when the mass ratio of baicalein to copper salt is 2.125:1, a reticular crosslinked material is formed, which is not a nanowire structure and loses the catalytic site; in Comparative Example 2, when the mass ratio of baicalein to copper salt is 0.5:1, a flaky or massive structure is formed, the size is not uniform, and the active site is not exposed enough, resulting in poor peroxidase-like clearance effect and free radical clearance effect; In Comparative Example 3, when the mass ratio of baicalein-copper nanowire to hyaluronic acid is 5:1, the HA coating is not complete, the nanowire structure is unstable, and the active site is easily damaged and inactivated; in Comparative Example 4, when the mass ratio of baicalein-copper nanowire to hyaluronic acid is 0.25:1, the HA layer is too thick to hinder the free radicals from contacting the active site in the core; in Comparative Example 5 (random flaky without NaOH regulation), the activity is significantly reduced, all of which result in poor peroxidase-like clearance effect and free radical clearance effect; In Comparative Example 6, when the coordination reaction pH is less than 10, amorphous particles are formed, resulting in disordered structure; when the coordination reaction pH is greater than 12, the strong base will destroy the coordination bond, resulting in agglomeration, all of which result in poor peroxidase-like clearance effect and free radical clearance effect; In addition, since the coordination of baicalein and copper enhances the electron transfer ability and synergistically improves the enzyme-like activity, the peroxidase-like clearance effect and the free radical clearance effect of the example are much higher than those of single copper salt, single baicalein and physical mixing; and a higher concentration of sample solution can provide more active sites, accelerate the free radical clearance kinetics, and thus improve the clearance effect.

[0093] Application Example 2 The application example provides an exploration of the ROS clearance and cell protection ability and the immune regulation activity of M1 macrophages of the samples prepared in the example and the comparative examples ①ROS in cells is considered as a key messenger to amplify inflammatory responses and regulate the production of inflammatory mediators. To evaluate whether baicalein-copper nanowire microgels prepared in Example 1 can protect cells from oxidative stress, RAW264.7 cells activated by 3 mmol / L hydrogen peroxide were used as an in vitro inflammation model. The 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe was used to visualize the ROS signal in cells, and the Calcein AM / PI cell live and dead double staining kit was used to detect the live and dead conditions of cells after oxidative stress. In the ROS detection, the stronger green fluorescence indicates the higher level of active oxygen in cells; in the cell live and dead double staining, the green fluorescence of Calcein AM represents live cells, and the red fluorescence of PI represents dead cells. The addition concentration of the comparative samples (including the blank group, single copper salt, physical mixture of Comparative Example 8, and single lipopolysaccharide) is the same as that of the example.

[0094] The test results are shown in Figs. a~b in the drawings: Fig. a is a confocal image of the detection of active oxygen in cells. As can be seen from the figure, compared with untreated cells, the green fluorescence signal of the hydrogen peroxide stimulation group is significantly enhanced. Compared with baicalein, copper ions, and a physical mixture of baicalein and copper ions at the same concentration, the baicalein-copper nanowire microgels prepared in Example 1 have higher ROS scavenging capacity. In addition, as can be seen from the cell live and dead staining image of Fig. b, based on its good ROS scavenging activity, the baicalein-copper nanowire microgels prepared in Example 1 can effectively protect cells from apoptosis, and this group also shows the most number of live cells (the strongest green fluorescence). Figure 12

[0095] ②The imbalance of inflammatory mediators seriously damages the intestinal barrier function of UC patients and triggers excessive immune responses. After stimulation by lipopolysaccharide (LPS), RAW264.7 cells will differentiate into pro-inflammatory M1 phenotype and release various pro-inflammatory mediators such as chemokines and cytokines. The effects of baicalein-copper nanowire microgels on the secretion of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 by M1 macrophages were determined by enzyme-linked immunosorbent assay (ELISA).

[0096] As shown in Figs. a~b in the drawings: Figure 13 ​The levels of TNF-a, IL-1 β and IL-6 in RAW264.7 cells treated with 0.2 μg / mL of LPS for 24 h were tested. As shown in the figure, compared with normal RAW264.7 cells, the expression of proinflammatory cytokines (IL-6, IL-1 β and TNF-a) in LPS-treated RAW264.7 cells was significantly increased. Different treatment groups (single copper salt, single baicalein and physical mixing group of Comparative Example 8) reduced the levels of inflammatory factors in LPS-stimulated RAW264.7 cells to a certain extent, but the baicalein-copper nanowire microgel prepared in Example 1 had the best inhibitory effect on the expression of proinflammatory cytokines among all the treatment groups, showing a stronger anti-inflammatory effect.

[0097] Application Example 3 This application example provides a safety and effectiveness determination of the baicalein-copper nanowire microgel prepared in Example 1 for ulcerative colitis ①Cytotoxicity determination experiment: 1) L929 cells in logarithmic growth phase were inoculated in 96-well plates, and after 24 h of culture, different concentrations of baicalein-copper nanowire microgels (copper ion concentrations were 3.175, 6.25, 12.5, 25, 50 and 100 μg / mL) were added, and after 24 h of further culture, toxicity detection was performed.

[0098] The test results are shown in Table 1. Figure 14 As the concentration of copper ions increased, the cell survival rate was maintained above 70%, indicating that the baicalein-copper nanowire microgel prepared in this example had good biocompatibility and did not have obvious toxicity to mouse fibroblasts.

[0099] 2) RAW264.7 cells in logarithmic growth phase were inoculated in 96-well plates, and after 24 h of culture, different concentrations of baicalein-copper nanowire microgels (copper ion concentrations were 1, 2.5, 5, 10 and 20 μg / mL) were added, and after 24 h of further culture, toxicity detection was performed.

[0100] The test results are shown in Table 2. Figure 15 As the concentration of copper ions increased, the cell survival rate was maintained at about 100%, indicating that the baicalein-copper nanowire microgel prepared in this example had good biocompatibility and did not have obvious toxicity to mouse macrophages.

[0101] The C57 mice were fed with 3% DSS water solution for 6 days to successfully model ulcerative colitis, and then the baicalein-copper nanogel prepared in Example 1 was used for continuous gavage treatment for 4 days from the 7th day, with a dose of 5 mg / kg, and the control group was gavaged with the same volume of normal saline. The mice were euthanized on the 12th day, and the colon tissue was removed for observation and measurement.

[0102] The test results are shown in Table 1. Figure 16~17 As shown in Table 1, the colon tissue of the mice in the model group was obviously congested and edematous, obvious blood was visible in the feces, and the colon was obviously shortened and thickened, with a colon length of about 5.2 cm; which was significantly shorter than the normal group (p<0.001). After drug treatment, the symptoms of colon length shortening and edema were improved, and the colon length of the treatment group was about 7.0 cm, close to the normal group. Therefore, it is shown that the baicalein-copper nanogel prepared in the embodiment of the present application has the potential to relieve the symptoms of ulcerative colitis, and it can restore the colon length to the normal level at a small dose (5 mg / kg).

[0103] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A baicalin-copper nanogel, characterized in that, The baicalein-copper nanogel comprises a nanowire core formed by baicalein-copper coordination and a hyaluronic acid coating layer covering it; the mass ratio of baicalein to copper in the nanowire core is 1~2:1; the mass ratio of the nanowire core to hyaluronic acid is 0.5~2.5:

1.

2. The baicalin-copper nanogel as described in claim 1, characterized in that, The copper in the nanowire core is in a mixed valence state, Cu. + / Cu 2+ The nanowire core is formed by the coordination of copper ions with baicalin.

3. The baicalin-copper nanogel as described in claim 1, characterized in that, The hydration radius of the nanowire core is 90~98 nm, and the hydration radius of the baicalin-copper nanogel is 100~120 nm. Preferably, the hydration radius of the nanowire core is 96 nm, and the hydration radius of the baicalin-copper nanogel is 110 nm; Preferably, the baicalin-copper nanogel exhibits stability for ≥24 hours in simulated gastrointestinal solutions at pH 1.5 and pH 6.8 without dissociation or aggregation.

4. A method for preparing baicalin-copper nanogel according to any one of claims 1 to 3, characterized in that, The process includes the following steps: mixing copper salt and surfactant in a mixed solvent, adding NaOH solution as a regulator and stirring to react, then adding baicalein solution to the reaction solution, adjusting the pH and reacting at room temperature to obtain baicalein-copper nanowire dispersion, and then mixing with hyaluronic acid aqueous solution to obtain baicalein-copper nanogel.

5. The preparation method according to claim 4, characterized in that, The copper salt is selected from CuSO4·5H2O and CuSO4; the surfactant is selected from one or more of polyvinylpyrrolidone and polyvinyl alcohol; the mass ratio of the copper salt to the surfactant is 0.2~1:

1.

6. The preparation method according to claim 4, characterized in that, The mixed solvent is obtained by mixing water and an organic solvent, wherein the organic solvent is selected from one or more of methanol and ethanol; the volume ratio of water to organic solvent is 1 to 5:

1. Preferably, the concentration of the NaOH solution is 0.5~1.0 M to adjust the pH to 8~12; Preferably, the temperature of the stirring reaction is 25~50℃ and the time is 0.5~3 h.

7. The preparation method according to claim 4, characterized in that, The baicalein solution is prepared by dissolving baicalein in an organic solution, wherein the organic solution is selected from one or more of methanol, ethanol, and acetone, and the concentration of the baicalein solution is 10~60 mg / mL.

8. The preparation method according to claim 4, characterized in that, The pH is adjusted to 10-12, the reaction time at room temperature is 12-24 h, and then the product is dispersed in deionized water after centrifugation and washing to obtain baicalin-copper nanowire dispersion; wherein the centrifugation speed is 8000-12000 rpm and the time is 10-15 min.

9. The preparation method according to claim 4, characterized in that, The volume ratio of the baicalin-copper nanowire dispersion to the hyaluronic acid aqueous solution is 5~10:1, preferably 5:1; the concentration of the hyaluronic acid aqueous solution is 2.5~10 mg / mL, and the concentration of the baicalin-copper nanowire dispersion is 1~5 mg / mL; the mixing reaction temperature is 25~35℃, and the time is 12~24 h.

10. The use of the baicalin-copper nanogel according to any one of claims 1 to 3 in the preparation of a drug for treating inflammatory diseases.

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